ISEV2024 Abstract Book (2024)

About ISEV

The International Society for Extracellular Vesicles is the leading professional society for researchers and scientists involved in the study of microvesicles and exosomes. With nearly 1,000 members, ISEV continues to be the leader in advancing the study of extracellular vesicles. Founded in 2012 in Sweden, ISEV has since moved its Headquarters to the United States. Through its programs and services, ISEV provides essential training and research opportunities for those involved in exosome and microvesicle research.

Mission Statement

Advancing extracellular vesicle research globally.

Vision

Our vision is to be the leading advocate and guide of extracellular vesicle research and to advance the understanding of extracellular vesicle biology.

ISEV2024 Annual Meeting

The International Society for Extracellular Vesicles is the is the premier international conference of extracellular vesicle research, covering the latest in exosomes, microvesicles and more. With an anticipated 1,000+ attendees, ISEV2024 will feature presentations from the top researchers in the field, as well as providing opportunities for talks from students and early career researchers.

ISEV2023 International Organizing Committee

IOC Chairs: Cherie Blenkiron (New Zealand), David Greening (Australia)

IOC Members: Randy Carney (USA), Leslie Cheng (Australia), Eisuke Dohi (Japan), Qing‐Ling Fu (China), Charles Lai (Taiwan), Metka Lenassi (Slovenia), Andreas Moeller (China), Jisook Moon (South Korea), Natalie Turner (Australia)

Journal of Extracellular Vesicles: Editors in Chief

Jan Lötvall (Sweden)

0T04.O02 Cellular interaction and uptake of human endogenous retrovirus (HERV) envelope‐displaying EVs

Dr. Zach Troyer, Sarah Marquez, PhD Olesia Gololobova, PhD Kenneth Witwer

0T04.O03 Functionalized engineered extracellular vesicles for targeted delivery to intervertebral disc cells

Ms Mia Kordowski, Dr Ana Salazar‐Puerta, Ms María Rincon‐Benavides, Mr Justin Richards, Dr Nina Tang, Dr Safdar Khan, Dr Elizabeth Yu, Dr Judith Hoyland, Dr Devina Purmessur, Dr Natalia Higuita‐Castro

0T04.O04 Phospholipid scrambling: a novel regulator of extracellular vesicle cargo packaging and function

Ms Akbar Marzan, Ms Monika Petrovska, Professor Suresh Mathivanan, Sarah Stewart

0T04.O05 Quantitative features of extracellular vesicle‐mediated crosstalk in multi‐cellular 3D tumor models

Dr. Maria Harmati, Akos Diosdi, Ferenc Kovács, Ede Migh, Gabriella Dobra, Timea Boroczky, Matyas Bukva, Edina Gyukity‐Sebestyen, Peter Horvath, Krisztina Buzas

FA01 Extracellular vesicles in human body fluids compete with virus particles for binding of phosphatidylserine receptors to prevent infection and transmission

Dr. Ruediger Gross, Hanna Reßin, Pascal von Maltitz, Dan Albers, Laura Schneider, Hanna Bley, Markus Hoffmann, Mirco Cortese, Dhanu Gupta, Miriam Deniz, Jae‐Yeon Choi, Jenny Jansen, Christian Preußer, Kai Seehafer, Stefan Pöhlmann, Dennis R Voelker, Christiane Goffiniet, Elke Pogge‐von Strandmann, Uwe Bunz, Ralf Bartenschlager, Samir El Andaloussi, Konstantin MJ Sparrer, Eva Herker, Stephan Becker, Frank Kirchhoff, Jan Münch, Janis A Müller

FA02 Machine learning models detect blood ‘fingerprints’ for accurate glioblastoma tumour surveillance

Dr Susannah Hallal, Dr Ágota Tűzesi, Dr Abhishek Vijayan, Dr Laveniya Satgunaseelan, Associate Professor Hao‐Wen Sim, Associate Professor Brindha Shivalingam, Associate Professor Michael Buckland, Associate Professor Fatemeh Vafaee, Dr Kimberley Alexander

FA03 Barcoding of small extracellular vesicles with CRISPR‐gRNA enables high‐throughput, subpopulation‐specific analysis of their release regulators

Prof. Dr. Ryosuke Kojima, Mr. Koki Kunitake, Professor Tadahaya Mizuno, Professor Yasuteru Urano

FA04 In vivo visualization of endothelial cell‐derived extracellular vesicle formation in steady state and malignant conditions

Dr Georgia Atkin‐Smith, Jascinta Santavanond, Amanda Light, Joel Rimes, Andre Samson, Jeremy Er, Joy Liu, Darryl Johnson, Melanie Le Page, Pradeep Rajasekhar, Raymond Yip, Niall Geoghegan, Kelly Rogers, Catherine Chang, Vanessa Bryant, Mai Margetts, Cristina Keightley, Trevor Kilpatrick, Michele Binder, Sharon Tran, Erinna Lee, Doug Fairlie, Dilara Ozkocak, Andrew Wei, Edwin Hawkins, Ivan Poon

LB01.O01 Fetal Exposure to Extracellular Vesicles. Is it safe?

Dr Ishmael Inocencio², Mr Naveen Kumar², A/Prof Rebecca Lim², Dr Tamara Yawno²

¹Hudson Institute Of Medical Research, Clayton, Australia, ²The Ritchie Centre, Clayton, Australia

LB01.O02 Engineered EVs as mRNA cancer vaccine delivery platform conferring immune modulation in HCC

Lecturer Cong He^1,2, Guangxin Shao², Dr. Yumin Li⁴, Dr. Xiao Yun⁵, Dr. Bo Sun⁴, Prof. Zhongdang Xiao⁴, Prof. Beicheng Sun³

¹Jiangsu Key Laboratory for Biofunctional Molecules, College of Life Science and Chemistry, Jiangsu Second Normal University, Nanjing, China, ²Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, China, ³Department of Hepatobiliary Surgery, The First Affiliated Hospital of Anhui Medical University, Hefei, China, ⁴State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China, ⁵Department of General Surgery, The Affiliated Changzhou No. 2 People's Hospital of Nanjing Medical University, Changzhou, China

LB01.O03 Extracellular vesicles derived from human umbilical cord mesenchymal stem cells attenuate septic acute kidney injury by delivering miR‐125a‐5p and miR‐125b‐5p to inhibit inflammation and glycolysis

Dr. Feng Chen^1,2, Dr Tao‐Tao Tang², Dr. Zhi‐qing Chen², Prof. Zhong Wang¹, Dr Bi‐Cheng Liu²

¹Tsinghua University, Beijing, China, ²Institute of Nephrology, Zhong Da Hospital, Southeast University School of Medicine, Nanjing, China

LB01.O04 Extracellular vesicles derived from clonally expanded, immortalized mesenchymal stromal cells lower Alzheimer's pathology in mice

Dr Lien Van Hoecke¹, Yanis Mouloud², Tobias Tertel², Prof Bernd Giebel², Prof Roosmarijn E Vandenbroucke¹

¹VIB‐UGent, Gent (Zwijnaarde), Belgium, ²University Hospital Essen, University Duisburg‐Essen, Essen, Germany

LB02.O01 Lipid droplets are packaged within extracellular vesicles during virus infection

Dr Ebony Monson¹, Miss Irumi Amarasinghe¹, Mr William Phillips², Dr Amy Baxter², Ms Camille Braganca¹, Ms Abbey Milligan², Dr Donna Whelan², Dr Eduard Willms², Professor Andrew Hill², Professor Karla Helbig¹

¹Department of Microbiology, Anatomy, Physiology & Pharmacology, La Trobe University, Melbourne, Australia, ²La Trobe Institute for Molecular Sciences, La Trobe University, Melbourne, Australia, ³Institute for Health and Sport, Victoria University, Melbourne, Australia

LB02.O02 Breast cancer sEVs binding to CCL2 and other cytokines directs cancer metastasis organotropism

Dr Luize Lima¹, Dr Sunyoung Ham^1,2,3, Professor Andreas Möller^1,2,3

¹Tumour Microenvironment Laboratory, QIMR Berghofer Medical Research Institute, Herston, Australia, ²Department of Otorhinolaryngology, Head and Neck Surgery, Chinese University of Hong Kong, Shatin, Hong Kong, ³School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Australia

LB02.O03 Tubule derived exosomal Integrin reside in ECM microenvironment activates latent TGF‐β1 in renal fibrosis

Phd Anran Shen^{Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China}, Phd Xin Zhong^{Institute ofNephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China}, Phd Ning Li^{Institute of Nephrology, Zhongda Hospital, SoutheastUniversity School of Medicine, Nanjing, China}, Phd Yuqi Fu^{Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China}, Professor Linli Lv^{Institute of Nephrology, Zhongda Hospital, Southeast University School of Medicine, Nanjing, China}

¹Medical School Of Southeast University, Nanjing, China

LB02.O04 Enhanced Packaging of U6 Small Nuclear RNA and Splicing‐Related Proteins into Extracellular Vesicles During HIV Infection

Dr Yiyao Huang^1,2, Ahmed Abdelgawad³, Dr Olesia Gololobova¹, Zhaohao Liao¹, Xinyu Cong⁴, Associate Professor Mona Batish³, Prof. Lei Zheng², Dr Kenneth Witwer^1,5,6

¹Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, Baltimore, United States, ²Department of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China, ³Department of Medical and Molecular Sciences, University of Delaware, Newark, United States, ⁴Division of Biostatistics and Bioinformatics, Department of Environmental and Public Health Sciences, University of Cincinnati, Cincinnati, United States, ⁵Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, United States, ⁶Richman Family Precision Medicine Center of Excellence in Alzheimer's Disease, Johns Hopkins University School of Medicine, Baltimore, United States

LB03.O01 Protein mutation identification and monitoring using exosomes‐SERS‐AI

Master's degree Kim Seungmin, Doctor of Philosophy (Ph.D.) ByeongHyeon Choi, Hyunku Shin, Master's degree Kihun Kwon, Doctor of Philosophy (Ph.D.) Sung Yong Lee, Doctor of Philosophy (Ph.D.) Hyun Koo Kim, Doctor of Philosophy (Ph.D.) Yeonho Choi

¹Department of Biomedical Engineering, Korea University, Seoul, South Korea, ²Korea Artificial Organ Center, Korea University, Guro, Republic of Korea, ³Department of Thoracic and Cardiovascular Surgery, Korea University, Guro, Republic of Korea, ⁴Exopert Corporation, Seoul, Republic of Korea, ⁵Department of Internal Medicine, Korea University, Guro, Republic of Korea, ⁶School of Biomedical Engineering, Korea University, Seoul, Republic of Korea

LB03.O02 Proteomic investigations of mechanisms underlying high‐dose sodium ascorbate in sepsis in circulating plasma extracellular vesicles

Dr Samantha Emery‐Corbin^{1. Division of Advanced Technology and Biology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria,Australia 2. Department of Medical Biology, The University of Melbourne, Parkville, VIC, Australia}, Dr Jumana Yousef^{1. Division of Advanced Technology andBiology Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia 2. Department of Medical Biology, The University of Melbourne,Parkville, VIC, Australia}, Professor Yugeesh R Lankadeva^{3. The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville,Victoria, Australia 4. Department of Critical Care, University of Melbourne, Parkville, Victoria, Australia}, Professor Rinaldo Bellomo^{4. Department of CriticalCare, University of Melbourne, Parkville, Victoria, Australia 5. Department of Intensive Care, Austin Hospital, Melbourne, Victoria, Australia 6. Australian and NewZealand Intensive Care Research Centre (ANZIC‐RC), Monash University, Melbourne, Victoria, Australia 7. Department of Intensive Care, Royal Melbourne Hospital,Parkville, Victoria, Australia 8. Data Analytics Research and Evaluation Centre, Austin Hospital, Melbourne, Victoria, Australia}, Dr Fumitaka Yanase^{5.Department of Intensive Care, Austin Hospital, Melbourne, Victoria, Australia 6. Australian and New Zealand Intensive Care Research Centre (ANZIC‐RC), MonashUniversity, Melbourne, Victoria, Australia}, Associate Professor Mark P Plummer^{9. Department of Intensive Care, Royal Adelaide Hospital, Adelaide, SouthAustralia, Australia}, Professor Clive N May^{3. The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, Australia4. Department of Critical Care, University of Melbourne, Parkville, Victoria, Australia}, Dr Laura F Dagley^{1. Division of Advanced Technology and BiologyDivision, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia 2. Department of Medical Biology, The University of Melbourne, Parkville,VIC, Australia}

¹Division of Advanced Technology and Biology Division, Walter And Eliza Hall Institute Of Medical Research, Melbourne, Australia, ²Department of Medical Biology, University of Melbourne, Melbourne, Australia, ³The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne, Australia, ⁴Department of Critical Care, University of Melbourne, Melbourne, Australia, ⁵Department of Intensive Care, Austin Hospital, Melbourne, Australia, ⁶Australian and New Zealand Intensive Care Research Centre (ANZIC‐RC), Monash University, Melbourne, Australia, ⁷Department of Intensive Care, Royal Melbourne Hospital, Melbourne, Australia, ⁸Data Analytics Research and Evaluation Centre, Austin Hospital, Melbourne, Australia, ⁹Department of Intensive Care, Royal Adelaide Hospital, Adelaide, Australia

LB03.O03 Framework for isolating EVs from neurons and measuring their cargo

Senior Staff Scientist Dmitry Ter‐Ovanesyan¹, Sara Whiteman, Tal Gilboa, Siddharth Iyer, Bogdan Budnik, Aviv Regev, George Church, David Walt

¹Wyss Institute, Harvard University, Boston, United States

LB03.O04 Identification of a circulating HNSCC proteomic biomarker signature from peripheral and local tumor plasma before/after tumor removal surgery

Dapi Menglin Chiang, Dr. Christina Ludwig, Dr. Chen Meng, PD Dr. Marlene Reithmair, Laura Benecke, Yannik da Silva, PD Dr. Laurent Müller, Prof. Dr. Michael W. Pfaffl

LB04.O01 Detection of EVs in hepatotoxicity using CD9‐mEmerald reporter mice

Section Chief Ryuichi Ono¹, Mie Naruse², Makiko Kuwagata¹, Yusuke Yoshioka³, Yoko Hirabayashi¹, Takahiro Ochiya³, Masahito Ikawa⁴, Satoshi Kitajima¹

¹National Institute Of Health Sciences, Japan, Kawasaki, Japan, ²National Cancer Center Research Institute, Tokyo, Japan, ³Tokyo Medical University, Tokyo, Japan, ⁴Osaka University, Osaka, Japan

LB04.O02 Drafting the proteome and lipidome atlas of circulating small EVs in humans: Universal protein and lipid features

Dr Alin Rai¹, Prof David Greening

¹Baker Heart and Diabetes Institute, MELBOURNE, Australia

LB04.O03 Enhanced endosomal escape by amphipathic‐charged engineered extracellular vesicles

Dr Hema Saranya Ilamathi^1,2, Dr Doste Mamand^1,2, Anna Maria Zimbo³, Dr Samir El Andaloussi^1,2, Dr Oscar Wiklander^1,2

¹Division of Biomolecular and Cellular Medicine, Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden, ²Center for Cell Therapy and Allogeneic Stem Cell Transplantation (CAST), Karolinska University Hospital, Stockholm, Sweden, ³Department of Experimental and Clinical Medicine, Magna Graecia University of Catanzaro, Italy

LB04.O04 Depletion‐zone isotachophoresis of extracellular vesicles: a separation method suitable for biomarker discovery applications that can increase purity over current techniques

Dr. Andrea Capuano¹, Meia Numan², Prof. Thomas Hankemeier¹

¹University of Leiden, Leiden, The Netherlands, ²EXIT071 B.V., Leiden, The Netherlands

OF09.O02 Enhancing breast cancer diagnosis and subtyping: multiplexed profiling of dual surface protein‐expressing single extracellular vesicles via nano‐flow cytometry

Phd Yunyun Hu, Haonan Di, PhD Ye Tian, Yiyin Weng, Jujiang Guo, Professor Xiaomei Yan

OF09.O03 Analyzing copy number variation status in extracellular vesicles as novel clinical biomarkers of high‐grade serous ovarian carcinoma

MD. Ryosuke Uekusa, Dr. Akira Yokoi, Dr. Kosuke Yoshida, Dr. Jyuntaro Matsuzaki, Dr. Yusuke Yamamoto, Dr. Hiroaki Kajiyama

OF09.O04 Extracellular vesicles as biomarkers of endometriosis – a comparison between peritoneal fluid and peripheral blood

Ms Chloe James, Ms Johanna Farley, Ms Natasha Borash, Dr Hannah Nazri, Ms Anna Tresso, Dr Shima Bayat, Dr Anup Shah, Dr Joel R Steele, Prof Ralf B Schittenhelm, Dr Shanti Gurung, Prof Caroline E Gargett, Prof Christian M Becker, Prof Beverley Vollenhoven, Dr Thomas Tapmeier

OF09.OWP01 Paracrine miRNA communication via extracellular vesicles: regulating glucose metabolism and fetal growth in gestational diabetes mellitus between maternal adipose tissue and placenta in vivo

Dr Nanthini Jayabalan, Dr Andrew Lai, Dr Dominic Guanzon, Dr Soumyalekshmi Nair, Mrs Katherin Scholz‐Romero, Valeska Ormazabal, Professor Aasa Handberg, Dr Flavio Carrion, Professor Harold McIntyre, A/ Professor Martha Lappas, Professor Carlos Salomon

OF09.OWP02 Profiling of single‐vesicle surface proteins via droplet digital immuno‐PCR for multi‐subpopulation extracellular vesicles counting towards cancer diagnostics

Dr. Huixian Lin, Dr. Chunchen Liu, Prof. Bo Li, Prof. Lei Zheng

OF09.OWP03 Proteomics of salivary exosomes: a potential breakthrough for screening of oral cancer among tobacco consumers

Ms Afsareen Bano, Dr. RASHMI BHARDWAJ

OF10.O02 Are the extracellular vesicles released by Parabacteroides goldsteinii anti‐inflammatory?

Dr Simon Swift, Yevetta Xiang, Dr Priscila Dauros‐Singorenko, Professor Hsin‐Chih Lai

OF10.O03 Physicochemical and immunomodulatory properties of Bifidobacterium‐ derived extracellular vesicles with anti‐allergic potential

Ms Dominika Kozakiewicz, Dr Agnieszka Razim, Dr Sabina Górska

OF10.O04 Bacterial growth conditions and mechanisms of biogenesis alter the composition and functions of bacterial extracellular vesicles

Dr Ella Johnston, Dr Lauren Zavan, Associate Professor David Greening, Professor Andrew Hill, Associate Professor Maria Kaparakis‐Liaskos

OF10.O05 Klebsiella pneumoniae‐derived extracellular vesicles facilitate bacterial translocation from the gastrointestinal tract to the liver by inducing the M2‐like macrophage phenotype

Hitoshi Tsugawa, Student Shogo Tsubaki, Dr Takuma Araki, Dr. Yusuke Yoshioka, Dr. Juntaro Matsuzaki, Dr Hitoshi Tsugawa

OF11.O02 Endothelial cell‐derived extracellular vesicles modulate bone marrow in cardiovascular disease

Mr Lewis Timms, Dr Daniel Radford Smith, Prof Daniel C. Anthony, Associate Prof Naveed Akbar, Prof Robin P. Choudhury

OF11.O03 Renal tubule‐derived EVs carrying complement 3 aggravate vascular calcification of CKD by downregulating autophagy in vascular smooth muscle cells

Yuxia Zhang, Associate Researcher Taotao Tang, Professor Rining Tang, Professor Bicheng Liu

OF11.O04 Small extracellular vesicles (sEV) mediate tubular ferroptosis propagation in the transition from acute to chronic kidney disease

Ms Xiangju Wang, A/Prof Chang Seong Kim, Mr Benjamin Adams, Dr Monica Ng, A/Prof Helen Healy, Dr Andrew Kassianos

OF11.O05 Role of cardiomyocyte‐derived extracellular vesicles in post‐ischemic cardiac remodeling

Phd Lélia Borowski, Cécile Devue, Paul Alayrac, Jean Sébastien Silvestre, Chantal M. Boulanger, Xavier Loyer, Stéphane Camus

OF12.O02 Ex vivo imaging of exosomes in Drosophila secondary cells reveals a novel membrane microdomain involved in exosome biogenesis

Mr Adam Wells, Dr Pauline Marie, Dr Claudia C. Mendes, Dr Shih‐Jung Fan, Dr Mark Wainwright, Dr. Preman Singh, Dr. Bhavna Verma, Professor Clive Wilson, Dr Deborah Goberdhan

OF12.O03 Mutation in ESCRT‐II component VPS25 alters small extracellular vesicle processing in lethal neonatal encephalopathy

Ioannis Isaioglou, Lama AlAbdi, Yossef Lopez de los Santos, Muhammad Tehseen, Mansour Aldehaiman, Gloria Lopez‐Madrigal, Norah Altuwaijri, Maya Ayach, Ashraf Al‐Amoudi, Rachid Sougrat, Vlad‐Stefan Raducanu, Amani Al‐Amodi, Hessa Alsaif, Firdous Abdulwahab, Amal Jaafar, Tarfa Alshidi, Adriana Montaño, Kara Klemp, Ellen Totten, Wesam Kurdi, Samir Hamdan, Stephen Braddock, Fowzan Alkuraya, Jasmeen Merzaban

OF12.O04 Expressing the human proteome in Saccharomyces cerevisiae as a model for advancing extracellular vesicle biology

Mr. Joseph Trani, Dr. Aashiq H. Kachroo, Dr. Christopher L. Brett

OF12.O05 The unexpected formation of the footprint of death during apoptosis

Ms Stephanie Rutter, Ms Amy Hodge, Ms Dilara Ozkocak, Dr Julian Ratcliffe, Dr Taeyoung Kang, Dr Niall Geoghegan, Dr Pradeep Rajasekhar, Dr Georgia Atkin‐Smith, Dr Ivan Poon

OF13.O02 Shear Stress‐Induced Extracellular Calcium Influx: A Pivotal Trigger Amplifying Mesenchymal Stem Cell‐derived Extracellular Vesicle Production

Ph.D candidate Youngju Seo¹, Ibio hyejin Kang, Ibio, Mechanical Engineering Jaesung Park

¹Postech, South Korea

OF13.O03 Acellular therapy with umbilical cord mesenchymal stem‐derived small extracellular vesicles or mitochondria, as a new treatment for osteoarthritis

Miss Cynthia Garcia Guerrero, Patricia Luz‐Crawford, Ana Maria Vega‐Letter, Carolina Pradenas, Alexander Ortoff, Jose Barraza, Fernando Figueroa, Maroun Khoury, Aliosha Figueroa, Yeimi Herrera

OF13.O04 Improved neurological recovery in a rodent ischemic stroke model using human GMP compatible embryonic vascular progenitor cell exosomes

Scientist Jieun Lee

OF13.O05 Examining the efficacy of Immortalised Human Amniotic Epithelial Derived Extracellular Vesicles in a rodent model of perinatal brain injury.

Mr Naveen Kumar¹, Dr Ishmael Inocencio, Dr Tamara Yawno, Dr Dandan Zhu, Associate Professor Rebecca Lim

¹Hudson Institute Of Medical Research, Clayton, Australia

OF13.O06 Small extracellular vesicles from metabolically reprogrammed mesenchymal stem/stromal cell as a potential immunosuppressive mechanism

Miss Eliana Lara Barba, Miss Yesenia Flores Elías, Mr Felipe Bustamente Barrientos, Miss María Jesús Araya, Miss Yeimi Herrera Luna, Miss Noymar Luque Campos, Ms Ana María Vega Letter, Ms Patricia Luz Crawford

OF14.O02 Biomarkers from neuronal‐enriched EVs predict resilience to Alzheimer's disease in the presence of APOE ε4 allele: findings from a large longitudinal study

Dimitrios Kapogiannis, Maja Mustapic, Carlos Nogueras‐Ortiz, Apostolos Manolopoulos, Francheska Delgado‐Peraza, Pamela Yao, Krishna Pucha, Mark A Espeland, Luigi Ferrucci, Stephen R. Rapp, Susan M. Resnick

OF14.O03 Identification of PECAM1+ and ITGB1+ plasma extracellular vesicle as biomarkers of unruptured intracranial aneurysm based on single extracellular vesicle proximity barcoding assay

Dr. Hao Tian, Dr. Yanling Cai, Mrs. Fang Wang, Professor Chuanzhi Duan, Dr. Haitao Sun

OF14.O04 A liquid biopsy approach: Neural networks‐based identification of brain tumor exosomes via theirSERSsignatures

Hülya Torun^{Stanford and Koç University}, PhD Ugur Parlatan, Chris Nguyen, BS Tim Valencony, MS Furkan Kaysin, PhD Ozgur Albayrak, MD Ibrahim Kulac, MD, PhD Candidate Oguz Baran, MD, PhD Candidate Goktug Akyoldas, MD Ihsan Solaroglu, PhD Utkan Demirci, PhD, DVM Demir Akin, PhD Mehmet Ozgun Ozen

OF14.O05 Nanoscale Flow Cytometry Quantification of Blood‐based Extracellular Vesicle Biomarkers Distinguishes MCI and Alzheimer's Disease.

Dr. Thamara Dayarathna, Dr. Austyn Roseborough, Dr. Janice Gomes, Dr. Reza Khazaee, Dr. Shaun Whitehead, Dr. Hon Sing Leong, Professor Stephen Pasternak

OF14.O06 Comparative analysis of plasma‐derived small extracellular vesicles and whole plasma‐derived miRNAs as biomarker targets for Parkinson's disease

Ms. Sanskriti Rai, Mr. Rishabh Singh, Dr. Prahalad Singh Bharti, Dr. Roopa Rajan, Dr. Saroj Kumar

OF15.O02 Encapsulate the components of CRISPR/Cas9 into extracellular vesicles by protein palmitoylation

Ph.d Yaoyao Lu, research assistant Nathalie Majeau, Ph.D Gabriel Lamothe, Research assistant Joel Rousseau, Professor Jacques‐P Tremblay

OF15.O03 Nanofluidic platform with ultrahigh‐throughput for versatile loading of small extracellular vesicles

Hui Yang, PhD Rui Hao, PhD Candidate Zitong Yu, PhD Candidate Shi Hu, PhD Yanhang Hong, Professor Yi Zhang, Sihui Chen

OF15.O05 Affinity‐based bategorization of antimicrobial and cell‐penetrating peptides in vesicle interactions

Phd Tamas Beke‐Somfai, Tasvilla Sonallya, PhD Imola Cs. Szigyarto, PhD Tunde Juhasz, Kinga Ilyes, Priyanka Singh, Delaram Khamari, DSc Edit Buzas, PhD Zoltan Varga

OF15.O06 Tonicity‐driven osmotic cargo loading for engineering extracellular vesicles

Professor Yoon‐Kyoung Cho, Chaeeun Lee, Sumit Kumar

OF16.O02 Cancer‐derived small extracellular vesicles reprogram the DNA methylome of normal epithelial cells adjacent to the primary cancer

Hanguo Jiang, Professor Zhijie Chang

OF16.O03 ULK1 enhances biogenesis of oncogenic small extracellular vesicles (sEV) to induce tumorigenesis and metastasis in hepatocellular carcinoma (HCC)

Mr Samuel Wan Ki Wong, Miss Claudia Wing Lam Tam, Mr Nicolas Cheuk Hang Lau, Dr Xiaowen Mao, Prof Judy Wai Ping Yam

OF16.O04 Extracellular vesicle encapsulated miR‐1307‐5p confers chemoresistance by modulating cancer stem cells in oral cancer

Mrs. Aditi Patel^{Ahmedabad University, Ahmedabad, Gujarat, India}, Dr. Shanaya Patel^{Ahmedabad University, Ahmedabad, Gujarat, India}, Ms. Vaishnavi Patel^{Ahmedabad University, Ahmedabad, Gujarat, India}, Dr. Vivek Tanavde^{Ahmedabad University, Ahmedabad, Gujarat, India}

OF16.O05 PTPN23 Downregulation by WDR4 Determines the Exosome Secretion Fate of MVB to Promote Cancer Metastasis and Immune Evasion

Phd Candidate Nai Yang Yeat, Li‐Heng Liu, Yu‐Hsuan Chang, PhD Kui‐Thong Tan, PhD Ruey‐Hwa Chen

OF16.O06 The elevated ECM1 protein in circulating sEVs is associated with integrin‐β2, and it mediates the enhanced breast cancer growth and metastasis under obesity conditions

Mr Keyang Xu

OS17.O02 Extracellular vesicles carrying tenascin‐C: a

Highly sensitive & specific, multi‐omics compatible, pan‐tumor liquid biopsy platform

Dr. Yanan Zhang, Dr. Alexander Koepp, Adela Brzobohata, Dr. Emanuele Puca, Dr. Roberto De Luca, Dr. Cesare Di Nitto, Dr. Teresa Hemmerle, Dr. Yingchao Meng, Dr. Stavros Stavrakis, Dr. Alexander Ring, Prof. Dr. Andreas Wicki, Prof. Dr. Julia Furtner, Dr. Caroline Hertler, Dr. Marcel Buehler, Prof. Dr. Michael Weller, Dr. Emilie Le Rhun, Prof. Dr. Dario Neri, Dr. Tobias Weiss

OS17.O03 Lectin microarray profiling of plasma EV glycosylation for gastric cancer diagnosis, prognosis, and prediction of immunotherapy response

Dr Fanqin Bu, Dr Guangyu Ding, Dr Yunzi Wu, Dr Chenjie Xu, Dr Liyi Bai, Professor Xintao Qiu, Professor Pengfei Yu, Professor Yibin Xie, Professor Li Min

OS17.O04 Higher concentration of small extracellular vesicles‐GCC2 in the pulmonary veins as a prognostic biomarker for patients with surgically resected lung adenocarcinoma

Dr Byeong Hyeon Choi, MD Jun Hee Lee, Dr Ok Hwa Jeon, Mr Chang Gun Kim, Professor Yeonho Choi, Professor Yong Park, Professor Ji‐Ho Park, Professor Sunghoi Hong, Professor Hyun Koo Kim

OS17.O05 Single EV protein and RNA expression detection via an in‐situ concurrent technology: sEV‐PREDICT for PD‐L1 positive extracellular vesicles analysis in plasma

Student Tong Liao, PhD Weilun Pan, Professor Lei Zheng, Professor Bo Li

OS18.O02 Deletion of P2RX7 ameliorates cognitive dysfunction and neurodegeneration in PS19 mice via suppression of extracellular vesicle mediated tau transfer

Seiko Ikezu, Post doctoral fellow Victor Santos, Postdoctoral fellow Mohammad Abdullah, Technician Justice Ellison, Research associate Zhi Ruan, Professor Tsuneya Ikezu

OS18.O03 Circulatory extracellular vesicles transport complement C1q for promoting neuronal amyloid‐beta production in alzheimer's disease

Dr Yang Yu, Dr Wenjun Xiao, Associate Professor Zhigang Li

OS18.O04 APOE genotype alters lipidomic and proteomic profiling of Alzheimer's disease brain‐derived extracellular vesicles reflecting inflammation and lipid dysbiosis

Dr. Zhengrong Zhang, Dr. Kaiwen Yu, Dr. Hanmei Bao, Dr. Michael DeTure, Ms. Clara Scholes, Dr. Yang You, Dr. Seiko Ikezu, Dr. Dennis Dickson, Dr. Xianlin Han, Dr. Junmin Peng, Dr. Tsuneya Ikezu

OS18.O05 Extracellular vesicle remodeling in response to mutant huntingtin

Natayme Rocha Tartaglia, Francesca Farina, Morgane Fontaine, Johanna Cormenier, Damarys Loew, Florent Dingli, Heike Rohweder, Chantal Bazenet, Emmanuel Brouillet, Lorena Martin‐Jaular, Frédéric Saudou, Clotilde Théry, Christian Neri

OS19.O02 Simultaneous tracking of big and small extracellular vesicles via multiplexed bioluminescence resonance energy transfer reporters

Dr. Anthony Yan‐Tang Wu, Ms. Wendy Wan‐Ting Wong, Ms. Shannon Yu‐Hsuan Yeh, Ms. Angela Yun‐Fei Zhang, Dr. Charles Pin‐Kuang Lai

OS19.O03 Nanoscale visualization and tracking of small extracellular vesicles and their DNA‐associated cargo in the recipient cells using single‐molecule localization microscopy

Dr Basant Kumar Thakur, Prof. Dr. Cremer Christoph, Dr Jamal Ghanam, Prof. Dr. Dirk Reinhardt, Dr. Xiaomin Liu, Xingfu Zhu, Dr. Venkatesh Kumar Chetty

OS19.O04 Endovesiclosis: a novel technology for quantum dot‐based extracellular vesicles labeling

Dr. Koushik Debnath^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Sadiq Umar^{Department of Oral Biology, College ofDentistry, UIC, Chicago, IL, USA}, Kasey Leung^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Chun‐Chieh Huang^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Miya Kang^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Yu Lu^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Prof. Praveen Kumar Gajendrareddy^{Department of Oral Biology, Collegeof Dentistry, UIC, Chicago, IL, USA}, Prof. Sriram Ravindran^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}

OS19.O05 Tracing extracellular vesicle subpopulations using HaloTag fusion proteins

Ms. Willemijn De Voogt, Dr. Sander Kooijmans, Mr. Kevin Harrijvan, Ms. Soultana Karakyriakou, Dr. Richard Wubbolts, Dr. Pieter Vader

OS20.O02 Bottom‐up assembly of synthetic extracellular vesicles for the regulation of immune activity in atopic dermatitis

Phd Student Amelie Chane, PhD Student Meline Macher, PhD Student Sarada Muduli, Doctor (Dr.) Ilia Platzman, Professor (Prof.) Joachim Spatz

OS20.O03 Precise and robust cell‐free synthesis of artificial extracellular vesicles

Mr. Tanner Henson, Alessandra Arizzi, Hyehyun Kim, David Wang, Neona Lowe, Conary Meyer, Keerthana Ananda, Dr. Erkin Seker, Dr. Randy Carney, Dr. Aijun Wang, Dr. Cheemeng Tan

OS20.O04 Extracellular vesicle‐iron oxide nanoparticle hybrid system: ExoFeR to induce ferroptosis and reverse therapeutic resistance in cancer

Assistant Professor Akhil Srivastava, Anjugam Paramanantham, Yariswamy Manjunath, Rahmat Asfiya, Siddharth Das, Grace McCully, Assistant Professor Jussuf Kaifi

OS20.O05 Novel noncoding RNA drugs bioinspired by therapeutic EV cargo

Professor And Executive Director Eduardo Marban, Dr Ahmed Ibrahim, Dr Russell Rogers, Dr Alessandra Ciullo, Dr Ke Liao

OS21.O02 Synergistic combination of extracellular vesicle formulations for the treatment of KRAS‐driven cancer

Dr. Cao Dai Phung, Thi Tuyet Trinh Tran, Brendon Zhi Jie Yeo, Gao Chang, Rebecca Carissa Prajogo, Migara Kavishka Jayasinghe, Thi Thanh Xuan Dang, Yuan Ju, Mai Trinh Nguyen, Boya Peng, Hong Anh Le, Eric Yew Meng Yeo, Bonney Glenn, Boon Cher Goh, Dahai Luo, Wai Leong Tam, Minh TN Le

OS21.O03 Fecal derivatives and extracellular vesicles enhance response to immune checkpoint blockade

Postdoctoral Fellow Golnaz Morad, Brenda Melendez, Sarah Johnson, Manoj Chelvanambi, Matthew Wong, Ashish Damania, Nadim Ajami, Jennifer Wargo

OS21.O04 Designed extracellular vesicles for therapeutic applications in neurofibromatosis type 1 (NF1)

Miss Maria Angelica Rincon‐Benavides, Miss Aarti Patel, Mrs. Tatiana Cuellar‐Gaviria, Mr. Ethan Stamas, Mr. Jad Hussein, Mr. Diego Alzate‐Correa, Miss Yuyan Yu, Miss Cintia Gomez, Mrs. Heather Powell, Mr. Daniel Gallego‐Perez, Mrs. Natalia Higuita‐Castro

OS21.O05 Extracellular vesicles‐mediated targeting of the glioma microenvironment

Miss Jacqueline YT Yeo, Dr Yuganthini Vijayanathan, Miss Janice HY Tan, Mr Fikri Mohamad, Ms Rachel LY Ho, Miss Nurashikin Abdul Halim, Dr Tatsuya Kozaki, Mr Zhi Wei Zhang, Dr Hai Tao Tu, Dr Jann Sarkaria, Dr Florent Ginhoux, Dr Li Zeng, Dr Ivy Ho

OS21.O06 Therapeutic exosomes targeting neuroendocrine prostate cancer

Associate Professor Sharanjot Saini, Dr Sandip Nathani, Ms Diana Asante, Ms Amritha Sreekumar, Dr. Matthew Simmons

OS23.O02 EXO‐CD24‐is a revolutionary immunomodulator that is smarter than steroids: the road from an idea to a ground‐breaking reality

Shiran Shapira, Prof., MD, MHA, CMO Nadir Arber

OS23.O03 In situ production of engineered extracellular vesicles for efficient delivery of protein biotherapeutics

Samantha Roudi, Post‐Doc Dhanu Gupta, Professor Samir El Andaloussi

OS23.O04 Treatment of NASH utilizing engineered extracellular vesicles with surface‐displayed FGF21 and encapsulated miR‐223

Associate Professor Kyungmoo Yea, Professor Moon‐Chang Baek

OS23.O05 Engineered red blood cells extracellular vesicles as therapeutic strategy for the treatment of renal diseases

Dr Alessia Brossa¹, Dr Michela Arena¹, Dr Alessandro Gori², Dr Marina Cretich², Dr Ilaria Giusti³, Prof Vincenza Dolo³, Benedetta Bussolati¹

¹Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy, ²Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM), Milano, Italy, ³Pathology Unit, Department of Life, Health and Environmental Sciences, University of L'Aquila, Italy

OS23.O06 Engineered let‐7a‐5p‐enriched extracellular vesicles

To alleviate inflammation for acute lung injury

Mr. Sin‐Yu Chen, Mr. Po‐Chen Li, Dr. Tai‐Shan Cheng, Ms. Hsin‐Tung Chen, Ms. Wei‐Ni Tsai, Dr. Hsiu‐Jung Liao, Professor Ly James Lee, Professor Chi‐Ying F. Huang

OS24.O02 Microfluidic isolation of complete platelet‐free plasma for enhanced detection of blood extracellular vesicles (EV) microRNAs and surface proteins

Sheng Yuan Leong, Ms. Wan Wei Lok, Ms Hui Min Tay, Mr. Hong Boon Ong, Dr. Poh Loong Soong, Dr. Roger Sik Yin Foo, Dr. Rinkoo Dalan, Dr. Han Wei Hou

OS24.O03 Optimized isolation of fecal bacterial extracellular vesicles and its application in colorectal cancer diagnostics

Student Yicong Xue, doctor Zihao Ou, Professor Bo Situ, Professor Lei Zheng

OS24.O04 A low‐cost kit for gentle, effective and timely extracellular vesicle (GET EV) isolation: accelerating development of RNA‐based liquid biopsies for neuroendocrine neoplasms

Mr. Boyang Su, Dr. Morteza Jeyhani, Dr. Xiaojing Yang, Jina Nanayakkara, Reese Wunsche, Dr. Neil Renwick, Dr. Scott Tsai, Dr. Hon Leong

OS24.O05 Light‐induced extracellular vesicle adsorption

Colin Hisey, Xilal Rima, Colin Hisey, Chiranth Nagaraj, Sophia Mayone, Kim Nguyen, Sydney Wiggins, Chunyu Hu, Divya Patel, David Wood, Zachary Schultz, Derek Hansford, Eduardo Reategui

OS24.O06 Hybrid microfluidic tangential flow filtration and herringbone microstructures for rapid extracellular vesicles isolation from blood plasma

Mr. Jia Yi Voo, Dr. Sheng Yuan Leong, Dr. Rinkoo Dalan, Prof. Han Wei Hou

OT01.O02 miR‐151a‐5p cargo in neuron‐derived extracellular vesicles is a biomarker and mediator of antidepressant treatment response

PhD Dariusz Żurawek, PhD Alice Morgunova, PhD Laura Fiori, M.S. Jennie Yang, PhD Claudia Belliveau, M.S. Pascale Ibrahim, M.S. Jean Francois Théroux, M.S. Ryan Denniston, Prof. Sidney H. Kennedy, Prof. Raymond W. Lam, Prof. Roumen Milev, PhD Susan Rotzinger, MD Claudio N. Soares, MD Valerie H. Taylor, MD Rudolf Uher, PhD Jane A. Foster, MD Benicio N. Frey, PhD Cecilia Flores, PhD Corina Nagy, MD Gustavo Turecki

OT01.O03 First‐in‐human clinical trial of allogeneic platelet extracellular vesicles as a potential therapeutic for chronic wound healing

Dr. Jancy Johnson, Dr. Gregor Lichtfuss

OT01.O04 Navigating the regulatory and commercial challenges of translating extracellular vesicle‐based biomarkers into clinical practice

Dr Olasehinde Olusanya

OT01.OWP01 Serum extracellular vesicle profiling to determine extracorporeal photopheresis response in graft versus host disease

Miss Kimberly Schell, Doctor Aisling Flinn, Professor Matthew Collin, Professor Andrew Gennery, Doctor Rachel Crossland

OT01.OWP02 Cracking the code: Understanding oncogenic small EVs in pancreatic cancer diagnostic landscape

Ms Arunima Panda, Mrs Ilaria Casari, Dr Abir Halder, Dr Walid Abu Shawish, Dr Danielle Dye, Prof Krish Ragunath, A/Prof David Greening, Prof Marco Falasca

OT01.OWP03 Hydrogel loaded with microalgae‐derived extracellular vesicles for preventing skin ultraviolet damage

Miss Jiarong Cui, Prof. Min Zhou

OT02.O02 Multi‐omic insights into extracellular vesicles mediating drug resistance in leishmania parasites

Associate Professor Christopher Fernandez‐Prada

OT02.O03 Common mechanisms of protection against pathogenic gram‐negative bacteria by host‐derived sEV

Dr. Adam Fleming, Mr. Graham Matulis, Ms. Heather Hobbs, Dr. Valentin Giroux, Mr. Hunter Mason, Dr. Weidong Zhou, Dr. Valerie Calvert, Dr. Nitin Agrawal, Professor Emanuel Petricoin, Dr. Rekha Panchal, Professor Igor Almeida, Dr. Sina Bavari, Professor Ramin Hakami

OT02.O04 Parasite extracellular vesicles selectively target human monocytes to induce T‐cell anergy and amelioration of DSS‐induced colitis in mice

Dr Anne Borup, Dr Farouq Mohammad Sharifpour, Dr Litten Sørensen Rossen, Dr Bradley Whitehead, MSc Anders Toftegaard Boysen, Dr Paul Giacomin, Mrs Kim Miles, Ms Maggie Veitch, Dr Andrea Ridolfi, Dr Marco Brucale, Dr Francesco Valle, Dr Lucia Paolini, Dr Paolo Bergese, Dr Alex Loukas, Professor Peter Nejsum

OT02.O05 Legionella pneumophila outer membrane vesicles promote macrophage survival while Legionella pneumophila induce inflammatory cell death pathways

Ms. ‐ Ayesha, Dr Franklin Wang Ngai Chow, Prof. Polly Hang‐mei LEUNG

OT03.O02 Extracellular vesicles as mediators of retinal homeostasis and immune modulation

Dr Yvette Wooff, Dr Adrian Cioanca, Miss Rakshanya Sekar, Associate Professor Riccardo Natoli

OT03.O03 Matrimeres: Cell‐secreted nanoscale mediators enabling systemic maintenance of tissue integrity and function

Dr. Koushik Debnath, Dr. Irfan Qayoom, Mr. Steven O'Donnell, Ms. Julia Ekiert, Ms. Can Wang, Mr. Mark Sanborn, Mr. Chang Liu, Ms. Ambar Rivera, Dr. Ik Sung Cho, Ms. Saiumamaheswari Saichellappa, Dr. Peter Toth, Prof. Dolly Mehta, Prof. Jalees Rehman, Prof. Xiaoping Du, Prof. Yu Gao, Jae‐Won Shin

OT03.O04 Elucidation of the mechanisms of participation of mesenchymal stromal cells extracellular vesicles in the regulation of myofibroblasts differentiation on 2D and 3D models of fibrosis

Ms Anastasiya Tolstoluzhinskaya, Ms Natalia Basalova, Ms Anastasiya Efimenko

OT03.O05 Mechanical overload‐induced extracellular mitochondria and particles release from tendon cells leads to inflammation in tendinopathy

Dr. Ziming Chen, Mengyuan Li, Peilin Chen, Andrew Tai, Jiayue Li, Euphemie Bassonga, Junjie Gao, Delin Liu, David Wood, Brendan Kennedy, Qiujian Zheng, Professor Minghao Zheng

OT05.O02 Interferon induced isoform of ADAR1 aids in Ewing sarcoma metastasis by fueling the pro‐inflammatory response in tumor microenvironment

Mr. Manideep Pachva, Dr. Peter Ruzanov, Dr. Valentina Evdokimova, Dr. Melanie Rouleau, Dr. Laszlo Radvanyi, Dr. Poul Sorensen

OT05.O03 Tumor ‐derived extracellular vesicles endogenously released by tumors are captured by resident and non‐resident cells in the pre‐metastatic niche and activate the inflammasome in macrophages

Dr. Laurence Blavier‐Sarte, Dr. Irina Matei, Dr. David Lyden, Professor Yves DeClerck

OT05.O04 Extracellular vesicles derived from plasma of exercise mice attenuated aggressive breast cancer tumour growth and metastasis

Dr Pamali Fonseka, Dr Sanjay Shahi, Prof Mark Febbraio, Prof Suresh Mathivanan

OT05.O05 RalA enhances hepatocellular carcinoma metastasis via upregulating protein cargos of small extracellular vesicles

Dr Lu Tian, Miss Jingyi Lu, Dr Karen Man‐Fong Sze, Dr Goofy Yu‐Man Tsui, Dr Daniel Wai‐Hung Ho, Prof Irene Oi‐Lin Ng

OT05.O06 CRISPR/Cas9‐based deletion of cortactin reduces the secretion of small extracellular vesicles, blocks cancer‐associated cachexia, and prolongs survival

Dr Sai Vara Prasad Chitti¹, Mrs Akbar L Marzan¹, Professor Suresh Mathivanan¹

¹La Trobe Institute For Molecular Science, La Trobe University, Melbourne, Victoria, 3086, Australia, Australia

OT06.04 Spirulina EVs induce pro‐inflammatory response by targeting innate immune cells, demonstrating potential as a vaccine adjuvant

Dr. Mohammad Farouq Sharifpour, Dr. Suchandan Sikder, Dr. Yide Wong, Dr. Na'ama Koifman, Dr. Matthias Floetenmeyer, Dr. Robert Courtney, Prof. Jamie Seymour, Prof. Alex Loukas

OT06.05 Targeting PD‐L1 in cholangiocarcinoma using milk‐derived nanovesicle‐based immunotherapy

Dr. Piyushkumar Gondaliya, Mr. Adil Ali Sayyed, Julia Driscoll, Irene K Yan, Dr. Tushar Patel

OT06.O02 Engineered extracellular vesicles mediate the in situ propagation of antibacterial signaling enabling biofilm eradication: in vitro and in vivo studies

Postdoctoral Scholar Tatiana Cuellar‐Gaviria, Maria Rincon‐Benavides, Hatice Topsakal, Ana Salazar‐Puerta, Mia Kordowski, Pranav Rana, Orlando Combita‐Heredia, Daniel Wozniak, Daniel Gallego‐Perez, Natalia Higuita‐Castro

OT06.O03 Celery exosome‐like nanovesicles as dual function anti‐cancer nanomedicine

Xin Lu, Qing Han, Professor Weiliang Xia¹

¹Shanghai Jiao Tong University, Shanghai, China

OT06.OWP01 Reshaping the landscape of prostate cancer treatment: FeS‐Functionalized OMVs as a promising nanodrug for immunotherapy

Doctor Xinxing Du, Doctor Huan Chen, Doctor Cong Hu, Doctor Yanhao Dong, Doctor Xinrui Wu, Doctor Jinyao Liu, Doctor Liang Dong, Doctor Wei Xue

OT06.OWP02 Bovine milk extracellular vesicles (mEVs)‐liposomes hybrid systems: a potential strategy for oral delivery of siRNA

Dr. Yunyue Zhang

OT06.OWP03 Turmeric‐derived extracellular vesicles laden polyphenol‐based hydrogel synergistically restores skin barrier in atopic dermatitis

Mingzhen Zhong, PhD Weilun Pan, Professor Lei Zheng

OT06.OWP1 Oral and rectal administration of bovine milk derived EVs in a colitis mouse model

Miss Nidhi Seegobin¹, Miss Marissa Taub¹, Dr Atheer Awad^1,2, Dr Sudax Murdan¹, Prof Abdul Basit¹

¹University College London, London, United Kingdom, ²University of Hertfordshire, Hatfield, United Kingdom

OT07.O02 EHD4 cooperates with Ral GTPase to drive multi‐vesicular body maturation and exosome secretion

Dr Vincent Hyenne, Dr Kuang‐Jin Huang, Dr Jacky G. Goetz

OT07.O03 Exogenous bacterial Cas9 expression alters small EV secretion and their protein cargo in p53 dependent manner

Professor Suresh Mathivanan¹

¹La Trobe University, Melbourne, Australia

OT07.O04 Screening for an inhibitor of EV secretion in ovarian cancer cells using a small molecule library

Dr. Yusuke Yoshioka, Dr. Akira Yokoi, Prof. Takahiro Ochiya

OT07.O05 Piezo1 activation increases release of therapeutic extracellular vesicles after mechanical stimulation in bioreactors

Phd André Cronemberger Andrade, Sarah Razafindrakoto, Lea Jabbour, Florence Gazeau, Amanda Silva Brun

OT07.O06 Regulation of EV biogenesis by ubiquitination and deubiquitination

Professor Sharad Kumar, Dr Ammara Farooq, Dr Natalie Foot, Dr Yoon Lim

OT08.O02 Detection of multi‐cancer signatures from extracellular vesicles using automated high‐performance liquid chromatography

Dr Andrew Lai, Dr Dominic Guanzon, Dr Carlos Palma, Dr Flavio Carrion, Dr Ryan Cohen, Prof Andreas Obermair, Prof Andreas Moller, Prof Carlos Salomon

OT08.O03 Double digital assay for single extracellular vesicle and single molecule detection

Dr. Jina Ko

OT08.O04 High‐sensitive rapid detection of urinary EVs with upconverting nanoparticle‐based lateral flow immunoassay

PhD Md Khirul Islam, Professor Urpo Lamminmäki, Adjunct professor Janne Leivo

OT08.O05 Optofluidic lab‐on‐a‐chip for point‐of‐need diagnostics and monitoring of treatment effectiveness by detection and quantification of EV subpopulations

Dr. Vasiliy Chernyshev, Mr. Alexey Kuzin, Dr. Vadim Kovalyuk, Dr. Pavel An, Mr. Alexandr Golikov, Mr. Sergey Svyatodukh, Mr. Stanislav Perevoschikov, Dr. Irina Florya, Dr. Alexey Schulga, Dr. Sergey Deyev, Dr. Gregory Goltsman, Dr. Dmitry Gorin

OT08.OWP01 Isolation and molecular characterization of exosomes from glioblastoma patients using a microfluidic device after ultrasound‐based opening of the blood brain barrier

Ms Abha Kumari, Dr Mark Youngblood, Andrew Gould, Dr Yoon‐Tae Kang, Li Chen, Karl Habashy, Thiago Reis, Dr Chris Amidei, Dr Rachel Ward, Cristal Gomez, Guillaume Bouchoux, Michael Canney, Dr Roger Stupp, Prof. Adam Sonabend, Prof. Sunitha Nagrath

OT08.OWP02 EV biomarker discovery for ultra‐early differential diagnosis of stroke

Lee‐Ann Clegg, MD, PhD Rolf A. Blauenfeldt, Bioinformatician, PhD Jesper Just, MSc in Engineering Rikke Bæk, Professor Peter Kristensen, Professor, MD Grethe Andersen, MSc, PhD Kim R. Drasbek, MSc, Ph.D Malene M. Jørgensen

PF01.01 Biomarkers for diagnosis of abdominal aortic aneurysm using small extracellular vesicle‐associated microRNA in human serum

Dr. Kazuki Takahashi^{Department of Molecular Cellular Medicine, Tokyo Medical University Institute of Medical Science, Shinjuku‐ku, Japan}, Dr. Yusuke Yoshioka^{Department of Molecular Cellular Medicine, Tokyo Medical University Institute of Medical Science, Shinjuku‐ku, Japan}, Dr. Naoya Kuriyama^{Department of Vascular Surgery, Asahikawa Medical University, Asahikawa, Japan}, Dr. Shinsuke Kikuchi^{Department of Vascular Surgery, Asahikawa MedicalUniversity, Asahikawa, Japan}, Professor Nobuyoshi Azuma^{Department of Vascular Surgery, Asahikawa Medical University, Asahikawa, Japan}, Professor Takahiro Ochiya^{Department of Molecular Cellular Medicine, Tokyo Medical University Institute of Medical Science, Shinjuku‐ku, Japan}

PF01.04 Enhancing concurrent chemoradiation outcome prediction for locally advanced cervical cancer patients through plasma extracellular vesicle proteomics analysis

Mr. Kittinun Leetanap*rn, Mr. Jitti Hanprasetpong, Miss Wararat Chiangjoing, Mr. Sitthiruk Roytrakul, Miss Piyatida Molika, Mrs. Raphatphorn Navakanitworakul

PF01.05 Extracellular vesicles as dual messengers: Deciphering microbial and host interaction for periodontitis

Miss Chun Liu, Dr Chaminda Jayampath Seneviratne, Prof Sašo Ivanovski, Dr Pingping Han

PF01.06 Glioblastoma‐derived salivary proteins in small extracellular vesicles as prognostic biomarkers

Dr Juliana Müller Bark, Dr Lucas Trevisan França de Lima, Dr Xi Zhang, Dr Daniel Broszczak, Dr Paul J. Leo, Dr Rosalind L. Jeffree, Dr Benjamin Chua, Dr Bryan W. Day, Professor Chamindie Punyadeera

PF01.08 Multicenter, prospective, observational study for urinary extracellular vesicle biomarkers of kidney allograft fibrosis

Professor Sung Shin, Dr. Hye Eun Kwon, Dr. Mi Joung Kim, Professor Heungman Jun, Professor Sang Jun Park, Professor Jun Gyo Gwon

PF01.09 Placental EVs enriched with Chromosome 19 and 14 cluster miRNAs as predictive biomarkers for Idiopathic Recurrent Pregnancy Loss

Ms. Chitra Bhardwaj, Dr. Priyanka Srivastava, Dr. Minakshi Rohilla, Dr. Seema Chopra, Dr. Anupriya Kaur, Dr. Inusha Panigrahi

PF01.10 Plasma‐derived EVs as biomarkers of sepsis in burn patients via label‐free Raman spectral analysis

Ms. Hannah O'Toole, Ms. Neona Lowe, Ms. Visha Arun, Ms. Anna Kolesov, Prof. Tina Palmieri, Prof. Nam Tran, Prof. Randy Carney

PF01.11 Proteome signature in serum extracellular vesicles reflects bronchial asthma pathophysiology

Md.PhD Yosh*to Takeda, MD Hanako Yoshimura, MD.PhD Yuya Shirai, MD Takahito Enomoto, PhD Jun Adachi, MD.PhD Atsushi Kumanogoh

PF01.13 Proteomic study of small extracellular vesicle protein biomarker profiles for breast cancer liquid biopsy

Ms Yu Jin Lee, Dr Jie Ni, Dr Valerie Wasinger, Mr Qi Wang, Dr Joanna Biazik, A/Prof Peter Graham, Prof Yong Li

PF01.14 Rapid and non‐invasive diagnosis of high PI‐RADS prostate cancer by high‐performance serum extracellular vesicles key metabolites

Postgraduate Zehong Peng, Postgraduate Yuning Wang, Postgraduate Xinrui Wu, Postgraduate Xingxing Du, Postgraduate Cong Hu, Postgraduate Yanhao Dong, Postgraduate Qi Chen, Postgraduate Yang Ge, Professor Kun Qian, Associate Research Fellow Liang Dong, Professor Wei Xue

PF01.16 Small extracellular vesicles in body fluids: promising prognostic biomarkers for head and neck cancer

Mr Abolfazl Jangholi, Dr Sarju Vasani, Prof Liz Kenny, Prof Sudha Rao, Prof Riccardo Dolcetti, Prof Chamindie Punyadeera

PF01.17 Spatiotemporal characteristics of tissue derived small extracellular vesicles is associated with tumor relapse and anti‐PD‐1 response

Doctor Qiu‐Yun Fu, Professor Gang Chen

PF01.18 Urinary exosomal miRNA biomarkers for antibody‐mediated

Rejection after kidney transplantation

Professor Sung Shin, Dr. Mi Joung Kim

PF01.20 Use of small RNAs from follicular fluid‐derived extracellular vesicles as biomarkers for predicting success rates of fertility treatment with assisted reproductive technology

Dr. Ayako Muraoka, Dr. Akira Yokoi, Dr. Kosuke Yoshida, Mrs. Masami Kitagawa, Dr. Hiroaki Kajiyama

PF01.21 A Pilot Study on Intra‐Articular Injection of Umbilical Cord‐derived Mesenchymal Stem Cell (UC‐MSC) Secretome in Temporomandibular Joint Dysfunction

drg. Dhanni Gustiana¹, Dr. Cynthia Retna Sartika², Mrs. Rima Haifa³, Ms Marsya Nilam Kirana³, Mrs Nisa Zulfani³, Ms Karina Kalasuba³, Mrs Ditta Kalyani Devi³, Mrs Vinessa Dwi Pertiwi³

¹RSUD Tangerang Selatan, Tangerang, Indonesia, ²Faculty of Pharmacy Universitas Padjadjaran, Jatinangor, Indonesia, ³Prodia StemCell Indonesia, Central Jakarta, Indonesia

PF01.22 Investigating the Therapeutic Effects of Umbilical Cord‐derived Mesenchymal Stem Cell and Umbilical Cord Mesenchymal Stem Cell‐derived Secretome in Chronic Ulcer Treatment: A Case Report

Dr Lisa Hasibuan¹, Dr Cynthia Retna Sartika^2,3, Mrs. Rima Haifa², Miss Atikah Anwar Hasibuan², Mrs. Ditta Kalyani Devi², Mrs. Adina Novia Permata Putri²

¹Immanuel Hospital, Bandung, Indonesia, ²Prodia StemCell Indonesia, Central Jakarta, Indonesia, ³Faculty of Pharmacy, Universitas Padjajaran, Jatinangor, Indonesia

PF01.23 International standardization concept to promote the technological development of extracellular vesicles

Senior Expert Ikuo Kawauchi¹

¹Fujifilm Holdings, Tokyo, Japan

PF01.23 A microfluidic device for isolation and quantitation of hepatocyte‐secreted extracellular vesicles and monitoring their exosomal cytochrome P450 activities on‐chip

Doctoral Researcher Ehsanollah Moradi, Ph.D. Päivi Järvinen, Ph.D. Markus Haapala, Associate Professor Tiina Sikanen

PF01.24 Fostering Consistency in EV‐Based Vaccine Development and Clinical Trials: Advancing Towards Standardization

Dr. Anis Larbi¹

¹Beckman Coulter Life Sciences, France

PF01.24 A single‐particle‐level detection of miRNA in extracellular vesicles using gold particle molecular beacons

ParvezMD SORWER ALAM, Takahiro Kochi, Prof Shin‐ichi Kano, Prof Atsuo Sasaki, Prof Kazuhiko Tabata, Dr. Eisuke Dohi

PF01.25 Aptasensor detection of infectious viral disease by targeting extracellular vesicles

Ms Harleen Kaur, Professor Nathan Bartlett, Doctor Renee V Goreham

PF01.26 Characterization of prostate‐specific antigen (PSA) associated with extracellular vesicles (EVs) from prostate cancer patients to develop a lateral flow diagnostic test

Kimberly Luke, Casey Scott‐Weathers

PF01.28 Detection of extracellular vesicles from bacteria or mammalian cells using aptasensor technology

Dr Renee Goreham

PF01.29 Detection of human immunodeficiency virus (HIV) proteins in extracellular vesicles (EVs) by immunocapture lateral flow method

Mr.Casey Scott‐Weathers, Ms. Kaitlyn King, Kimberly Luke

PF01.30 Fluorescent Nanoparticle‐Based Glycoprofiling of Colorectal and Pancreatic Cancer‐Derived Extracellular Vesicles for Early Detection

Mr. Rufus Vinod, Ms. Priyadharshini Parimelazhagan Santhi, Mrs. Erica Routila, Ms. Marina Alexeeva, Dr. Kjetil Søreide, Dr. Kim Pettersson, Dr. Janne Leivo

PF01.33 Optimizing diagnostic accuracy: a comprehensive standardization approach for CL‐ELISA with extracellular vesicles isolated from toxoplasma gondii

Master Letícia Pedrini, Master Paula Meneghetti, Doctor Vera Lúcia Chiocolla, Doctor Ana Claudia Torrecilhas, Doctor Blima f*ck

PF01.34 Rapid assessment of single extracellular vesicles using ultrathin nanoporous membranes for ‘catch and display’ of surface biomarkers

Samuel Walker^{Department of Biomedical Engineering, University of Rochester, Rochester, NY, USA}, PhD James McGrath^{Department of BiomedicalEngineering, University of Rochester, Rochester, NY, USA}, MD, MBA Jonathan Flax^{Department of Biomedical Engineering, University of Rochester, Rochester,NY, USA; Department of Urology, University of Rochester Medical Center, Rochester, NY, USA}

PF01.36 Single‐particle analysis of circulating bacterial extracellular vesicles reveals their biogenesis, changes in blood and links to intestinal barrier

Ph.d Zihao Ou

PF01.38 Surface modification of cellulose acetate membrane for fabrication of microfluidic platforms for express extracellular vesicle‐based liquid biopsy

Ms. Ekaterina Moiseeva, Dr. Vasiliy Chernyshev

PF01.39 Transferrin‐conjugated magnetic nanoparticles for the isolation of brain‐derived blood exosomal microRNAs: a novel approach for parkinson's disease biomarker

Associate professor Eun‐jae Lee, Professor Yong Shin

PF01.42 Mapping the Multi‐omics of Small Extracellular Vesicles in Diffuse Intrinsic Pontine Gliomas Reveals Biomarker Composition with Diagnostic Impact

Mr. Gaoge Sun¹, M.D. Tian Li³, Ying Zhang¹, Hang Yin^1,2

¹School of Pharmaceutical Sciences, Tsinghua University, Beijing, China, ²Tsinghua‐Peking Center for Life Sciences, Tsinghua University, Beijing, China, ³Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, China.

PF01.43 Impact of diabetes in proteomic profile of serum extracellular vesicles in obese patients after bariatric surgery

Dr. Jae‐a Han¹, M.D. Haekyung Lee², Hee‐Sung Ahn³, Dr. Soon Hyo Kwon³, Dr. Kyunggon Kim³, Dr. Seongho Ryu¹

¹Soonchunhyang Institute of Med‐Bio Science (SIMS), Soonchunhyang University, Cheonan‐si, South Korea, ²Division of Nephrology, Department of Internal Medicine, Seoul, South Korea, ³Asan Institute for Life Sciences, Asan Medical Center, Seoul, South Korea

PF01.44 Exploring small extracellular vesicles as a new biomarker to monitor duodeno‐pancreatic neuroendocrine tumors (DPNET) in the PRODIGE 31‐ REMINET cohort

Valentin Vautrot¹, Isen Naiken¹, Carmen Garrido¹, Pr Côme Lepage², Dr Jessica Gobbo³

¹INSERM 1231, Label “Ligue National contre le Cancer” and Label d'Excellence LipSTIC, DIJON, France, ²Federation Francophone de Cancérologie Digestive (FFCD), EPICAD INSERM 1231, DIJON, France, ³INSERM 1231, Label “Ligue National contre le Cancer” and Label d'Excellence LipSTIC, Department of Medical Oncology, Early phase unit INCa CLIP²; Center Georges‐François Leclerc, DIJON, FRANCE

PF01.46 Development of RNA sequencing platform for extracellular vesicles for identification of RNA markers for pancreatic cancer diagnosis

Visiting Researcher Yuta Shimizu^1,2, Researcher Fumi Asai², Researcher Keidai Miyakawa², Assistant Professor Kenji Takahashi³, Director Tatsutoshi Inuzuka²

¹Baylor Genetics, Houston, United States, ²H.U. Group Research Institute, Akiruno, Japan, ³Asahikawa Medical University, Asahikawa, Japan

PF01.47 Spectral flow cytometry of plasma EVs for detection of endometrial stromal cell markers CD10, CD90 and CD140b for endometriosis biomarkers

Ms Emily Paterson¹, Dr Simon Scheck^1,2, Dr Simon McDowell², Dr Nick Bedford², Associate Professor Jane Girling³, Dr Claire Henry¹

¹University of Otago, Wellington, New Zealand, ²Te Whatu Ora ‐ Capital and Coast, Wellington, New Zealand, ³University of Otago, Dunedin, New Zealand

PF01.48 miR‐15a from tear‐derived EVs in diabetic retinopathy

Professor Tengku Ain Fathlun Kamalden¹, 2 Nur Musfirah Mahmud¹, 3 Ying Jie Liows¹, 4 Sujaya Singh¹, 5 Samarjit Das²

¹UM Eye Research Centre, Department of Ophthalmoogy, Universiti Malaya, Kuala Lumpur, Malaysia, ²Department of Anaesthesiology and Department of Pathology, Johns Hopkins School of 37 Medicine, Baltimore, United States of America

PF01.49 Proteomics discovered differential extracellular vesicle enriched protein cargo for hepatocellular carcinoma early diagnosis.

Zhenxun Wang¹, Ph.D Bodeng Wu², Qiaoting Wu¹, Jiawei Li¹, Jiaming Chen¹, Quan Zhong¹, Phd Xin Zhang², Prof. Lei Zheng², Prof. Yu Wang¹

¹Department of Hepatobiliary Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China, ²Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou, China

PF01.50 EV‐derived circular RNAs as biomarkers for pleural mesothelioma

Dr Ben Johnson¹, Mr Winston Lay¹, Dr Tamkin Ahmadzada², Mr Richard Zelei¹, Dr Anthony Linton¹, Dr Elham Hosseini‐Beheshti¹

¹Asbestos And Dust Diseases Research Institute, Concord, Sydney, Australia, ²The University of Sydney, Camperdown, Sydney, Australia

PF01.53 Circadian Changes in mouse plasma miRNAs

Dr. Eisuke Dohi¹

¹National Center Of Neurology And Psychiatry, Kodaira city, Japan

PF01.54 Exploring the utility of exosome subpopulations for biomarker discovery

CEO, R&D Se‐Hwan Paek¹, Associate Research Engineer Taekmin Kim¹, Research Engineer Dayeon Choi¹, Research Director Seung‐Cheol Choi¹

¹SOL Bio Corporation, Seoul, South Korea

PF01.55 Radiation‐induced miR‐126‐5p in extracellular vesicles suppresses cholesterol efflux by targeting ABCG5

Min Eon Park¹, You Yeon Choi¹, Ki Moon Seong¹

¹Korea Institute of Radiological & Medical Sciences (KIRAMS), Seoul, KOREA

PF01.56 Screening of Exosomal miRNAs in Radiation‐induced AKR/J leukemia mice model

Min Eon Park¹, You Yeon Choi¹, Ki Moon Seong¹

¹Korea Institute of Radiological & Medical Sciences (KIRAMS), seoul, KOREA

PF01.57 Enhanced characterization of extracellular vesicles using oni nanoimager: a comparative analysis of isolation techniques for jurkat cell‐derived extracellular vesicles

Diane Nelson¹, Mahir Mohiuddin, Investigator Jennifer Jones, Jeffrey fa*gan, Jerilyn Izac, Sumeet Poudel, Bryant Nelson, Lili Wang

¹Nist, United States

PF01.58 Harnessing extracellular vesicles for precise drug delivery across CNS barriers

Dr. Marie Pauwels¹, Dr. Nele Plehiers¹, Dr. Charysse Vandendriessche¹, Prof. Matthew JA Wood², Dr Lien Van Hoecke¹, Prof Roosmarijn E Vandenbroucke¹

¹VIB‐UGent, Gent (Zwijnaarde), Belgium, ²University of Oxford, Oxford, UK

PF01.59 Benzo[a]pyrene exposure detection by Raman spectroscopy of large extracellular vesicles

Ms. Geetika Raizada, Dr. Benjamin Brunel, Mr. Joan Guillouzouic, Dr. Eric Le Ferrec, Dr. Eric Lesniewska, Dr. Wilfrid Boireau, Dr. Céline Elie‐Caille

¹FEMTO‐ST Institute, CNRS, University of Franche‐Comté, Besançon, France

PF01.61 Label‐free biomarker detection in advanced colorectal cancer plasma exosomes

Dr Rana Rahmani¹, Dr. Sanduru Thamarai Krishnan^1,2, Dr. David Rudd^1,2, Ehud Hauben^4,5, Prof. Nicolas H. Voelcker^1,2,3

¹Monash Institute of Pharmaceutical Sciences, Monash University, Australia, ²Melbourne Centre for Nanofabrication, Victorian Node of the Australian National Fabrication Facility, Clayton 3168, Australia, ³Commonwealth Scientific and Industrial Research Organization (CSIRO), Australia, ⁴The Basil Hetzel Institute for Translational Health Research, Australia, ⁵Discipline of Surgery, Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Australia

PF01.64 Profiling single extracellular vesicle phenotypes in cancer using advanced nanotechnologies

Dr Richard Lobb¹, Dr Alain Wuethrich¹, Associate Professor David Fielding², Professor Andreas Möller³, Professor Matt Trau¹

¹University of Queensland, Brisbane, Australia, ²Department of Thoracic Medicine, Royal Brisbane and Women's Hospital, Brisbane, Australia, ³5JC STEM Lab, Li Ka Shing Institute of Health Sciences, Department of Otorhinolaryngology, Faculty of Medicine, Chinese University of Hong Kong, Hong Kong, China

PF01.65 Title: Proteomics‐based Molecular Signatures of Alzheimer's Disease – From Blood to Extracellular Vesicles

Associate Prof. Shona Pedersen¹

¹College of Medicine, Qatar University, Doha, Qatar

PF02.01 A novel circulating extracellular vesicle miRNA panel regulates tumour cell migration, chemoresistance and patient survival outcomes in ovarian cancer

Dr Soumyalekshmi Nair, Anas Emerizal, Dominic Guanzon, Andrew Lai, Flavio Carrion, Yaowu He, Aase Handberg, Lewis Perrin, Gregory Rice, John Hooper, Carlos Salomon

PF02.03 Acquisition of cancer stem cell‐like characteristics in tumor cells by application of EVs derived from 5‐FU‐treated human gastric cancer cells

Ms. Akane Sato, Mr. Kyo Okita, Dr. Etsuro Ito

PF02.05 Alteration of bone marrow‐derived mesenchymal stem cell exosomes on cervical cancer spheroids

Miss Piyatida Molika, Assoc. Prof. Dr. Raphatphorn Navakanitworakul

PF02.06 Anti‐cancer effects of extracellular vesicles from a Lacticaseibacillus paracasei strain in triple negative breast cancer cells

Professor Wen‐wei Chang, Miss Hui‐Yu Jiang, Dr. Wan‐Hua Tsai, Professor Hsueh‐Te Lee

PF02.07 Application of extracellular vesicles in 3D cell culture model of primary hepatocyte and liver tumoral cells

PhD Student Silvia López‐Sarrió, PhD student Clara Garcia‐Vallicrosa, PhD Student Guillermo Bordanaba‐Florit, PhD Maria Azparren‐Angulo, Postdoctoral researcher Félix Royo, Principal investigator Juan Manuel Falcón‐Pérez

PF02.09 Blocking the secretion of small extracellular vesicles prevents muscle atrophy, lipolysis and cancer‐induced cachexia

Dr Sai Vara Prasad Chitti, Akbar Marzan, Prof Suresh Mathivanan

PF02.10 Carcinoma‐associated fibroblast‐derived lysyl oxidase‐rich extracellular vesicles mediate collagen crosslinking and promote epithelial‐mesenchymal transition

Xue Liu

PF02.11 Clathrin light chain A‐enriched small extracellular vesicles remodel microvascular niche to induce hepatocellular carcinoma metastasis

Dr. Yi Xu

PF02.12 Comparative proteomics analysis of small EVs derived from mouse oral cancer cells

Research Fellow Adnan Shafiq, Shinya Sato, Alissa Weaver

PF02.13 CXCR4‐mediated ciliogenesis controls cancer cell death

Mr Tae‐Kyu Jang, Dr Eunyi Moon

PF02.14 Devising integrin β4‐enriched small extracellular vesicle as drug delivery vehicle for targeting pulmonary metastasis of hepatocellular carcinoma

Dr Tung Him Ng, Ms Aijun Liang, Prof Judy Wai Ping Yam

PF02.15 Endocytosis of EV into HNSCC cancer cells is required for increased sensitization to tyrosine kinase inhibitor

Dr Darren Toh, Ms Hui Sun Leong, Ms Fui Teen Chong, Ms Mengjie Ren, Dr Gopalakrishna Iyer

PF02.16 Exosomal G6PD drives metabolic reprogramming and reshape the pre‐metastastic niche to facilitate metastasis in hepatocellular carcinoma.

Dr. Xiaoxin Zhang

PF02.20 Exploring exosome‐mediated mechanisms in gefitinib‐resistant lung adenocarcinoma

Mr. Chun‐FanLung, Ph. D Student Chun Fan Lung

PF02.21 Exploring the role of obesity‐induced extracellular vesicles secretion and associated oncogenic proteins in endometrial cancer pathogenesis

Dr Takahiko Sakaue, Kalpana Deepa Priya Dorayappan, Dr Wafa Khadraoui, Dr Muralidharan Anbalagan, Dr Adrian Suarez, Dr Casey Cosgrove, Dr Larry J. Maxwell, Dr Hironori Koga, Dr David O'Malley, Dr David Cohn, Dr Selvendiran Karuppaiyah

PF02.22 Extracellular vesicle‐dependent inhibition effects of EGR1 on HCC metastasis

Phd Xin Zhang, Quan Zhong, Jiaming Chen, Zhenxun Wang, Bin Xu, Boyan Boyan Huang, Jinsheng Zheng, Tianyu Wu, Yu Wang, Lei Zheng

PF02.25 Extracellular vesicles secreted during oncolytic viral therapy with hmgb1 promotes melanoma outgrowth

PhD Darshak Bhatt, Msc Annemarie Boema, PhD Silvina Bustos, PhD Andreia Otake, PhD Alexis Carrasco, Professor Patricia Reis, Professor Roger Chammas, Professor Toos Daemen, PhD Luciana Andrade

PF02.26 Functional assessments of extracellular vesicles coronated with human epidermal growth factor receptor 2 (HER2) protein

Extracellular vesicles and breast cancer Mina Mobin Rahni, Immunology Marzieh Ebrahimi, Extracellular Vesicles Faezeh Shekari

PF02.27 GRP78‐rich extracellular vesicles derived from gastric cancer cells promote gastric cancer stemness and chemoresistance

Dr. Jen‐Lung Chen, Ms. Hsin‐Yi Tsai, Assistant Professor Ming‐Wei Lin

PF02.30 Impeding the secretion of tumor cell‐derived small extracellular vesicles attenuates breast cancer progression and metastasis

Dr. SANJAY SHAHI, Prof. Suresh Mathivanan

PF02.31 Interrogation of the spatial tissue architecture and miRNA sequencing of extracellular vesicles in matched epithelial ovarian cancers

Dr Andrew Lai, Dr Priyakshi Kalita‐de Croft, Dr Dominic Guanzon, Dr Soumyalekshmi Nair, Mr Nihar Godbole, Dr Flavio Carrion, Dr Shayna Sharma, A/Prof Margaret Cummings, Prof Lewis Perrin, Prof John Hooper, Prof Ken O'Byrne, Prof Sunil Lakhani, A/Prof Fernando Guimaraes, Dr Arutha Kulasinghe, Prof Andreas Moller, Prof Carlos Salomon

PF02.33 Macrophage function is modulated by EVs derived from plasma of HNSCC patients through the NF‐κB signaling pathway

Ms Diana Huber, Mrs Tsima Abou Kors, PhD Linda Hofmann, Prof Monika Pietrowska, PhD Marta Gawin, Prof Ramin Lotfi, Prof Thomas K Hoffmann, Prof Cornelia Brunner, Prof Marie‐Nicole Theodoraki

PF02.34 Melanoma secreted melanosomes induce immune tolerance in lymphatic endothelial cells through overexpression of CEACAM‐1

Ms Daniela Likonen

PF02.35 Metabolic reprogramming into a glycolysis phenotype induced by extracellular vesicles derived from prostate cancer cells

Professor Yoon‐Jin Lee, Ms. Shinwon Chae, Ms. Haekang Yang, Mr. Chul Won Seo, Mr. Chang Yeol Lee, Professor Sang‐Han Lee, Dongsic Choi

PF02.36 MiR‐195‐5p‐loaded tumor‐derived extracellular vesicles restrains melanoma spheres growth and radiotherapy‐induced resistant phenotype

Ms Nathalia Leal Santos, Roger Chammas, Luciana Andrade

PF02.37 Monitoring cancer extracellular vesicle transfer within tumor tissue context

Nao Nishida‐Aoki

PF02.40 Pancreatic cancer cell derived extracellular vesicles enriched microRNAs play a critical role in macrophage reprograming

Dr. Baldev Singh, Dr. Pankaj Gaur, Dr. Jeyalakshmi Kandhavelu, Mr. Yanjun Zhang, Mr. Zihao Zhang, Dr. Shivani Bansal, Mr. Meth Jayatilake, Mr. Yaoxiang Li, Dr. Pritha Bose, Dr. Seema Gupta, Dr. Partha Banerjee, Dr. Vivek Verma, Dr. Baldev Singh

PF02.41 Pancreatic cancer cell‐derived EVs promote monocyte differentiation towards immunosuppressive tumor‐associated macrophages

Mr. Yanjun Zhang, Dr. Baldev Singh, Dr. Pritha Bose, Dr. Jeyalakshmi Kandhavelu, Mr. Zihao Zhang, Dr. Shivani Bansal, Dr. Sunil Bansal, Mr. Meth Jayatilake, Mr. Yaoxiang Li, Dr. Shu Wang, Dr. Baldev Singh

PF02.44 Proteomic analysis of Butyrate‐resistant colorectal cancer‐derived exosomes reveals potential resistance to anti‐cancer drugs

Kesara Nittayaboon, Kittinun Leetanap*rn, Prof. Surasak Sangkhathat, Prof. Sittiruk Roytrakul, Assoc. Prof. Raphatphorn Navakanitworakul

PF02.46 Proteomics analysis of the small extracellular vesicles and soluble secretory proteins from cachexia‐inducing cells and their effect on C2C12 myotubes

Mrs Akbar Marzan, Dr. Sai Chitti, Prof Suresh Mathivanan

PF02.47 Quantification and characterization of circulating extracellular vesicles in cervical cancer patients before, during, and after treatment

Prof Muriel Meiring, Ms Noluthando Gasa

PF02.49 Small but mighty: CD‐sEV cargos promote pancreatic cancer metastasis and stem cell reprogramming

Mr Harrison Rudd, Dr Geeta Upadhyay

PF02.50 Small extracellular vesicle PD‐1 leads to senescence‐initiated epithelial‐mesenchymal transition in oral cancer through intrinsic PD‐L1‐p38 MAPK signaling

Doctor Lin‐Zhou Zhang, Professor Gang Chen

PF02.51 Small extracellular vesicles derived from cancer cells modulate breast cancer patients’ immune system via affecting Th1/Th2 and T‐reg cells

Mr Abdulwahab Teflischi Gharavi, Ms Raheleh Tahmasvand, Dr Amirabbas Rahimi, Dr Saeed Irian, Prof Mona Salimi

PF02.52 The enrichment of death associated miRNAs in placental explant culture promoted cervical tumor tissue undergoing necrosis

Lin Wang

PF02.53 The role of extracellular vesicle‐contained CD155 during cancer progression

Li‐Ying Wu, Dr. Luize Lima, Dr. Sunyoung Ham, Student Mina Lim, Dr. Edna Chai, Prof. Yong‐Soo Choi, Prof. Andreas Möller

PF02.54 Three‐dimensional matrix stiffness drives piezo1 activation in cancer spheroid‐derived small extracellular vesicles

Maulee Sheth, Dr Manju Sharma, Maulee Sheth

PF02.55 Title: Osteoclasts educated by prostate cancer cells promote bone destruction via EV‐mediated communication networks in bone metastatic site

M.D. Takaaki Tamura, Ph.D. Tomofumi Yamamoto, Ph.D. Akiko Kogure, Ph.D. Yusuke Yoshioka, M.D., Ph.D. Shinichi Sakamoto, M.D., Ph.D. Tomohiko Ichikawa, M.D. Takahiro Ochiya

PF02.56 Tracking the EMT‐like phenotype switching during targeted therapy in melanoma by analyzing extracellular vesicle phenotypes

Mr Quan Zhou, Prof. Jing Wang, Dr. Zhen Zhang, Dr. Alain Wuethrich, Dr. Richard Lobb, Prof. Matt Trau

PF02.57 Tumor cell‐derived extracellular vesicles promote ROS‐induced DNA damage in hepatocellular carcinoma

Mr. Zhixian Chen, Prof. Judy Yam

PF02.58 Uncovering extracellular vesicle microRNA and protein cargo from chemoresistant osteosarcoma: Shedding light on the potential transfer of therapy resistance

Mr. Joaquín Jurado‐Maqueda, Alessandra De Feo, Prof. Katia Scotlandi

PF02.59 Understanding the role of mesothelioma cell‐derived extracellular vesicles in modulating fibroblast functions

Mr Vivek Dharwal, Dr Vivek Dharwal, Mr Jiawei Chang, Dr Zaklina Kovacevic, Dr Elham Hosseini‐Beheshti

PF02.60 Unraveling the role of CD133 in Breast cancer‐Extracellular Vesicles in invasion and metastasis

Mireia Gomez

PF02.61 Y‐box binding protein 1 in small extracellular vesicles reduces the osteogenic differentiation of bone marrow‐derived mesenchymal stem cells – significance in acute myeloid leukemia

Dr Venkatesh Kumar Chetty, Dr Jamal Ghanam, Prof. Dr Dirk Reinhardt, Dr Basant Kumar Thakur

PF02.62 Comparison of profile and functional activities of EVs from fresh tumor biopsies and decellularized tumor tissue in colorectal cancer

Dr Sarah Tassinari¹, Dr Federica Collino², Benedetta Bussolati¹

¹Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy, ²8 Department of Clinical Sciences and Community Health, University of Milano, Milano, Italy

PF02.63 Adipocyte‐origin exosomes induce metastasis in models of TNBC

Graduate Student Yuhan Qiu¹, Undergraduate Student Rebecca Yu¹, Graduate Student Andrew Chen¹, Postdoc Matt Lawton¹, Graduate Student Pablo Llevenes¹, Lab Manager Manohar Kolla¹, Postdoc Naser Jafari¹, Kiana Mahdaviani², Naomi Ko², PI Stefano Monti¹, PI Gerald Denis¹

¹Boston University School Of Medicine, Boston, United States, ²Boston Medical Center, Boston, United States

PF03.02 Are mouse blood EV‐derived miRNA profiles consistent across studies? systematic reviews

Parvez MD　SORWER　ALAM, Dr. Eisuke Dohi

PF03.03 Characterization of human follicular fluid extracellular vesicle subtypes and their impact on human granulosa‐like tumor cell line KGN

Ms Inge Varik, Ms Katariina Johanna Saretok, Dr Ileana Quintero, Dr Maija Puhka, Dr Aleksander Trošin, Ms Kristine Roos, Dr Paolo Guazzi, Dr Agne Velthut‐Meikas

PF03.04 Comprehensive and specific analysis of surface glycans on extracellular vesicles (EVs) for understanding EV diversity

Ph.D. Asako Shimoda, Professor Emeritus Kazunari Akiyoshi

PF03.05 Extracellular particles are present in platelet concentrates

Lauren Litchfield, Dr Rebecca Wellburn, Dr Sarah Bajan, A/Prof Yoke Lin Fung, A/Prof John‐Paul Tung

PF03.06 Extracellular vesicle associated markers present on lipid droplets

Miss Irumi Amarasinghe, Dr Ebony Monson, Dr Eduard Wilms, Mr William Phillips, Dr Shuai Nie, Miss Abbey Milligan, Dr Donna Whelan, Prof Andrew Hill, Prof Karla Helbig

PF03.07 Extracellular vesicles in fresh frozen plasma and cryoprecipitate

Ji Hui Hwang, A/Prof John‐Paul Tung, Prof Damien Harkin, Prof Robert Flower, Dr Natalie Pecheniuk

PF03.08 Large extracellular vesicles subsets and contents discrimination: the potential of morpho mechanical approaches at single EV level

Ms.Geetika Raizada, Mr. Joan Guillouzouic, Dr. Eric Le Ferrec, Dr. Eric Lesniewska, Dr. Wilfrid Boireau, Dr. Céline Elie‐Caille

PF03.09 MBsomes and other EVs intercellular communication in skin wound healing

Phd Student Mariane Shouky, Graca Raposo

PF03.10 Nano‐flow cytometry‐based discrimination of extracellular vesicles and non‐vesicular particles: insights into extracellular carriers of specific biomolecules

Xiaomei Yan, Yunyun Hu, Haonan Di, Dr. Ye Tian

PF03.11 Quantitative profiling of single exosome heterogeneity using single‐molecule binding assay

Ms Jiyoung Goo, Ms Somi Park, Ms Hyeyeong Ku, Ms Jeongmin Lee, Mrs Jeong Hee Kim, Mr In‐San Kim, Mr Cherlhyun Jeong

PF03.12 Quantitively mapping the EV field and its trajectory through scientometrics

Mr Liam Hourigan, Mr William Phillips, Mr Chaomei Chen, Mr Amirmohammad Nasiri Kenari, Mr Krishna Chaitanya Pavani, Mrs Lesley Cheng, An Hendrix, Mr Andrew Hill

PF03.13 Spatial diversity of intraperitoneal extracellular vesicles and potential tumor‐suppressive roles of liver‐surface extracellular vesicles in the development of high‐grade serous ovarian carcinoma

Dr Kosuke Yoshida, Dr Akira Yokoi, Dr Kazuhiro Suzuki, Dr Yukari Nagao, Dr Ryosuke Uekusa, Ms Masami Kitagawa, Dr Eri Inami, Dr Takao Yasui, Dr Hiroaki Kajiyama

PF03.14 The effects of acute and chronic hypoxia on EV production and phenotype in cancer cells

Dr Chris Pridgeon, Ms Julia Monola, Ms Kerttu Airavaara, Dr Daniel Palmer, Prof. Marjo Yliperttula, Dr Riina Harjumäki

PF03.15 Matrix‐bound nanovesicles: biogenesis and ties to the ECM

Marley Dewey, Assistant Professor George Hussey, Professor Stephen Badylak

PF03.17 The impact of follicular fluid small and large extracellular vesicles on the gene expression of human granulosa‐like tumor cell line KGN

Dr Agne Velthut‐Meikas¹, Inge Varik¹, Katariina Johanna Saretok¹, Kristine Rosenberg^1,2, Aleksander Trošin³, Maija Puhka⁴, Ileana Quintero⁴, Paolo Guazzi⁵

¹Tallinn University Of Technology, Tallinn, Estonia, ²Nova Vita Clinic, Tallinn, Estonia, ³East Tallinn Central Hospital, Tallinn, Estonia, ⁴University of Helsinki, Helsinki, Finland, ⁵HansaBioMed Life Sciences Ltd, Tallinn, Estonia

PF03.18 Towards development of detergent‐based strategy for the enrichment of extracellular particle subpopulations and subdomains

Dr. Igor V Kurochkin¹, Lausonia Ramaswamy

¹Central Research Laboratory, Sysmex Co., Kobe, Japan

PF03.22 Exosomes Isolation by Ultracentrifugation: Novel Subpopulations reveal Extracellular Vesicle Heterogeneity and Diverse Functional Signatures

Director, Computational Oncology Unit Ahmed Fadiel², Process Development Lead Shuaizhen Yuan¹, Associate Scientist Eileah Loda¹, Ceo Adam Koster¹, Chair, Medical Scientific Advisory Board Frederick Naftolin¹, Director, Medical Affairs Matthew Peterson

¹Interactome Biotherapeutics, Grand Rapids, United States, ²University of Chicago, Chicago, USA

PF04 Cell‐derived nanovesicles as a scalable production of extracellular vesicles‐mimetics for therapeutic applications

Dr Wei Heng Chng¹, Mr Ram Pravin Kumar Muthuramalingam¹, Dr Yub Raj Neupane¹, Dr Chenyuan Huang¹, Dr Wei Jiang Goh¹, Dr Choon Keong Lee¹, Bertrand Czarny², Assistant Professor Jiong‐Wei Wang¹, Associate Professor Giorgia Pastorin¹

¹National University of Singapore, Singapore, ²Nanyang Technological University, Singapore

PF04.03 Advancements in lung cancer immunotherapy using engineered exosome to deliver PD‐L1 siRNA

Dr. Farrukh Aqil, Raghuram Kandimalla, Disha Moholkar, Margaret Wallen, Chuanlin Ding, Ramesh Gupta

PF04.05 Engineered exosomes for HLA‐G‐targeted co‐delivery of MSI1 siRNA and chemotherapeutics to reduce the tumor progression

PhD Chih‐Ming Pan, MS Yu‐Ting Liao, PhD Shao‐Chih Chiu

PF04.07 Extracellular vesicle‐mediated delivery of customized ASOs targeting driver mutants for personalized Non‐Small Cell Lung Cancer treatment

PhD student Trinh Tran, Doctor Dai Phung, Brendon Yeo, Rebecca Prajogo, Migara Jayasinghe, Yuan Ju, Eric Yeo, Doctor Boon Cher Goh, Doctor Wai Leong Tam, Doctor Minh Le

PF04.08 FDA‐approved ETA antagonist regulates cellular and exosomal B7‐H4 through N‐glycosylation inhibition

Ms Sua Kim, Dr. Dokyung Jung, Professor Moon‐Chang Baek

PF04.09 Hydrogel‐encapsulated exosome vaccine as a novel immunotherapeutic approach and its role in enhancing immunotherapy for prostate cancer

Liang Dong, QI Chen

PF04.10 In vivo CAR‐T generated by CD3ɛ nanobody‐engineered exosomes eliminates solid tumors and promotes the immunological memory formation

Dr. Shi‐Wei Huang, Dr. Mei‐chih Chen, Dr. Yu‐Chuan Lin, Dr. Chih‐Ming Pan, Dr. Chung‐Chun Wu, Miss Chen‐Yu Lin, Miss Pei‐Ying Lin, Miss Yu‐Ting Chiang, Miss Yu‐Han Huang, Miss Wan‐Yu Mao, Miss Steffany Rusli, Professor Shao‐Chih Chiu, Professor Der‐Yang Cho

PF04.11 Novel personalized cancer vaccine using attenuated tumor extracellular vesicles with enhanced immunogenicity

Graduate student Jihoon Han, Graduate student Yeongha Hwang

PF04.12 Redirecting pre‐existing noncancer immunity to cancer cells using tumor‐targeting extracellular vesicles for delivery of MHC‐I‐compatible peptides for cancer immunotherapy

Yang Lu, Songbo Qiu, Professor Zhen Fan

PF04.16 Surface‐engineered NK cell‐derived small extracellular vesicles induce potent anti‐tumor effects in lung cancer cells

Dr. Sung‐Min Kang, Dr. Dokyung Jung, Ms Soojeong Noh, Ms Sanghee Shin, Ms Minju Kim, Professor Byungheon Lee, Professor Kyungmoo Yea, Professor Moon‐Chang Baek

PF04.17 Synthetic immunogenicity‐induced DNA accumulation in colorectal cancer extracellular vesicles enhances T cell stemness

Ms Seong A Kim, Ms Yeji Lee, Dr. In‐San Kim

PF04.18 Therapeutic plasma exchange as a method to combat extracellular vesicle‐mediated immunotherapy resistance in melanoma

Dr. Jacob Orme, Henan Zhang, Prashanth Lingamaneni, Yohan Kim, Roxane Lavoie, Jacob Hirdler, Elizabeth Bering, Joanina Gicobi, Heather Dale, Lisa A Kotschade, Matthew S. Block, Svetomir N. Markovic, Haidong Dong, Fabrice Lucien, Annie T. Packard, Jeffrey L. Winters, Sean S. Park

PF04.21 Vitamin B enhances anti‐tumor immunity by inhibiting CD47 on cellular and extracellular vesicles

MS course Na‐Eun Kim, Dr. Dokyung Jung, Professor Moon‐Chang Baek

PF04.24 Synthetic biology‐based bacterial extracellular vesicles displaying BMP‐2 and CXCR4 to ameliorate postmenopausal osteoporosis

Associate Professor Han Liu

PF04.25 TNFα‐bearing small extracellular vesicles synergize with SMAC mimetics to eradicate tumor cells

Dr. Rostyslav Horbay, Daniel Panting, Michaela van der Meerwe, Maria Dimancheva, Dr Eric LaCasse, Dylan Burger, Dr Shawn Beug

¹Apoptosis Research Centre, Children's Hospital of Eastern Ontario Research Institute and University of Ottawa, 401 Smyth Rd, Ottawa, Ontario, K1H 8L1, Canada, Ottawa, Canada, ²Kidney Research Centre, The Ottawa Hospital Research Institute and University of Ottawa, 401 Smyth Rd, Ottawa, ON, K1H 8L1, Canada,

PF04.26 Therapeutic poxviruses trigger the secretion of anti‐tumor extracellular vesicles with immunomodulatory potential

Lucas Walther³, Jacky Goetz¹, Karola Rittner², Dr Vincent Hyenne¹

¹INSERM U1109, Strasbourg, France, ²Transgene SA, Illkirch‐Graffenstaden, France, ³INSERM U1109 and Transgene SA, Strasbourg, France

PF04.27 CAR‐T derived extracellular vesicles demonstrate in vitro therapeutic efficacy in breast and blood cancer cells

Dr Kartini Asari¹, Siena Barton¹, Sadman Bhuiyan¹, Kol Thida Mom¹, Amirah Fitri¹, Dr Mozhgan Shojaee¹, Dr Carlos Palma¹, Dr Sara Nikseresht¹, Dr Ramin Khanabdali¹, Professor Gregory Rice^1,2

¹INOVIQ Ltd, Notting Hill, Australia, ²UQ Centre for Clinical Research, Herston, Brisbane City, Australia

PF05.02 Watermelon alleviates IBD by modulating intestinal lactobacillus plantarum colonisation and the release of bacterial extracellular vesicles

Phd Qianbei Li, Professor Lei Zheng

PF05.03 L. amazonensis amastigotes release unique extracellular vesicles in a calcium and pH dependent manner

Deborah Brandt Almeida, Ms Jenicer Kazumi Umada Yokoyama Yasunaka, Doctor Verônica Feijoli Santiago, Doctor Simon Ngao Mule, Ms Paula Menegheti, Doctor Giuseppe Palmisano, Doctor Ana Claudia Torrecilhas, Doctor Mauro Cortez

PF05.05 A human host‐defense peptide LL‐37 ameliorates mouse sepsis by orchestrating the chemotaxis of neutrophils and secretion of anti‐inflammatory extracellular vesicles

Assistant Professor Yumi Kumagai^1,2, Special Appointed Professor Isao Nagaoka^1,3, Professor Etsuo Susaki¹

¹Dept. of Biochemistry and Systems Biomedicine, Graduate School of Medicine, Juntendo University, Bunkyo‐ku, Japan, ²Biomedicine Research Core Facility, Graduate School of Medicine, Juntendo University, Bunkyo‐ku, Japan, ³Faculty of Medical Science, Urayasu, Japan

PF05.06 Induction of proinflammatory response in bystander macrophages by extracellular vesicle‐delivered SARS‐CoV‐2 accessory protein ORF3a

Dr Sin‐Yee Fung¹, Kam‐Leung Siu¹, Man Lung Yeung², Prof Judy Wai Ping Yam³, Prof Dong‐Yan Jin¹

¹School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong, ²Department of Microbiology, The University of Hong Kong, Pokfulam, Hong Kong, ³Department of Pathology, The University of Hong Kong, Pokfulam, Hong Kong

PF05.07 Redox‐active outer‐membrane vesicles boost extracellular electron uptake in marine sedimentary bacteria under energy starvation conditions

Dr. Xiao Deng¹

¹National Institute For Materials Science, Tsukuba, Japan

PF05.08 “Strain‐Based Comparison and Pharmacological Investigation of Bacterial Extracellular Vesicles”

Master Seoah Park¹, Jongsoo Mok², Junghoon Choi¹, Hye‐Min Yu³, Hye‐Jin An³, Ga‐Hyun Choi³, Yeon‐Seon Lee³, Ki‐Jin Kwon³, Sung‐Jun Choi³, Soo‐Jin Kim³, Joonghoon Park^1,2

¹Graduate School of International Agricultural Technology, Seoul National University, Korea, ²Institute of Green Bio Science & Technology, Seoul National University, Korea, ³Schofield Biome Research Lab, HK inno.N, Korea

PF05.09 Cracking the egg: probing Schistosoma mansoni eggs for tolerogenic products

Mx Madeleine Rogers¹, Dr Athena Andreosso¹, Dr Jagan Billakanti², Dr Sandip Kamath³, Prof Donald McManus¹, Prof Malcolm Jones¹, Dr Catherine Gordon¹, A/Prof Severine Navarro¹, A/Prof Severine Navarro⁴

¹QIMR Berghofer Medical Research Institute, Herston, Australia, ²Cytiva, Brisbane, Australia, ³Medical University of Vienna, Vienna, Austria, ⁴Centre for Childhood Nutrition Research, Brisbane, Australia

PF06.01 Evaluating the immunogenicity of circulating extracellular vesicles from gestational diabetes patients: insights for therapeutic use

Professor Flavio Carrion, Dr Soumyalekshmi Nair, Katherin Scholz‐Romero, Dr Carlos Palma, Dr Andrew Lai, Dr Dominic Guanzon, Professor Bernardo Morales, Associate Professor Martha Lappas, Professor Carlos Salomon

PF06.03 Pancreatic cancer‐derived small extracellular vesicles alter immune cell behaviour via the sphingosine‐1‐phosphate signalling pathway

Miss Jordan Fyfe, Dr Danielle Dye, Dr Pat Metharom, Professor Marco Falasca

PF06.04 The HLA‐I immunopeptidome of platelet‐derived extracellular vesicles

Dr Caitlin Boyne, Mr Jordan Marsh, Dr Sally Shirran, Dr Alan Stewart, Dr Simon Powis

PF06.05 Therapeutic rescue of sepsis induced liver damage by immune‐regenerative HIF1α enriched extracellular vesicles

Miss YEJI LEE, Miss Jiyoung Goo, Mr In‐San Kim

PF06.06 Unfolding the role of placental small extracellular vesicles in preeclampsia in the development of the fetal immune system

Ms Michaela Klaczynski, Ms Birgit Hirschmugl, Ms Barbara Darnhofer, Ms Katharina Eberhard, Mr Harald Köfeler, Mr Karl Kashofer, Mr Christian Wadsack

PF06.07 Aerobic fitness levels can alter the secretion of circulating extracellular vesicles during moderate intensity exercise

Dr Mee Chee Chong, Dr Anup D. Shah, Associate Professor Ralf B. Schittenhelm, Dr Anabel Silva, Dr Patrick F. James, Professor Jason Howitt

PF06.08 Akkermansia muciniphila alleviates lipid metabolism disorders in mice via delivery of Amuc_1100‐Containing vesicles

Phd Qianbei Li, Professor Lei Zheng

PF06.10 Calpeptin alters insulin‐mediated glucose uptake and extracellular vesicle secretion in human adipocytes

Msc Johanna Matilainen, Viivi Berg, Maija Vaittinen, Janne Tampio, Ville Männistö, Jussi Pihlajamäki, Tanja Turunen, Marjo Malinen, Pirjo Käkelä, Dorota Kaminska, Veera Luukkonen, Anne‐Mari Mustonen, Uma Thanigai‐Arasu, Kristiina Huttunen, Reijo Käkelä, Sanna Sihvo, Petteri Nieminen, Kirsi Rilla

PF06.11 Changes in insulin sensitivity across gestation is associated with changes in the profile of maternal circulating extracellular vesicle protein and miRNA: A Longitudinal study.

Dr Soumyalekshmi Nair, Lilian Kessling, Dominic Guanzon, Andrew Lai, Flavio Carrion, David Simmons, Mireille Van Poppel, Harold David McIntyre, The Dali Core Investigator Group, Gernot Desoye, Carlos Salomon

PF06.12 Early pregnancy serum maternal and placenta‐derived exosomes miRNAs vary based on pancreatic β‐cell function in gestational diabetes

MD, PhD Melissa Razo‐Azamar, PhD Rafael Nambo‐Venegas, PhD Iván Rafael Quevedo, PhD Gregorio Juárez‐Luna, PhD Carlos Salomon, MD, PhD Martha Guevara‐Cruz, PhD Berenice Palacios‐González

PF06.13 Functional implications of hepatic EV alteration in NAFLD and T2DM

Pin Hsuan Chu, Dr. Han‐Yi E. Chou, Dr. Tien‐Jyun Chang, Dr. Shiau‐Mei Chen

PF06.14 Induction of renal damage by methylglyoxal‐lysine dimer (MOLD) through exosome‐mediated miR‐130a‐3p

Principal Researcher Eun Hee Han, Ms. Hye Min Kim, Dr. Jin Young Min, Mr. Min Sung Park1

PF06.15 Multifaceted action of stem cell‐derived extracellular vesicles for nonalcoholic steatohepatitis

Ph.D. Jimin Kim, M.S. Seul Ki Lee, M.S. Haedeun You, M.S. Sang‐Deok Han, Ph.D. Tae Min Kim, Ph.D. Soo Kim

PF06.17 Podocyte‐derived urinary extracellular vesicles in membranous nephropathy

PhD student Karen Lahme, PhD Wiebke Sachs, PhD Desiree Loreth, Stephanie Zielinski, Johannes Brand, PhD Kristin Surmann, Professor Uwe Völker, Thorsten Wiech, Professor Tobias N. Meyer, Lars Fester, Professor Catherine Meyer‐Schwesinger

PF06.18 Primary pancreatic ductal cells from normal cadaveric donors are responsive to type 1 diabetes‐mimicking proinflammatory cytokines in vitro and secrete extracellular vesicles

Neslihan Erdem, Nathaniel Hansen, Min Talley, Heather Zook, Kevin Jou, Jose Ortiz, Nagesha Guthalu Kondegowda, David Arribas‐Layton, Fouad Kandeel, Enrique Montero, Helena Reijonen, Rupangi Vasavada, Patrick Pirrotte, Tijana Jovanovic‐Talisman, Hsun Teresa Ku

PF06.19 Small extracellular vesicles inhibit NLRP3 inflammasome activation in diabetic retinopathy

Henry Louie, Ilva D. Rupenthal, Odunayo O. Mugisho, Lawrence W. Chamley

PF06.22 Investigating the impact of extracellular vesicles in obese pregnancies: Are EVs from obese

Pregnant dams during early pregnancy sufficient to cause obesity in offspring?

Phd Student Taylor Hollingsworth¹, Pharm.D., Ph.D. Thea Golden¹, M.D. Rebecca Simmons¹

¹University Of Pennsylvania, Philadelphia, United States

PF06.23 Characterizing plasma‐derived EVs from pregnant Black cis‐women as a potential tool to predict adverse pregnancy outcomes

Ms. Kobe Abney¹, Pharm.D., Ph.D. Thea Golden¹, Dr. Yu‐Chin Lien¹, Dr. Nadav Schwartz¹, Dr. Rebecca Simmons¹

¹University Of Pennsylvania, United States

PF06.24 Human seminal fluid extracellular vesicles induce immune responses in female cervical cells in vitro

Miss Cottrell Tamessar^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia.2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308,Australia.}, Miss Chishan Burch^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia.2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308,Australia.}, Miss Piper Miller^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia.2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308,Australia.}, Miss Jane Durbidge^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia.2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308,Australia.}, Miss Tegan Bryde^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia.2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308,Australia.}, Miss Shanu Parameswaran^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW 2305,Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan,NSW 2308, Australia.}, Associate Professor Geoffry De Iuliis^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, NewLambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle,University Drive, Callaghan, NSW 2308, Australia.}, Doctor Judith Weidenhofer^{3 Precision Medicine Research Program, Hunter Medical Research Institute,New Lambton Heights, NSW 2305, Australia. 4 School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, The University of Newcastle,Ourimbah, NSW 2258, Australia.}, Dr Hui‐ming Zhang^{5 Central Analytical Facility, Research and Innovation Division, The University of Newcastle, UniversityDrive, Callaghan, NSW 2308, Australia.}, Professor Sarah Robertson^{7 Robinson Research Institute and School of Biomedicine, The University of Adelaide, SA5005, Australia.}, Doctor Elizabeth Bromfield^{1 Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, NSW2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, University Drive, Callaghan,NSW 2308, Australia. 6 Bio21 Institute, School of BioSciences, The University of Melbourne, VIC 3010, Australia.}, Doctor David Sharkey^{7 Robinson ResearchInstitute and School of Biomedicine, The University of Adelaide, SA 5005, Australia.}, Professor Brett Nixon^{1 Infertility and Reproduction Research Program,Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Science andEnvironment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}, Doctor John Schjenken^{1 Infertility and Reproduction ResearchProgram, Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia. 2 School of Environmental and Life Sciences, College of Engineering, Scienceand Environment, The University of Newcastle, University Drive, Callaghan, NSW 2308, Australia.}

¹Infertility and Reproduction Research Program, Hunter Medical Research Institute, New Lambton Heights, Newcastle, Australia, ²School of Environmental and Life Sciences, College of Engineering, Science and Environment, The University of Newcastle, Callaghan, Newcastle, Australia, ³Precision Medicine Research Program, Hunter Medical Research Institute, New Lambton Heights, Newcastle, Australia, ⁴School of Biomedical Sciences and Pharmacy, College of Health, Medicine and Wellbeing, The University of Newcastle, Ourimbah, Central Coast, Australia, ⁵Central Analytical Facility, Research and Innovation Division, The University of Newcastle, Callaghan, Newcastle, Australia, ⁶Bio21 Institute, School of BioSciences, The University of Melbourne, Parkville, Melbourne, Australia, ⁷Robinson Research Institute and School of Biomedicine, The University of Adelaide, North Adelaide, Adelaide, Australia

PF06.25 Local treatment of inflammatory bowel disease by EV drug carriers

Miss Nidhi Seegobin¹, Miss Victoria Chris^1,2, Miss Marissa Taub¹, Dr Sudaxshina Murdan¹, Prof Abdul Basit¹

¹University College London, London, United Kingdom, ²University of Oxford, Oxford, United Kingdom

PF06.27 The induction of pro‐inflammatory extracellular vesicles in the progression of metabolic‐associated fatty liver disease (MAFLD)

Dr. Allen Wei‐Lun Huang¹, Ms. Tzu‐Ching Kao², Dr. Sin‐Tian Wang², Ms. Yi‐Wen Chiu², Dr./Prof. Pin‐Nan Cheng³, Dr./Prof. Chi‐Yi Chen⁴, Prof. Kung‐Chia Young²

¹Center of Applied Nanomedicine, National Cheng Kung University, Tainan, Taiwan, ²Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ³Department of Internal Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ⁴Division of Gastroenterology and Hepatology, Department of Internal Medicine, Chia‐Yi Christian hospital, Chiay, Taiwan

PF06.28 Stem Cell‐Derived Nano Vesicles' Impact on Muscle Regeneration in Cachexia Models

Postgraduate student Keren Esther Kristina Mantik^1,2, Researcher Sohee Moon¹, Sujin Kim¹, Researcher, MS Bon‐Sang Gu¹, Postgraduate student Jubi Lee^1,2, Postgraduate student Chan‐Young So^1,2, CEO Shingyu Bae⁴, Professor, MD, PhD Ju‐Hee Kang^1,2,3

¹Department of Pharmacology and Research Center for Controlling Intercellular Communication, College of Medicine, Inha University, 22212, South Korea, ²Program in Biomedical Science and Engineering, Inha University, 22212, South Korea, ³Institute of Sports & Arts Convergence (ISAC), Inha University, 22212, South Korea, ⁴BioDrone Research Institute, MDimune Inc, South Korea

PF07.01 19F‐MRI‐labeled extracellular vesicle mimetics for specific monitoring of targeted drug delivery

Dr. Andrea Galisova, Dominik Havlicek, Ayca Tunca, Ondrej Sedlacek, Daniel Jirak

PF07.02 A dynamic, label‐free, and efficient small EVs sensing platform for assessment of EV based drugs —‐ case study of MSC‐sEVs for Cardiac Oxidative Stress Injury Therapy

Dr Chunlian Qin, Dr Danyang Li, Dr Ning Hu, Dr Lizhou Xu

PF07.04 A novel pathogen sensing platform for detection of Escherichia Coli

Miss Shiana Malhotra, Dr Renee Goreham, Dr Thomas Nann

PF07.05 Analysis of tumor‐derived small extracellular vesicles with spectral flow cytometry

Dr Linda Hofmann, Dr Annika Betzler, Prof Thomas Hoffmann, Prof Cornelia Brunner, Prof Marie‐Nicole Theodoraki

PF07.10 Fueling strategy‐based self‐sacrificed MOF@DNAzyme integrated chip for the isolation and detection of tumor‐derived extracellular vesicles

Student Zehan Zeng, PhD Weilun Pan, Professor Jinxiang Chen

PF07.11 Highly sensitive detection of extracellular vesicles using the fluorescence molecular projection imaging system

Highly sensitive detection of extracellular vesicles using the fluorescence molecular projection imaging system Yulin Cao, Highly sensitive detection of extracellular vesicles using the fluorescence molecular projection imaging system Yuxuan Jiang, Highly sensitive detection of extracellular vesicles using the fluorescence molecular projection imaging system Qiubai Li, Highly sensitive detection of extracellular vesicles using the fluorescence molecular projection imaging system Yong Deng

PF07.12 Improvement of extracellular vesicle detection sensitivityon a surface‐functionalized power‐free microchip

Associate Professor Ryo Ishihara, Hinako Yokohari, Ren Ogata, Kotomi Katori, Kentaro Doi, Kurumi Omiya, Tadaaki Nakajima, Eri Shimura, Takeshi Baba

PF07.14 Multiparametric analysis of single small extracellular vesicles using nanoflow cytometry (nFCM): optimized experimental design and implementation

Research Scientist Prashant Kumar, Brian Dobosh, Rabindra Tirouvanziam

PF07.19 Robotic fluidic force microscopy (robotic FluidFM)‐based nanoinjection of extracellular vesicles into individual living cells

Dr. Tamás Visnovitz, Ms Kinga Dóra Kovács, Dr. Tamás Gerecsei, Dr. Beatrix Péter, Dr. Sándor Kurunczi, Ms Anna Koncz, Dr. Krisztina Németh, Ms Dorina Lenzinger, Dr. Krisztina V Vukman, Ms Anna Balogh, Ms Imola Rajmon, Dr. Péter Lőrincz, Dr. Inna Székács, Prof. Edit I Buzás, Dr. Róbert Horváth

PF07.20 Single vesicle flow cytometry with enhanced small particle detection using spectral flow cytometry

Dr. Maria Gracia Garcia Mendoza, Dr. John Nolan, Erika Duggan, Patrick Nolan, Kate Pilkington, Dr. Haley Pugsley

PF07.22 Tracking system of CD63‐positive extracellular vesicles under in vitro coculture conditions

Lecturer Yutaka Naito, Professor Kazufumi Honda

PF07.24 Visualizing intercellular transfer of extracellular vesicle RNA cargo using an improved metabolic labelling approach

Ms. Willemijn de Voogt, Dr. Richard Wubbolts, Dr. Pieter Vader

PF07.25 The comparison and optimalization of sEV s staining protocols for visualization of cellular uptake in super‐resolution microscopy

Mr Jakub Tomaszewski¹, Bsc Wiktoria Klimek², PhD Hanna Kozłowska³, PhD Małgorzata Czystowska‐Kuźmicz¹

¹Department of Biochemistry, Medical University of Warsaw, Warsaw, Poland, ²Faculty of Biology and Biotechnology, Warsaw University of Life Sciences, Warsaw, Poland, ³Laboratory of Advanced Microscopy Techniques, Mossakowski Medical Research Institute Polish Academy of Sciences, Warsaw, Poland

PF07.26 Fluorescence Polarization Utilizing Aptamers for Targeted Sensing of sEVs

Mr Satendra Jaysawal^1,2, Dr. Rocky Chowdhury^1,2, Mr. Rajindra Napit^1,2, Ms. Jasmine Catague^1,2, Mr. Haben Melke^1,2, Dr Cuong Pham³, Dr. Wei Duan^1,2

¹School of Medicine, Deakin University, Geelong, Australia, ²Institute of Mental and Physical Health and Clinical Translation (IMPACT), Deakin University, Geelong, Australia, ³Molecular Imaging and Theranostics Laboratory, Baker Heart and Diabetes Institute, Melbourne, Australia

PF07.27 A flow cytometry approach for the characterization and isolation of extracellular vesicles

Dr. Anis Larbi¹

¹Beckman Coulter Life Sciences, Lyon, France

PS01.02 Apple‐derived nanovesicles influence bone regeneration by acting on THP‐1‐derived macrophage polarization and mesenchymal stem cell osteogenic differentiation

Martina Trentini, Dr. Luca Lovatti, Prof. Dr. Kathrin Becker, Dr. Giulia Brunello, Prof. Dr. Barbara Zavan

PS01.03 Atractylodes macrocephala derived EV‐like particles alleviate ulcerative colitis by modulating intestinal flora and TH17 signaling pathway

Professor Kewei Zhao, Xuejun Tan, Bowen Gao, Yukun Xu, Yue Cao, Qing Zhao, Tianxin Qiu, Mingzhen Zhang

PS01.04 Bacteria Gram+ derived nanovesicles and mimetics as new vaccine for Streptococcus pneumoniae (Pn)

Bertrand Czarny

PS01.05 Bacteria‐derived mimetic vesicles: their role in the invitro immune response against Streptococcus pneumoniae infection

Dr Dinesh Kesavan

PS01.08 Delineating the pro‐osteogenic potential of Lactobacillus rhamnosus derived extracellular vesicles in ameliorating glucocorticoid induced osteoporosis ex vivo

Ms. Megha Sharma^{All India Institute of Medical Sciences (AIIMS), New Delhi, India}, Dr. Rupesh K. Srivastava^{All India Institute of Medical Sciences(AIIMS), New Delhi, India}

PS01.09 Delivery of nucleic acids using red blood cell‐derived extracellular vesicles to the central nervous system

Ms Melissa Tan, Dr Brenda Wan Shing Lam, Dr Waqas Muhammad Usman, Dr Thach Tuan Pham, Dr Chang Gao, Dr Harwin Sidik, Ms Rachel Tan, Dr Minh TN Le

PS01.100 Therapeutic potential of IL‐1β‐primed mesenchymal stromal cells‐derived soluble factors and extracellular vesicles in wound healing

PhD Marina Trouillas, Mrs Marine De Taddeo, Mr Pierre Maincourt, Mrs Muriel Nivet, MD Guillaume Valade, Mrs Claire Langle, Mrs Marion Grosbot, Mrs Sylvie Goulinet, PhD Philippe Mauduit, MD, PhD Sébastien Banzet, PhD Juliette Peltzer

PS01.102 Therapeutic role of MSC exosomes in rabbit temporomandibular joint model of osteoarthritis

Dr Yuanyuan Jiang, Dr Shipin Zhang, Dr Sai Kiang Lim, Dr Wei Seong Toh

PS01.105 Umbilical cord mesenchymal stromal cells‐derived small extracellular vesicles: advancing knee osteoarthritis therapeutics

Mr. Aliosha I. Figueroa‐Valdés¹, Mr. Nicolás Georges², Ms. Catalina Adasme‐Vidal¹, Ms. Yeimi Herrera‐Luna³, Ms. Patricia Luz‐Crawford^{1, 3}, Mr. Maroun Khoury^1,2,4,5,6, Ms. Francisca Alcayaga‐Miranda^1,2,4,5,6

¹IMPACT, Center of Interventional Medicine for Precision and Advanced Cellular Therapy, Santiago, Chile, ²Universidad de los Andes, Centro de Investigación e Innovación Biomédica (CiiB), Laboratory of Nano‐Regenerative Medicine, Santiago, Chile, ³Universidad de los Andes, Centro de Investigación e Innovación Biomédica (CiiB), Laboratory of Molecular and Cellular Immunology, Santiago, Chile, ⁴Consorcio Regenero, Chilean Consortium for Regenerative Medicine, Santiago, Chile, ⁵Universidad de los Andes, Faculty of Medicine, School of Medicine, Santiago, Chile, ⁶Cells for Cells, Santiago, Chile,

PS01.107 Unveiling the power of adipose tissue stem cell‐derived peptide‐engineered CD81+/Tsg101+ extracellular vesicles for precision targeting and neural stem cell rejuvenation

Mr. Satyajit Ghosh, Dr. Surajit Ghosh

PS01.108 Urine derived stem cells: A unique robust production platform for autologous immunomodulatory EVs

Anders Boysen, Doctor Bradley Whitehead, Doctor Anne Louise S. Revenfeld, Doctor Anna Karina Juhl, Doctor Reza Yarani, Doctor Yonglun Luo, Doctor Thor Petersen, Doctor Peter Nejsum

PS01.108 An acellular targeted therapeutic approach using extracellular vesicles from human endometrial mesenchymal stem cells

Dr Shanti Gurung^1,2, Ms Diem‐Mai Pham Diem‐Mai Pham¹, Ms Molly McLaughlin^1,2, Dr Jill C. Danne⁴, Dr Joel R. Steele³, Professor Ralf B. Schittenhelm³, Professor Jerome A. Werkmeister^1,2, Professor Caroline E. Gargett^1,2

¹The Ritchie Centre/Hudson Institute Of Medical Research, Clayton, Australia, ²Obstetrics and Gynaecology, Monash University, Clayton, Australia, ³Proteomics and Metabolomics Platform, Monash University, Clayton, Australia, ⁴Monash Ramaciotti Centre for Cryo‐Electron Microscopy, Clayton, Australia

PS01.109 Extracellular vesicles as a treatment for metabolic dysfunction‐associated steatohepatitis (MASH) and hepatocellular carcinoma (HCC)

Miss Mihiri Goonetilleke^1,2, Ms Jeanne Correia¹, Dr Yuan Chen¹, Ms Hannah McDonald¹, Dr Siow Teng Chan¹, Mr Ian Simpson⁵, Dr Ishmael Inocencio¹, Prof. William Sievert^3,4, A/Prof Rebecca Lim¹

¹Hudson Institute Of Medical Research, Clayton, Australia, ²Obstetrics and Gynaecology, Monash University, Clayton, Australia, ³Gastroenterology and Hepatology Unit, Monash Health, Clayton, Australia, ⁴Centre for Inflammatory Disease, Monash University, Clayton, Australia, ⁵Monash Health, Clayton, Australia

PS01.11 Development of microbial nanovesicle‐based (OMVs) multivalent Vaccine formulation against human/animal diseases

Duvvada Srinivas

PS01.114 Effect of extracellular vesicles isolated from osteoblast differentiation medium of dedifferentiated adipocytes on osteogenic differentiation.

4th grade in Ph.D Yusuke Nishiguchi¹, Ph.D Mamoru Ueda², Ph.D Hirohito Kubo², Ph.D Junichiro Jo³, Ph.D Yoshiya Hashimoto³, Ph.D Toshihiko Takenobu²

¹Graduate school of dentistry department of oral and maxillofacial surgery, Osaka Dental University, Osaka/Chuoku Otemachi, Japan, ²Second Department of Oral and Maxillofacial Surgery, Osaka Dental University, Osaka/Chuoku Otemachi, Japan, ³Department of Biomaterials Osaka Dental University, Hirakatashi/Kuzuhahanazonocho, Japan

PS01.115 Acute Toxicity Effect of UC‐MSC Secretome in Different Route of Administration

Dr. Angliana Chouw^1,2, Dr. Cynthia Retna Sartika^1,2, Miss Geofanny Facicilia¹, Miss Annisa Nur Arofah¹, Miss Riska Agustina¹, Miss Zulfa Maulidah¹

¹Prodia Stemcell Indonesia, Jakarta, Indonesia, ²Universitas Padjajaran, Sumedang, Indonesia

PS01.116 Therapeutic Potential of Umbilical Cord Mesenchymal Stem Cell‐Derived Extracellular Vesicles on Atopic Dermatitis: A Comparative Study

Mrs Maimonah Al‐Masawa¹, Associate Professor Dr Angela Min Hwei Ng¹, Dr Jhi Biau Foo², Dr Chee Wun How³, Dr. Jia Xian Law¹

¹Centre For Tissue Engineering and Regenerative Medicine, Faculty of Medicine, National University Malaysia, Kuala Lumpur, Malaysia, ²School of Pharmacy, Faculty of Health and Medical Sciences, Taylor's University, Malaysia, ³School of Pharmacy, Monash University, Malaysia

PS01.118 Intravenous Administration of Extracellular Vesicles Derived from Mesenchymal Stem Cells (MSC‐EVs) Mitigates Tendon and Cartilage Degeneration in Type II Diabetes (T2D) Rats

Dr Sik Loo Tan¹, Dr Zahrah Shamim¹, Omar Maged¹, Nik Aizah¹, Dr Qi Hao Daniel Looi², Dr JhiBiau Foo³, Professor Tunku Kamarul^1,4

¹Department of Orthopaedic Surgery, National Orthopaedic Center for Excellence in Research and Learning (NOCERAL), Faculty of Medicine, Universiti Malaya, 50603, Kuala Lumpur, Malaysia., Malaysia, ²My CytoHealth Sdn. Bhd., Lab 6, DMC Level 2, Hive 5, Taman Teknologi MRANTI, Bukit Jalil, Kuala Lumpur 57000, Malaysia, Bukit Jalil, Malaysia, ³School of Pharmacy, Faculty of Health and Medical Sciences, Taylor's University, Subang Jaya 47500, Selangor, Malaysia, Subang Jaya, Malaysia, ⁴Advanced Medical and Dental Institute (AMDI), Universiti Sains Malaysia, Bertam 13200 Kepala Batas, Pulau Pinang, Malaysia., Bertam, Kepala Batas, Malaysia

PS01.12 Edible plant‐derived nanovesicles to systemic deliver nucleic acid medicine for oral administration

Dr. Tomohiro Umezu, Mamoru Yanagimachi, Ph.D. Masakatsu Takanashi, MD, Ph.D. Yoshiki Murakami, MD, Ph.D. Masahiko Kuroda

PS01.13 Effects of ginger‐derived extracellual vesicles on preadipocyte differentiation: implications for obesity

Ph.d Student Diksha Choudhary

PS01.130 Comprehensive characterization of olive‐derived nanoparticles (ODNPs) as a new drug delivery system.

Graduate Student Zhu Zhao¹, Dr. Jerome Lacombe¹, Dr. Frederic Zenhausern¹

¹Univ Of Arizona, Phoenix, United States

PS01.14 Engineering plant‐derived extracellular vesicles for targeted inflammatory therapy in colitis

Su Jin Kang, Ph.D Won Jong Rhee

PS01.15 Enhanced chemo‐resistance in liver and breast cancer cells: synergistic interplay of seaberry and garlic‐derived extracellular vesicles with cisplatin

Mr. Yasir Mohamed Riza, Dr. Faisal Alzahrani, Dr. Rami Mosaoa

PS01.16 Exosome‐delivered curcuminoids to target the brain: Modulation of key markers of Alzheimer's disease (AD)

Dr. Ramesh Gupta, Dr. Raghuram Kandimalla, Ms. Disha Moholkar, Dr. Margaret Wallen, Mr. Jeyaprakash Jeyabalan, Dr. Wendy Spencer, Dr. Neetu Tyagi, Dr. Farrukh Aqil

PS01.18 Extracellular vesicles from bovine milk loaded miR146a‐5p prevented group 2 innate lymphoid cells‐dominant allergic airway inflammation

Li Chan‐gu, Professor Fu Qing‐Ling

PS01.19 Extracellular vesicles‐derived from VGBR induces cell cycle arrest and apoptosis in human colon cancer cells through p53‐ pathway

PhD Student Abhinay Kumar Singh, Dr. Win‐Ping Deng

PS01.20 Gut commensal bacteria derived outer membrane vesicles tamp down skin inflammatory in psoriasis

Dandan Su, Manchun Li, Professor Hongbo Chen, Fang Cheng

PS01.22 Investigating the therapeutic potential of human amniotic epithelial cell derived extracellular vesicles for inflammatory fetal brain and lung injury in a large animal model

Dr Ishmael Inocencio, Mr Naveen Kumar, A/Prof Rebecca Lim, Dr Tamara Yawno

PS01.23 Isolation, characterization, and functional studies of the Gardenia‐derived EVs for potential Parkinson's disease treatment

Haobo Wang, Dr Wen Chen, Dr Yan Wang, Dr Danyang Li, Dr Lizhou Xu

PS01.25 Morinda officinalis‐derived extracellular vesicle‐like particles anti‐osteoporosis by regulating MAPK signaling pathway

Professor Kewei Zhao, Doctor Yue Cao, Master Xuejun Tan

PS01.26 Oral delivery of dihydroartemisinin for the treatment of melanoma via bovine milk‐derived small extracellular vesicles (sEVs)

Mr. Dulla Naveen Kumar, Ms. Aiswarya Chaudhuri, Ms Deepa Dehari, Dr. Dinesh Kumar, Dr. Ashish Kumar Agrawal

PS01.27 Parabacteroides goldsteinii‐derived exosomes alleviate acute lung injury by regulating gut microbiota

Ms Wensi Zhu, Ms Linxiao Han, Ms Ludan He, Dr Chih‐Jung Chang, Jian Zhou

PS01.28 Polygonum cuspidatum derived nanoparticles and acupuncture combined to alleviate rheumatoid arthritis via immunomodulation

Dr Ningcen Li, Prof Bo Li, Prof Lei Zheng

PS01.29 Polygonum cuspidatum derived nanovesicles accelerate wound healing of deep second‐degree burn by Nrf 2‐Keap pathway

Qi Xiu, Prof. Bo Li, Prof. Lei Zheng

PS01.30 Polyphenol‐rich cocoa supplementation elevates neuroactive compounds in escherichia coli nissle 1917 membrane vesicles

PhD Student Amelie Legare, Miss Michele Iskandar, Andre Marette, Stan Kubow

PS01.32 Potential of milk‐derived extracellular vesicles for oral drug delivery

HoChung Jang, Dr. Yoosoo Yang

PS01.33 Recombinant extracellular vesicles as vaccines within animal health

Professor Hanne Winther‐Larsen

PS01.34 Rhizoma Drynariae‐derived nanovesicles reverse osteoporosis by potentiating osteogenic differentiation of human bone marrow mesenchymal stem cells via targeting ERα signaling

Ph.d Qing Zhao, Ph.D Junjie Feng, Ph.D Lei Zheng, Ph.D Kewei Zhao

PS01.37 Turmeric‐derived nanoparticles functionalized aerogel regulates multicellular networks to promote diabetic wound healing

Ph.D Bodeng Wu, Ph.D Weilun Pan, Ph.D Shihua Luo, MD Mingzhen Zhong, Professor Bo Li, Professor Lei Zheng

PS01.38 Two‐photon responsive microneedles loaded with engineered turmeric‐derived extracellular vesicles for detection and treatment of subcutaneous infections

Dr Weilun Pan, Master Mingzhen Zhong, Prof Lei Zheng

PS01.40 Harnessing the delivery potential of milk extracellular vesicles as innovative therapeutic tools for treating bacterial infections

Jitendra Kumar¹

¹ICAR‐National Dairy Research Institute, Karnal‐132001, India

PS01.40 Amplifying the regenerative and immunomodulatory potential of mesenchymal stem cell‐derived small extracellular vesicles via apoptotic induction

Professor Sujata Mohanty, Ms Meenakshi Mendiratta, Ms Mohini Mendiratta, Dr Suchi Gupta

PS01.41 Assessing the cellular effects of ASC‐EVs in the context of autologous fat grafting

Dr Emma Symonds, Rachelle Smith, Mr Alexander Brown, Associate Professor Margaret Currie, Dr Kathryn Hally, Dr Kirsty Danielson

PS01.42 Large‐scale preparation of milk‐derived extracellular vesicles for medical cosmetics application

Xue Wu, Jiuheng Shen, Youxiu Zhong, Xian Zhao, Peifen Gao, Wantong Zhou, Xudong Wang, Professor Wenlin An

¹National Vaccine & Serum Institute (NVSI), China National Biotech Group (CNBG), Sinopharm Group, Beijing, China

PS01.44 Characterization of extracellular vesicles in mesenchymal stem cell co‐cultures derived from different tissue origins and potential therapeutic applications

Tsuyoshi Kawaharada, Daisuke Watanabe, Shuhei Iida, Amaka Watanabe, Akio Mizushima

PS01.44 Strain‐Based Comparison and Pharmacological Investigation of Bacterial Extracellular Vesicles

Extracellular Vesicles Seoah Park¹

¹Seoul National University, South Korea

PS01.45 Counter‐selection of EVs using Kupffer cells alters protein corona and EV biodistribution in vivo

Dr Chintan Bhavsar¹, Dr Rui Chen¹, Ms Elaina Coleborn¹, Ms Shuying Li¹, Ms Sarah Wilkey¹, Mr Trent Neilson¹, Dr Katharine Irvine², Dr James Cuffe¹, Dr Sherry Wu¹

¹The University Of Queensland, Brisbane, Australia, ²Mater Research, Brisbane, Australia

PS01.45 Charting cardiac cell repair: dual therapy combining nanovesicles and biomaterials

Phd Student Auriane Drack, Doctor Alin AR Rai, Hien A Tran, Associate professor Jelena Rnjak‐Kovacina, Associate professor David Greening

PS01.46 Characterization and biological activity potential of extracellular vesicles from industrial bovine milk: a comparative analysis of multiple sources

Mr. Aliosha I. Figueroa‐Valdés¹, Ms. Catalina Adasme‐Vidal¹, Dr. Maroun Khoury¹, Dra. Francisca Alcayaga‐Miranda¹

¹IMPACT, Center of Interventional Medicine for Precision and Advanced Cellular Therapy, Santiago, Chile

PS01.46 Comparative analysis of the therapeutic potential of extracellular vesicles from aged and young bone marrow‐derived mesenchymal stem cells in osteoarthritis pathogenesis

Ms sh*tal Wakale, Dr Antonia Sun, Dr Yang Chan, Dr Jennifer Gunter, Dr Chamikara Liyanage, Prof Ross Crawford, Dr Song Wu, Dr Hai Hu, Dr Indira Prasadam

PS01.47 Comparison of the therapeutic effect of fetal and perinatal MSCs derived EVs on inflamed chondrocytes in vitro

Karyna Tarasova, MSc Belen Arteaga, PhD Harini Nivarthi, MSc Johanna Gamauf, MSc Angkana Kidtiwong, PhD Sinan Gültekin, PhD Mathias Hackl, PhD Regina Grillari, Prof. PhD Christopher Gerner, Ass. Prof. Dr. Iris Gerner, Prof. Dr. Florien Jenner

PS01.49 Effects of bone marrow mesenchymal stem cell‐derived small extracellular vesicles (BM‐MSC‐sEVs) on H2O2‐induced oxidative damage in human retinal pigment epithelial cells

Dr. Nithikan Suthumchai, Miss Panjaree Siwaponanan, Miss Payalak Sudcharee, Dr. Siripakorn Sangkitp*rn, Miss Acharap*rn Dambua, Miss Patcharap*rn Boonchu, Miss Phatcharaphon Nopprang, Prof.Dr. Kovit Pattanapanyasat, Prof. La‐ongsri Atchaneeyasakul

PS01.50 Effects of hypoxia precondition on enhancing the anti‐inflammatory effects of mesenchymal stem cells derived exosomes may involve microRNA‐21‐5p

MD Kuan‐Wen Chen, PhD Chao‐Yuan Chang, MD,PhD Chun‐Jen Huang

PS01.51 Engineering adipose‐derived stem cell‐derived extracellular vesicles by calcium silicates activated for chronic wound healing

Associate Professor Jian‐Jr Lee, Dr En‐Wei Liu, Dr Yen‐Hong Lin, Ms Min‐Hua Yu, Associate Professor Ming‐You Shie

PS01.52 Enhancing extracellular vesicles yields and functionality for cardiac repair through scalable bioreactor production of human‐induced pluripotent stem cells

PhD Student Ana Meliciano, Pedro Vicente, Ana Filipa Louro, Cláudia Diniz, João Jacinto, Paula Marques Alves, Margarida Serra

PS01.53 EVs from hiPSC‐derived NSCs are proficient in inhibiting traumatic brain injury‐induced NLRP3‐p38/MAPK, cGAS‐STING activation, and IFN‐I signaling

Ashok Shetty, Dr Maheedhar Kodali, Dr Leelavathi N Madhu, Dr Shama Rao, Dr. Raghavendra Upadhya, Ms Sahithi Attaluri, Dr Bing Shuai

PS01.54 Exosome derived from 3D‐cultured hADSCs exhibited enhanced osteogenesis capacity via intravenous injection

Dr. Ruijing Chen, Dr. Taojin Feng, Dr. Ming Chen, Dr. Ruijing Chen

PS01.56 Exploring induced pluripotent stem cell‐derived exosomes as a potent antimicrobial and immunomodulatory agent against vibrio vulnificus

Phd Pei‐Ling Chi

PS01.57 Exploring the immunomodulatory and wound‐healing potential of Extracellular vesicles derived from iMSCs

Denise Zujur, MSc William Theoputra, PhD Makoto Ikeya

PS01.58 Exploring the therapeutic potential of extracellular vesicles derived from human mesenchymal stem cells in tumor‐bearing mice

Dr. Prapatsorn Charoenyingpaisal, Dr. Hsien‐Hen Lin, Dr. Toru Okubo, Mr. Hayato Kurata, Mr. Tetsuo Koike, Mr. Yoichi Honma

PS01.59 Extracellular vesicles derived from iMSC primed with hyaluronic acid enhance cardiac function.

Ph.D. Seon‐Yeong Jeong, Ph.D. Jimin Kim, M.S. Seul Ki Lee, M.S. Haedeun You, Ph.D. Soo Kim

PS01.60 Extracellular vesicles derived from mesenchymal stem cells and cartilage tissue to promote cartilage regeneration

Dr. Jia Xian Law, Ms. Chiew Yong Ng, Assoc. Prof. Min Hwei Ng, Prof. Ying Yang, Assoc. Prof. Jhi Biau Foo, Dr. Chee Wun How, Assoc. Prof. Kien Hui Chua, Assoc. Prof. Kok Yong Chin, Dr. Rizal Abdul Rani, Prof. Nor Hamdan bin Mohamad Yahaya

PS01.61 Extracellular vesicles derived from mesenchymal stem cells reduce inflammation and restore intestinal barrier integrity in a new in vitro co‐culture model of intestinal inflammation

Miss Mona Belaid, Giorgia Pastorin, Driton Vllasaliu

PS01.63 Extracellular vesicles of senescent mesenchymal stromal cells lose their antifibrotic potential both in vitro and in vivo

Mr. Maksim Vigovskii, Ms. Nataliya Basalova, Ms. Olga Grigorieva, Ms. Uliana Dyachkova, Mr. Vladimir Popov, Ms. Anastasia Tolstoluzhinskaya, Ms. Anastasia Efimenko

PS01.65 First European Medicines Agency approved study with Umbilical Cord Mesenchymal Stromal Cell Extracellular Vesicles in the prevention of Bronchopulmonary Dysplasia: merit of a scalable GMP production platform

Ms. Sofia Baptista, Ms. Cristina Manfredi, Dr. Marcin Jurga, Mr. Gabrielis Kundrotas, Mr. Dimitri Stevens, Mr. Domenico Mancuso, Ms. Elisabetta Gramegna, Mr. Rudra Kashyap, Ms. Sandrine Mores, Prof Eugenio Baraldi, Prof Maurizio Muraca, Dr. Beatrice De Vos

PS01.66 Human platelet lysate enhanced angiogenic potential of extracellular vesicles derived from mesenchymal stem cells

Dr Yue Zhang, Professor Tao‐tao Tang, Professor Lin‐li Lv, Professor Bi‐cheng Liu

PS01.67 Hypoxia primed WJ‐MSCs‐sEVs exhibit enhanced immunomodulatory & regenerative activity in wound milieu primarily via miR125b‐5p/IL‐6R axis

Ms Yashvi Sharma, Dr Sujata Mohanty

PS01.68 Immunomodulatory potential of IL‐1β‐primed mesenchymal stromal cells‐derived extracellular vesicles and soluble factors to prevent organ dysfunction after a traumatic hemorrhagic shock

PHD Student Guillaume Valade, MD Clément DEVAUTOUR, Mrs Marion GROSBOT, Mrs Muriel NIVET, Phd Student Marine DE TADDEO, PHD Ahmad HAIDAR, PHD Patrice DECKER, Mrs Sylvie GOULINET, PHD Philippe MAUDUIT, MD, PHD Sébastien BANZET, PHD Marina TROUILLAS, PHD Juliette PELTZER

PS01.69 Immunomodulatory properties of dental pulp stem cell derived EVs

Dr Sadiq Umar^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Koushik Debnath^{Department of Oral Biology, College of Dentistry,UIC, Chicago, IL, USA}, Chun‐Chieh Huang^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Kasey Leung^{Department of OralBiology, College of Dentistry, UIC, Chicago, IL, USA}, Miya Kang^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Yu Lu^{Departmentof Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Praveen Gajendrareddy^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Sriram Ravindran^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}

PS01.70 Intranasal delivery of NAMPT‐MSC‐sEV improves cognitive function in mice with repeated mild traumatic brain injury by reducing acetylated tau

Prof. Qing‐Ling Fu

PS01.71 Locoregional intra‐arterial delivery of MSC‐derived extracellular vesicles directly into the pancreas maintains glycemic regulation in diabetic rats

Dr. Reza Yarani, Dr. Rosita Primavera, Dr. Shashank Chetty, Dr. Jing Wang, Prof. Flemming Pociot, Dr. Avnesh Thakor

PS01.72 Lyophilization of engineered EVs for regenerative medicine

Dr. Chun‐chieh Huang, Dr Miya Kang, Dr Koushik Debnath, Ms Yu Lu, Dr Sriram Ravindran

PS01.73 Mechanism of miR‐155‐5p in hiMSC‐sEV in treating androgenic alopecia by activating AKT/β‐catenin/GSK3β signal pathway

Post‐doctor Ruiyun Tian, Professor Furong Li

PS01.74 Mensenchymal stem cell exosomes and its effect on facial skin regeneration and rejuvenation

Ms. Ching‐fen Yang, Ms. Hoei Ser Chong, Dr. Takaaki Matsuoka

PS01.75 Mesenchymal stem cells activate cellular autophagy by delivering exosomes to alleviate LPS‐induced endothelial inflammatory injury in sepsis

Shiyue Lu, Zhe Li, Yuqing Xu, Yuxiao Deng

PS01.76 Mesenchymal stromal cells‐derived small extracellular vesicles For corneal wound healing

Seyedmohammad Moosavizadeh, PhD Student Jiemin Wang, Dr. Ellen Donohoe, PhD Student Aoife Canning, Dr. Aideen Ryan, Professor Thomas Ritter

PS01.77 MicroRNA‐100‐5p mediates the therapeutic efficacy of mesenchymal stem cell‐derived exosomes in a murine psoriasis model

Dr Yu Chen Huang, Dr. Chao Yuan Chang, Dr. Chun Jen Huang

PS01.79 MSC‐small extracellular vesicles alleviated Th2‐airway inflammation by regulating the metabolism of DCs in mice

Prof. Qing‐Ling Fu, Lifen Wen, Longxin Huang

PS01.81 pcMSCs‐derived exosome promoting stem cell reprogramming and suppressing inflammatory condition in LPS‐induced ARDS/ALI model

Phd Student Kajal Singh, Mr. Abhinay Kumar Singh, Dr. Yen‐Hua Huang

PS01.83 Potential therapeutic effect of human dental stem cells‐derived exosomes enhances neurological function and cerebral blood flow after ischemic stroke in rats

Assistant Professor Sukonthar Ngampramuan, Doctor Anyapat Atipimonpat, Associate Professor Hathaitip Sritanaudomchai, Assistant Professor Paranee Yatmark

PS01.84 Purification and characterization of extracellular vesicles derived from induced pluripotent mesenchymal stem cells for treatment of vagin*l prolapse

Dr. Olivia Cardenas‐ Trowers, Ralph Perkerson, Tammee Parsons, Nabanita Halder, Nisha Durand, Abba Zubair, Jing Zhao, Takahisa Kanekiyo

PS01.86 Rejuvenation by OCT4/SOX2 in extracellular vesicles through regulation of the toll‐like receptor 4 (TLR4) pathway

Professor Jisook Moon

PS01.87 Safety evaluation of Wharton's Jelly Mesenchymal Stem Cells (WJMSCs) derived small extracellular vesicles (sEVs) on healthy Sprague Dawley rats

Mr Illayaraja Krishnan, Associate Professor Dr Min Hwei Ng, Dr. Jia Xian Law, Ms Shathiya Rajamanickam, Dr Baskar Subramani, Associate Professor Dr Yogeswaran Lokanathan

PS01.88 Secretome derived from wharton jelly‐mesenchymal stem cells mitigate acute graft‐versus‐host‐disease: impact of hypoxia and apoptosis

Ms Mohini Mendiratta, Ms Meenakshi Mendiratta, Dr. Sandeep Rai, Professor Ritu Gupta, Dr. Sabyasachi Bandyopadhyay, Dr. Hariprasad GuruRao, Professor Sujata Mohanty, Dr. Ranjit Sahoo

PS01.89 Senolytic CD38 antigen receptor‐modified mesenchymal stem cell‐derived extracellular vesicles slowing age‐associated degeneration

Dr Yaoying Long, Dr. Bianlei Yang, Prof. Zhichao Chen, Prof. Qiubai Li

PS01.93 Small extracellular vesicles derived from human chemically induced liver progenitors (hCLiPs) improve liver fibrosis via inactivation of hepatic stellate cells.

Ms Tomoko Yamaguchi, Dr Juntaro Matsuzaki, Dr Takeshi Katsuda, Ms Noi Tokuda, Mr Yuzhi Tan, Dr Masaki Kimura, Dr Takahiro Ochiya, Dr Yoshimasa Saito

PS01.94 Small extracellular vesicles derived from human mesenchymal stem cells prevent Th17‐dominant neutrophilic airway inflammation via immunoregulation on Th17 cells

PhD. Bi‐Xin He, Prof. Qing‐Ling Fu, PhD. Shu‐Bing Fang, PhD. Chan‐Gu Li

PS01.95 Stem Cell‐Derived Extracellular Vesicles: a potential therapy for premature lung disease

Dr Hala Saneh, Heather Wanczyk, Joanne Walker, Dr Christine Finck

PS02.01 3D culture of human adipose stem cells in physiological oxygen for therapeutic extracellular vesicle production

Doctoral Researcher Julia Monola, Postdoctoral Researcher Chris Pridgeon, Alisa Jokela, Principal investigator Riina Harjumäki

PS02.02 Biomanufacturing of immature cardiomyocytes derived extracellular vesicles in 2L stirred tank bioreactor

MSc João Jacinto, MSc Ana Meliciano, MSc Lara Inocêncio, MSc Pedro Vicente, PhD Margarida Serra

PS02.03 Bioprocess optimization for extracellular vesicles derived from mesenchymal stem cells

Aslan (mehdi) Dehghani, Senior Scientist Eric Black, Senior Scientist Zheng Zhao, Senior Scientist Namitha Haridas, Senior Manager of Process Development Sunandan Saha, Senior Manager of Process Development David Splan, Head of Process Development Services Mark Szczypka, Head of Advanced Bioprocessing David Pollard

PS02.04 Development of extracellular vesicles collect media for naïve and engineered HEK293 cells

Dr Kartini Asari, Kol Thida Mom, Amirah Fitri, Sadman Bhuiyan, Dr Ramin Khanabdali, Professor Gregory Rice

PS02.05 Extracellular vesicles bioprocess design and economic modeling

Aslan (mehdi) Dehghani

PS02.06 High‐yield exosome production from cells‐laden 3D auxetic scaffolds with cyclic mechanical stimulation for an effective drug delivery

Associate Professor Ming‐You Shie

PS02.07 Impact of donor variability on the therapeutic potential of platelet‐derived extracellular vesicles in regulating endothelial cell permeability

Malvika Gupta, Dr. Mandeep Kaur, Dr. Sowmya Shree Gopal, Dr. Jessica Cardenas, Dr. Amit Srivastava

PS02.10 Scalable production of modified HEK293T extracellular vesicles using adherent packed bed bioreactor culture system

Dr Choon Keong Lee^{Esco Aster Pte Ltd}, Ms Claudine Ming Hui Lim^{Esco Aster Pte Ltd}, Ms Winnie Faustinelie^{Esco Aster Pte Ltd}, Dr Desy Silviana^{Esco Aster Pte Ltd}, Mr Xiangliang Lin^{Esco Aster Pte Ltd}

PS02.11 A decision‐making tool to navigate through extracellular vesicle research and product development

FrancescaLoria, Sabrina Picciotto, Giorgia Adamo, Andrea Zendrini, Samuele Raccosta, Lucia Paolini, Mauro Manno, Paolo Bergese, Giovanna L. Liguori, Paolo Guazzi, Antonella Bongiovanni, Nataša Zarovni

PS02.12 Analytical toolbox for reliable characterization of extracellular vesicles

Aslan (mehdi) Dehghani, Paul Keselman, Prabuddha Mukherjee, Meng Chai, michael Olszowy, Jordan Speidel, Thomas Gaborski, Nick Luey

PS02.13 In vivo and in vitro studies on the role of sEVs as a drug delivery system in breast cancer; a systematic review

Mr Abdulwahab Teflischi Gharavi, Prof Keykavous Parang, Dr Saeed Irian, Prof Mona Salimi

PS02.14 Orthogonal measurement of number concentration standards for NTA calibration

Julie Chen, Product Line Manager ‐ Particle Characterization Jeffrey Bodycomb, Ph.D.

PS03.01 Effects of exosomes derived from skeletal muscle of senescent mice on bone metabolism

Dr Mingming Zhang, Dr Ran Li, Dr Zhongqi Wang

PS03.02 Elucidating the role of extracellular vesicles in mediating reprogramming processes: connecting in vitro insights to in vivo applications for enhanced peripheral nerve tissue repair

PhD Ana Salazar Puerta, Neil Ott, Sara Kheirkhah, Jon Stranan, Grant Barringer, Samuel Cortes, Roxanne Vermette, Emily Moser, MS Hallie Harris, PhD William Lawrence, PhD Devleena Das, MD PhD Mana Saffari, MD Amy Moore, PhD Daniel Gallego‐Perez

PS03.04 Extracellular vesicles secreted by mesenchymal stromal cells may regulate the pool of activated stromal cells during the development of fibrosis

Ms NATALIYA BASALOVA, Ms Olga Grigorieva, Ms Anastasiya Tolstoluzhinskaya, Ms Uliana Dyachkova, Mr Maxim Vigovsky, Ms Maria Kulebyakina, Mr Vladimir Popov, Ms Natalia Kalinina, Ms Zhanna Akopyan, Ms Anastasia Efimenko

PS03.05 Extracellular vesicles secreted by multipotent mesenchymal cells contribute to the suppression of macrophage proinflammatory phenotype and reduce their profibrotic properties

A. E. Tolstoluzhinskaya, Ms Uliana Dyachkova, Mr Maksim Vigovskiy, Dr Nataliya Basalova, Ms Anna Gardzhuk, Dr Anastasia Efimenko, Dr Olga Grigorieva

PS03.06 Facilitating muscle formation via bone‐derived extracellular vesicles induced by HDAC‐inhibition

Dr. Ming Chen, Dr. Taojin Feng, Dr. Mingming Zhang, Dr. Ruijing Chen, Prof. Yi Li, Prof. Licheng Zhang, Prof. Pengbin Yin, Prof. Peifu Tang

PS03.08 Plasma EV‐miR‐887‐3p levels reflect the therapeutic effect of the antifibrotic agent in patients with liver cirrhosis

Dr. Juntaro Matsuzaki, Ms. Mayu Yoshida, Dr. Koji Fujita, Dr. Masamichi Kimura, Ms. Noi Tokuda, Ms. Tomoko Yamaguchi, Dr. Masahiko Kuroda, Dr. Takahiro Ochiya, Dr. Yoshimasa Saito, Dr. Kiminori Kimura

PS04.01 Cardiomyocytes‐derived EVs for the treatment of COVID‐19‐induced cardiac damage

Dr Marta Prieto‐Vila, Dr Yusuke Yoshioka, Professor Takahiro Ochiya

PS04.03 Elucidating pathophysiology of hypertrophic cardiomyopathy (HCM): Proteomics in extracellular vesicles (EVs) of HCM patient tissue reveals altered metabolic state and increased cardiac EV release

Msc Sarah Hilderink^{Physiology, Amsterdam UMC, Vrije Universiteit Amsterdam, De Boelelaan 1118, Amsterdam, the Netherlands}, Rita Najor^{Skaggs Schoolof Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA}, Richard Goeij‐de Haas^{Department of Medical Oncology,Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, the Netherlands}, Berend Gagestein, Jaco Knol^{Department of Medical Oncology,Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, the Netherlands}, Thang V Pham^{Department of Medical Oncology, Cancer CenterAmsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, the Netherlands}, Kenneth C Bedi Jr^{Cardiovascular Research Institute, Perelman School of Medicine,University of Pennsylvania, Philadelphia, Pennsylvania, USA}, Kenneth B Marguiles^{Cardiovascular Research Institute, Perelman School of Medicine, University ofPennsylvania, Philadelphia, Pennsylvania, USA}, Michelle Michels^{Department of Cardiology, Erasmus MC, Rotterdam, The Netherlands}, Connie R Jimenez^{Department of Medical Oncology, Cancer Center Amsterdam, Amsterdam UMC, Vrije Universiteit Amsterdam, the Netherlands}, Asa Gustafsson^{Skaggs Schoolof Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA, USA}, Jolanda van der Velden^{Physiology, Amsterdam UMC, VrijeUniversiteit Amsterdam, De Boelelaan 1118, Amsterdam, the Netherlands}, Diederik WD Kuster^{Physiology, Amsterdam UMC, Vrije Universiteit Amsterdam, DeBoelelaan 1118, Amsterdam, the Netherlands}

PS04.04 Endothelial cell derived extracellular vesicles contribute to laminar shear stress adaptation

Research Specialist II Amber Eliason, Graduate Student Santiago Moreno, Assistant Professor David Marciano

PS04.05 Epithelial cell‐derived extracellular vesicle mediated inflammation, infection, and cellular senescence in chronic obstructive pulmonary disease

Miss Georgia Bateman, Professor Cliff Taggart

PS04.06 Inflammatory endothelial cell‐derived apoptotic bodies modulate innate and adaptive immune processes

Dr Amy Baxter, Ms Caitlin Vella, Dr Pamali Fonseka, Dr Tien Nguyen, Dr Emma Grant, Prof Suresh Mathivanan, Prof Stephanie Gras, Prof Mark Hulett, A/Prof Ivan Poon

PS04.06 Extracellular Vesicles mediated communication between fat and heart during heart failure

Achala Moncy, Assistant Professor Sam Das

PS04.08 Lipogenic lung fibroblast‐derived extracellular vesicles attenuate cigarette smoke‐induced COPD pathology by enhancing alveolar type II cell stemness

Dr Yu Fujita, Dr Shota Fujimoto, Dr Reika Kaneko, Dr Jun Araya

PS04.11 Reduced CD63+ extracellular vesicle levels associate with atherosclerosis in hypercholesteraemic mice and humans

Mr Brachyahu Kestecher

PS04.14 Small EV‐associated miR‐145 is a driver in mitral valvular interstitial cell transition in mitral valve prolapse

Associate Professor Vicky Yang, Dawn Meola, Nicole Moyer, Runzi Zhou, Sally Carnevale, Guoping Li, Saumya Das

PS04.15 VCAM‐1+ endothelial cell derived extracellular vesicles mediate the acute phase response following myocardial infarction

Naveed Akbar, Mr Daan Paget, Mr Lewis Timms, Dr Daniel Radford Smith, Ms Rebecca Rooney, Ms Heleah Soulati, Ms Carla De Villiers, Professor Paul Riley, Professor Robin Choudhury, Professor Daniel Anthony

PS04.17 Hypoxia induced extracellular vesicles of the neurovascular unit in a model of blood‐brain barrier disruption.

Miss Rebecca Raven¹, Doctor Jessica Williams¹, Professor Keith Morris¹, Professor Philip James¹

¹Centre for Cardiovascular Health and Aging, Cardiff Metropolitan University, Cardiff, United Kingdom

PS04.18 Extracellular vesicles in aging cold‐stored whole blood do not seem to compensate for the decreasing hemostatic function

Petra Ilvonen¹, Sanna Susila^1,2, Reetta Pusa¹, Ulla Impola¹, Tuukka Helin³, Lotta Joutsi‐Korhonen³, Saara Laitinen¹, Jouni Lauronen¹, Minna Ilmankunnas^1,4,5

¹Finnish Red Cross Blood Service, Helsinki, Finland, ²Emergency Medical Service and Emergency Department, Päijät‐Häme wellbeing services county, Lahti, Finland, ³Department of Clinical Chemistry, HUS Diagnostic Center, Helsinki University Hospital and University of Helsinki, Helsinki, Finland, ⁴Department of Anesthesiology and Intensive Care Medicine, Helsinki University Hospital and University of Helsinki, Helsinki, Finland, ⁵Meilahti Hospital Blood Bank, Department of Clinical Chemistry, HUS Diagnostic Center, Helsinki University Hospital and University of Helsinki, Helsinki, Finland

PS05.01 A novel approach of T cell engineering by targeted exosomes delivering CRISPR/Cas9 system for PD‐1 knock‐out

Ms, Ph.D. candidate Mahboubeh Shahrabi Farahani, Ph.D. Elham Hosseini‐Beheshti, Ph.D. Mehdi Forouzandeh Moghadam, Ms, Ph.D. candidate Leila Darzi

PS05.01 Production of a targeted delivery system for T cell modifications by engineering exosomes to express ICAM‐1

Ms, Ph.D. candidate Mahboubeh Shahrabi Farahani, Ph.D. Elham Hosseini‐Beheshti, Ph.D. Mehdi Forouzandeh Moghadam, Prof Seyed Mohammad Moazzeni, Ms Leila Darzi

PS05.02 Advanced SIRPα‐enhanced extracellular vesicles: a novel approach in fibrosis treatment

Advanced Sirpα‐enhanced Extracellular Vesicles: A Novel Approach In Fibrosis Treatment Minjeong Kwon, Advanced SIRPα‐Enhanced Extracellular Vesicles: A Novel Approach in Fibrosis Treatment Min Kyoung Jo, Advanced SIRPα‐Enhanced Extracellular Vesicles: A Novel Approach in Fibrosis Treatment Seohyun Kim, Advanced SIRPα‐Enhanced Extracellular Vesicles: A Novel Approach in Fibrosis Treatment Dong‐U Shin, Advanced SIRPα‐Enhanced Extracellular Vesicles: A Novel Approach in Fibrosis Treatment Gi Beom Kim, Advanced SIRPα‐Enhanced Extracellular Vesicles: A Novel Approach in Fibrosis Treatment Gi‐Hoon Nam, Advanced SIRPα‐Enhanced Extracellular Vesicles: A Novel Approach in Fibrosis Treatment In‐San Kim

PS05.03 Alleviating lung inflammation via effective miRNA delivery to alveolar macrophages using extracellular vesicles conjugated surfactant protein A

Student Miji Kim, Student Sujeong Park, Student Nayeong Lee, Student Dohyun Kim, Student Dongwoo Kim, Ph.D Seon‐Jin Lee, Ph.D DVM Jung Joo Hong, Ph.D (Professor) Heedoo Lee

PS05.04 Antigen binding extracellular vesicles for targeted drug delivery

Mr Madhusudhan Bobbili, Nuria Gimeno, Mr Stefan Vogt, Florian Rüker, Gordana Wozniak‐Knopp, Johannes Grillari

PS05.05 Cassette‐like modification of biofunctional peptides on extracellular vesicles (PepEVs) for on‐demand intracellular delivery

Dr. Ikuhiko Nakase

PS05.07 Development of an EV‐based siRNA delivery platform for targeting metastatic cancers

Chia‐Ling Hsieh, Doctor Anh Duy Do, Miss Mafewu Olga Raboshakga, Professor Shian‐Ying Sung

PS05.09 Development of DC‐targeting exosome‐based drug delivery platform

Ph.D. Student Sheng‐Yun Hsu, Undergraduate Hsi‐Ming Chiang‐Hsieh, M.S. Chen‐Guang Zhang, Ph.D. Chen‐Yun Yeh, Ph.D. Pi‐Hui Liang, Ph.D. Han‐Chung Wu, Ph.D. Yungling Leo Lee

PS05.10 Development of Dendritic cells derived exosomes based novel vaccine formulation against Latent tuberculosis

Dr Saima Naz, Dharani bandi, farhan ahmed

PS05.11 Development of exosome‐based antibiotic transport for enhanced intracellular efficacy

Miss Ayaulym Nurgozhina, Shynggys Sergazy, Madiyar Nurgaziyev, Laura Chulenbayeva, Mohamad Aljofan

PS05.12 Development of lung‐directed siRNA‐carrier using autologous serum‐derived small EVs for lung metastases of melanoma

Dr. Mai Hazekawa, Dr. Dasuke Watase, Dr. Takuya Nishinakagawa, Dr. Masato Hosokawa, Dr. Daisuke Ishibashi

PS05.13 Development of targeted exosome as plasmid delivery vehicles to HER2‐expressing breast cancer cells

Miss Leila Darzi, Dr Mehdi Forouzandeh Moghadam, Dr Mehdi Shamsara, DrElhamHosseini‐Beheshti

PS05.14 Development of targeted exosomes as CRISPR/Cas9 delivery platforms to HER2‐expressing breast cancer cells

Miss Leila Darzi, Dr Mehdi Forouzandeh Moghadam, Dr Mehdi Shamsara, DrElhamHosseini‐Beheshti

PS05.15 Doxorubicin‐loaded therapeutic EVs as effective drug delivery vehicles to neuroblastoma cells

Doctoral Candidate Marc Liébana, Doctoral Candidate Silvia López, PhD Esperanza González, PhD Juan Manuel Falcón

PS05.16 Endogenous protease mediated delivery of engineered immunomodulatory extracellular vesicles

Ms. Kasey Leung^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Miya Kang^{Department of Oral Biology, College of Dentistry,UIC, Chicago, IL, USA}, Dr. Koushik Debnath^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Chun‐Chieh Huang^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Sadiq Umar^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Mrs. Yu Lu^{Department of Oral Biology, College of Dentistry, UIC, Chicago, IL, USA}, Dr. Sriram Ravindran^{Department of Oral Biology, College of Dentistry,UIC, Chicago, IL, USA}

PS05.17 Engineered exosomes loaded with let‐7i‐5p microRNA allay acute lung injury in mice with gastric content aspiration

Professor Chun‐Jen Huang, Doctor Chao‐Yuan Chang, Doctor Ching‐Wei Chuang

PS05.18 Engineered extracellular vesicles for targeting and activation of lymphatic VEGFR‐3

Dr. Wolf Holnthoner

PS05.20 Engineered MSC‐derived exosomes alleviate radiation‐induced lung injury via transferring mitochondrial component to improve homeostasis of lung epithelial cells

Distinguished professor KS Clifford Chao, Attending Physician Chi‐Hsien Huang, Research Assistant Shi‐Xuan Yan, Research Assistant Hsin‐Yu Chang, Research Assistant Pei‐Chen Yang, Associate Professor Kevin Chih‐Yang Huang

PS05.21 Engineered MSC‐EVs scavenge self‐antigen for alleviating psoriasis via modulating metabolic and immunological disorders

Mr Xin Zhou, Dr Jiancheng Wang, Dr Danyang Li

PS05.22 Engineered targeting extracellular vesicles as nano‐carriers loaded with chemo‐drug for cancer therapeutics

Professor Yiwen Chen, Seiner Engineer Kai‐Wen Kan, Professor Ming‐You Shie, Professor Shao‐Chih Chiu, Superintendent Der‐Yang Cho

PS05.24 Enhancing the targeting and regenerative efficacy of mesenchymal stem cell‐derived small extracellular vesicles via dual modification strategies

Ms Meenakshi Mendiratta, Dr Sujata Mohanty

PS05.25 Enveloped protein nanocages (EPN) as a versatile and controllable engineered EV platform

Dr Daniel Humphrys

PS05.26 Ex vivo T cell editing as a therapeutically relevant model to evaluate EV cargo delivery

Juliette Suermondt, PhD Xiuming Liang, Guannan Zhou, Houze Zhou, Oskar Gustafsson, PhD H. Yesid Estupiñan, PhD Yang Liu, Professor Samir EL Andaloussi, Assistant professor Joel Nordin

PS05.27 Exploring HER2 isoform in secreted EV as a co‐treatment for HNSCC cells to tyrosine kinase inhibitors

Ms Fui Teen Chong, Ms Hui Sun Leong, Ms Mengjie Ren, Dr Shen Yon Toh, Prof N Gopalakrishna Iyer

PS05.28 Exploring the loading of cell penetrating peptides (CPPs) into extracellular vesicles (EVs) for therapeutic applications

Ms. Neona Lowe, Rachel Mizenko, Dr. Alyssa Panitch, Dr. Randy Carney

PS05.31 Generalizable anchor aptamer strategy for loading and targeted delivery of nucleic acid therapeutics on exosomes

PhD Gang Han

PS05.32 Generating engineered EVs with targeting properties against EGFR+ triple‐negative breast cancers

Ragnar Axel Adolfsson^{Faculty of Pharmaceutical Sciences, School of Health Sciences, University of Iceland}, Erna Jonsdottir^{Faculty of PharmaceuticalSciences, School of Health Sciences, University of Iceland; Biomedical Center, University of Iceland, Iceland}, Dr. Jens Guðmundur Hjörleifsson^{Department of Biochemistry, Science Institue, University of Iceland}, Dr. Berglind Eva Benediktsdóttir^{Faculty of Pharmaceutical Sciences, School ofHealth Sciences, University of Iceland; Biomedical Center, University of Iceland, Iceland}

PS05.34 In silico protein design with cyclization facilitates efficient delivery into cells and extracellular vesicles

Ms Yeonju Lee, Mr Kyung‐Min Kim, Mr Young‐Pil Kim

PS05.35 Intraarticularly delivered mRNA‐encapsulating extracellular vesicles for osteoarthritis therapy

Researcher Hsiu‐Jung Liao, Dr. Tai‐Shan Cheng, Miss Yi‐Shan Shen, Mr. Sin‐Yu Chen, Professor Chih‐Hung Chang, Professor Ly Lee, Professor Chi‐Ying Huang

PS05.37 mRNA loading of extracellular vesicles for the treatment of neurological disorders

Miss Patricia Wongsodirdjo, Dr Ya Hui Hung, Dr Fazel Shabanpoor, Dr Laura Vella, Dr Rebecca Nisbet

PS05.38 Nanofluidic constriction enables encapsulation of biomacromolecule in small extracellular vesicles for efficient intracellular delivery

Zitong Yu, Huitao Zhang, PhD Rui Hao, PhD Candidate Shi Hu, Sihui Chen, Professor Hui Yang

PS05.39 Optimization of mRNA loading into extracellular vesicles for in vivo therapeutic delivery

Ph.D Candidate Liouba Le Roux, Ph.D. Adityas Purnianto, Ph.D. Laura Vella, Ph.D. Ya Hui Hung

PS05.42 RBCEVs: A promising platform for safe and efficient gene therapy, mitigating risks and enhancing expression

Ms Melissa Tan, Dr Brenda Wan Shing Lam, Dr Harwin Sidik, Dr Tenzin Gocha, Dr Ronne Wee Yeh Yeo, Dr Minh TN Le, Dr Waqas Muhammad Usman

PS05.43 Reactive oxygen species responsive multifunctional fusion extracellular nanovesicles: prospective treatments for acute heart transplant rejection

Professor Hongbo Chen, Xingyu Lu, Dr Fang Cheng

PS05.44 Reprogramming of T cell‐derived small extracellular vesicles using IL2 surface engineering induces potent anti‐cancer effects through miRNA delivery

Dr. Dokyung Jung, Sanghee Shin, Dr. Sung‐Min Kang, Inseong Jung, Suyeon Ryu, Soojeong Noh, Dr. Sung‐Jin Choi, Jongwon Jeong, Beom Yong Lee, Kwang‐Soo Kim, Dr. Christine Seulki Kim, Dr. Jong Hyuk Yoon, Dr. Chan‐Hyeong Lee, Dr. Felicitas Bucher, Dr. Yong‐Nyun Kim, Prof. Sin‐Hyeog Im, Dr. Byoung‐Joon Song, Prof. Kyungmoo Yea, Prof. Moon‐Chang Baek

PS05.45 Research on the treatment of knee osteoarthritis with CXCR7 delivered by engineered extracellular vesicles

Bin Zeng, Duan Li

PS05.46 Sensitisation of EGFR‐driven cancers to EGFR tyrosine kinase inhibitors by application of exosomal EGFR isoform D as a co‐drug

Ms Hui Sun Leong, Dr Shen Yon Toh, Ms Fui Teen Chong, Ms Mengjie Ren, Prof N. Gopalakrishna Iyer

PS05.48 Slow controlled release of extracellular vesicles with hydrogel based nanoparticles

Ms. Reese Wunsche, Dr. Morteza Jeyhani, Mr. Boyang Su, Dr. Hon Sing Leong, Dr. Scott Tsai

PS05.49 Small extracellular vesicles as a superior targeted drug delivery system compared to liposomes

Dr. Diem Nguyen, Thieu Nguyen, Nhan Vo, Dr. Lan N Tu

PS05.50 Strategic loading of epitopes onto EV subtypes using Craftgen@EV for vaccine of cellular immunity

Ph.D. student Shota Shinagawa, Technical Staff Tamiko Minamisawa, Technical Staff Saki Matsumoto, Project Leader Kazuma Kiyotani, Kiyotaka Shiba

PS05.51 Targeted cargo delivery to mouse lower limb by exosome carrying a muscle targeting moiety with intravenous injection

Mr. Minghao Sun, Associate Director Mafalda Cacciottolo, Principal Scientist Yujia Li, Senior Scientist Mahrou Sadri, Senior Scientist Michael LeClaire, Research Associate David Tran, Chief Scientific Officer Kristi Elliott

PS05.53 The development of engineered exosome‐conjugated nanobody for nuclei acids/drug delivery in glioblastoma treatment

Dr. Shao‐chih Chiu, Dr. Ming‐You Shie, Dr. Shi‐Wei Huang, Dr. Chih‐Ming Pan, Dr. Der‐Yang Cho

PS05.54 Using autologous blood‐derived extracellular vesicles as precision therapeutics for retinal degenerations

Ms Rakshanya Sekar, Dr Yvette Wooff, Dr Adrian Cioanca, Associate Professor Riccardo Natoli

PS05.55 Utilising engineered stem cell‐derived nanovesicles (scNVs) as a scalable, cell reprogramming therapeutic for cardiac repair

Phd Candidate Jonathan Lozano, Dr. Jarmon G Lees, Dr. Alin Rai, Dr. Kyah Grigolon, Dr. Helen Kiriazis, Ren Jie Phang, Jonathon Cross, Haoyun Fang, Dr. Daniel Donner, Shiang Y Lim, Dr. David W. Greening

PS05.56 Engineered serum extracellular vesicles deliver CRISPR‐Cas9 ribonucleoproteins to modify the dystrophin gene

Ph.d Yaoyao Lu, Research assistant Nathalie Majeau, Ph.D Camile Bouchard, Professor Jacques‐P Tremblay

PS05.58 Engineered extracellular vesicles for delivery of therapeutic small RNA

PhD student Julia Anna Rädler, Giulia Corso, Antje Zickler, Noriyasu Kamei, Wenyi Zheng, Dhanu Gupta, Samir El Andaloussi

PS05.63 Multifunctional aggregation‐induced emission‐based extracellular vesicles to remodel microenvironment for infected wound healing

Doc. Pingping Wang¹, Professor Hang Zou¹, Prof. Lei Zheng¹

¹Department Of Laboratory Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China

PS05.64 Arginine‐rich cell‐penetrating peptide‐modified microvesicles for macropinocytosis induction and enhanced intracellular delivery

Dr. Ikuhiko Nakase¹, Kenta Morimoto¹, Jojiro Ish*tobi¹, Dr. Kosuke Noguchi¹, Ryoichi Kira¹, Dr. Tomoka Takatani‐Nakase², Dr. Ikuo Fujii¹, Dr. Shiroh Futaki³, Dr. Masamitsu Kanada⁴

¹Graduate school of Science, Osaka Metropolitan University, Sakai, Japan, ²School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women's University, Nishinomiya, Japan, ³Institute for Chemical Research, Kyoto University, Uji, Japan, ⁴Department of Pharmacology and Toxicology, Michigan State University, East Lansing, USA

PS05.65 Extracellular Vesicle Sorting Motif Platform for Natural Multiplex Cargo Delivery

Laboratory Director Gibeom Kim¹, Senior Researcher Inkyu Lee^1,3, CEO Gi‐hoon Nam^1,2, Professor In‐San Kim^3,4

¹Department of Research and Development, SHIFTBIO INC., Seoul, South Korea, ²Department of Biochemistry & Molecular Biology, Korea University College of Medicine, Seoul, South Korea, ³KU‐KIST Graduate School of Converging Science and Technology. Korea University, Seoul, South Korea, ⁴Chemical and Biological Integrative Research Center, Korea Institute of Science and Technology, Seoul, South Korea

PS06.01 Airway basal stem cell‐derived extracellular vesicles: a promising strategy for fibroblasts regulation

Dr. Lisi Luo, Dr. Huijie Yang, Dr. Junfeng Huang, Dr. Shiyue Li

PS06.06 Evaluating the role of GM‐1 ganglioside in neuronal uptake

Miss Thitikan Jirakittisonthon, Dr. Orman Snyder, Dr. Hong He, Dr. Mark Weiss

PS06.08 Hyaluronan coat enhances the targeting of extracellular vesicles into CD44 overexpressing cells

MSc Heikki Kyykallio, BSc Kirsti Härkönen, PhD Martina Hanzlíková, PhD Tatu Lajunen, Professor Tapani Viitala, PhD Kirsi Rilla

PS06.09 Identifying proteins that impact differential uptake of extracellular vesicles from ovarian follicular fluids collected at early and late in follicle growth phases

Assistant Professor Wei‐Ting Hung, Professor John S. Davis, Professor Lane Christenson

PS06.10 In vivo differential kinetic distribution of extracellular vesicles affected by oncogenic RAS and RAF transformation

Ms Shinwon Chae, Mr Chul Won Seo, Ms Haekang Yang, Professor Yoon‐Jin Lee, Professor Dongsic Choi

PS06.11 NaTaLi: Nanobody‐Tag Ligand click strategy for targeted multicolor EVs

Dr. Andrea Galisova, Dr. Jiri Zahradnik, Dr. Daniel Jirak

PS06.12 NHE7 upregulation potentiates the uptake of small extracellular vesicles by enhancing maturation of macropinosome in hepatocellular carcinoma

Dr Yao Yue, Dr Xu Yi, Dr Judy Wai Ping Yam

PS06.13 Nodal flow transfers polycystin to determine mouse left‐right asymmetry

Dr. Yosuke Tanaka, Dr. Ai Morozumi, Dr. Nobutaka Hirokawa

PS06.16 Study on the bio‐distribution of autologous serum‐derived small EVs in a melanoma spontaneous metastasis mice model for the development of nucleic acid carriers for cancer metastasis

Dr. Daisuke Watase, Mai Hazekawa, Ayano Yamada, Mitsuhisa Koga

PS06.17 Using single molecule microscopy to measure EV uptake, sub‐cellular localization and dynamics

Dr James Rhodes, Dr Stefan Balint, Mr Andras Miklosi, Dr Nina Jajcanin‐Jozic, Mr Andrei Traista, Dr Pradeep Kumar, Dr Grace DeSantis

PS06.18 Cancerous Extracellular Vesicles Induced Platelet mRNA Degradation by RNaseL Activation

Mr. Gaoge Sun¹, Zihan Liu¹, Ying Zhang¹, Hang Yin^1,2

¹School of Pharmaceutical Sciences, Tsinghua University, Beijing, China, ²Tsinghua‐Peking Center for Life Sciences, Tsinghua University, Beijing, China

PS06.19 Forward‐Thinking Insights: Exploration of future therapeutics through HNF4‐AS1 regulation of HNF4a promoter selection

Dr Pevindu Abeysinghe¹, Ms Breanna Humber², Mr Riccardo Cecchin², Prof. Kevin Morris¹

¹Centre for Genomics and Personalised Health, School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Australia, ²Menzies Health Institute Queensland, School of Pharmacy and Medical Science, Griffith University, Gold Coast, Australia

PT01.01 A High‐efficiency isolation system combined with proteomics in studying urinary small extracellular vesicles proteins for improving prostate cancer diagnosis

Dr. Cheng Zhou, Ms. Jie Gong, Mr. Baokun Fan, Ms. Xuan Ding, Dr. Bairen Pang, Prof. Yong Li, Dr. Junhui Jiang, Dr. Zejun Yan, Dr. Yue Cheng, Mr. Yingzhi Chen, Dr. Zhaohui Jiang, Mr. Tiannan Guo

PT01.02 A novel bladder cancer liquid biopsy using mutated proteins in urinary extracellular vesicles

M.D., Ph.D. Yuji Hakozaki, M.D., Ph.D. Yuta Yamada, M.D., Ph.D. Haruki Kume, Ph.D. Koji Ueda

PT01.03 A validated workflow and bioinformatic analysis pipeline for extracellular vesicle‐based RNA biomarker signature discovery in molecular diagnostics

Christian Grätz, Dr. Benedikt Kirchner, PD Dr. Marlene Reithmair, Dr. Florian Brandes, Dr. Agnes S. Meidert, Prof. Dr. Gustav Schelling, Prof. Dr. Michael W. Pfaffl

PT01.04 Advancements in biomarker development for toxicology and safety assessment studies

Tasvilla Sonallya, Annamária Minus, Ferenc Fekete, Dr. Anikó Gaál, Kinga Ilyés, Dr. Tamás Beke‐Somfai, Dr. Zoltán Varga, Dr. Katalin Monostory

PT01.05 Advancing precision: Development of extracellular vesicles protein‐based panel for validation of endometrial cancer biomarkers

Dr Anastasiia Artuyants, Martin Middleditch, Deanna Shea, Bianca Nijmeijer, Sophia Bebelman, Dr Cherie Blenkiron

PT01.06 Analysis of secreted small extracellular vesicles from activated human microglial cell line reveals distinct pro‐ and anti‐inflammatory proteomic profiles

Miss Xueming Niu, Dr Zhen Zhang, Mr Quan Zhou, Dr Alain Wuethrich, Dr Richard Lobb, Professor Matt Trau

PT01.07 Aquaporin 3 detection in placental extracellular vesicles in normal human pregnancy and preeclampsia

PhD Natalia Szpilbarg, Matías Nicolás Sierra, MD Juan Sebastián Sar, PhD Alicia Ermelinda Damiano

PT01.08 Automated high‐throughput isolation of extracellular vesicles (EVs) and small RNA sequencing profile in serum of breast cancer patients

Dr Ramin Khanabdali, Dr Scott Zhu, Dr Mathew Moore, Dr Gregory Rice

PT01.09 Cargo content in extracellular vesicles from a murine cell model of organotropic metastatic breast cancer

Graduate Student Amélie Nadeau, Graduate Student Thupten Tsering, PhD Kyle Dickinson, PhD Daniela Quail, PhD Peter Siegel, PhD Julia V Burnier

PT01.10 Changes to small and large urinary extracellular vesicles in glioblastoma

Dr Susannah Hallal, Mr Liam Sida, Dr Agota Tűzesi, Dr Elissa Xian, Dr Daniel Madani, Dr Krishna Muralidharan, Dr Brindha Shivalingam, Associate Professor Michael Buckland, Dr Laveniya Satgunaseelan, Dr Kimberley Alexander

PT01.11 Circulating EVs as diagnostic biomarkers of indeterminate thyroid nodules

Dr Nada Ahmed, Dr Kevin Beatson, Dr Jigisha Patel, Dr Mohammad Eddama, Dr Tarek Abdel‐Aziz, Professor Lucie Clapp

PT01.13 Detection of MTA1 in plasma sEVs derived from cancer patients

Graduate Research Assistant Kritisha Bhandari, Laboratory Technician Jeng Shi Kong, Physician Scientist Haoyao Sun, Professor Jinchang Wu, Assistant Professor Bethany Hannafon, Professor William Dooley, Professor Wei‐Qun Ding

PT01.14 Developing metabolomic approach in profiling extracellular vesicle biomarkers for prostate cancer diagnosis and progression risk stratification

Mr Mahmoud Hamed, Dr Valerie Wasinger, Mr Qi Wang, Associate Professor Peter Graham, Dr David Malouf, Dr Joseph Bucci, Professor Yong Li

PT01.15 Early cancer detection made easy: liquid biopsy analysis of low‐concentration EGFR mutations in NSCLC using large‐volume plasma and urine

Young‐Hye Seo, Sung‐Kyung Bong, Beomhee Ahn, Hanna Kim, Hwanghee Ryu, Myunghee Jang, Ph.D Seung‐Hak Choi, Ph.D Vijaya Sunkara, Juhee Park, Ph.D Yoon‐Kyoung Cho, Ph.D Kyusang Lee, Ph.D Beomseok Lee

PT01.16 Effect of X‐ray irradiation on quantity and tetraspanin markers expression of extracellular vesicles (EVs) derived from peripheral blood mononuclear cells (PBMCs) and plasma from patients undergoing total‐body irradiation (TBI)

Zi Huai Chew, Senior Research Scientist Christelle Chua

PT01.17 Establishing the capacity of liver derived extracellular vesicle cargo to reflect variability in drug exposure and response

Ms Lauren Newman, Dr Zivile Useckaite, Associate Professor Andrew Rowland

PT01.21 Exploring plasma‐derived small extracellular vesicles as novel biomarkers for early‐stage detection of pancreatic neuroendocrine tumors

Ms Priya Kumari Gorai, Ms Simran Rastogi, Dr Surabhi AS, Dr Seema Singh, Dr Shipra Agarwal, Dr Sujoy Pal, Dr Tapas Chandra Nag, Prof Renu Dhingra, Prof Mehar Chand Sharma, Prof Rakesh Kumar, Dr Saroj Kumar, Dr Neerja Rani

PT01.22 Extracellular vesicle‐derived RNA profiling predicts melanoma and non‐small cell lung cancer (NSCLC) response to immune checkpoint inhibitors

Ms Lidia Medhin, Doctor Lydia Warburton, Professor Benhur Amanuel, Doctor Leslie Beasley, Professor Elin Gray

PT01.23 Extracellular vesicles are diagnostic and predictive of blood pressure before and during exercise in people with hypertension

Samantha Upson, Dr. Sabrina LaSalvia, Eric Trillaud, Dr. Emily Heiston, Nathan Stewart, Dr. Steven Malin, Dr. Uta Erdbrügger

PT01.24 Extracellular vesicles as potential biomarkers for non‐alcoholic fatty liver disease (NAFLD)

Malene Joergensen, Anders Askeland, Rikke Bæk, Charlotte Sten, Rikke Wehner Rasmussen, Morten Hjuler Nielsen, Nahuel Garcia, Maiken Mellergaard, Aase Handberg

PT01.25 Forecasting post‐COVID syndrome: leveraging molecular signatures of extracellular vesicles for pedictive analysis

Dr Edina Gyukity‐Sebestyen, Gabriella Dobra, Matyas Bukva, Dr Maria Harmati, Timea Boroczky, Dr Szabolcs Nyiraty, Dr Barbara Bordács, Dr Margareta Korsos, Dr Zoltan Szabo, Dr Gabor Kecskemeti, Prof. Dr Tamas Varkonyi, Prof. Dr Zoltan Konya, Prof. Dr Marta Szell, Dr Peter Horvath, Dr Krisztina Buzás

PT01.26 Glioblastoma biomarkers in urinary extracellular vesicles reveal the potential for a ‘liquid gold’ biopsy

Dr Susannah Hallal, Dr Agota Tuzesi, Mr Liam Sida, Dr Elissa Xian, Dr Daniel Madani, Dr Krishna Muralidharan, Associate Professor Brindha Shivalingam, Associate Professor Michael Buckland, Dr Laveniya Satgunaseelan, Dr Kimberley Alexander

PT01.27 Glycosignatures of small extracellular vesicles secreted by breast cancer cells

Lifang Yang, Benjamin Johnson, Caleb Smack, Professor Eric Feliberti

PT01.28 High‐throughput and automated isolation of plasma derived extracellular vesicles to identify microRNAs with diagnostic potential for ovarian cancer

Dr Ramin Khanabdali, Dr Carlos Palma, Miss Siena Barton, Professor Greg Rice

PT01.30 Identification of extracellular vesicles and particles derived proteins as novel biomarkers for prostate cancer diagnosis, risk stratification and monitoring metastasis

Mr Qi Wang, Dr Bairen Pang, Dr Cheng Zhou, Dr Meng Han, Jie Ni, David Malouf, Joseph Bucci, Peter Graham, Tiannan Guo, Junhui Jiang, Yong Li

PT01.32 Investigation of the immunopeptidome carried by MHC class I molecules on extracellular vesicles (EV) released from lung cancer cells

Miss Debra Lennox, Dr Caitlin Boyne, Dr Sally Shirran, Dr Simon Powis

PT01.33 Isocitrate dehydrogenase 1 is increased in urinary extracellular vesicles from type 2 diabetic model rats

Ph.D. Student Haruka Sei, M.S. Naoya Hirade, Ph.D. Fumie Nakashima, Ph.D. Takahiro Shibata

PT01.34 Isolation and characterisation of extracellular vesicles from tumour and non‐tumour lung tissues for next generation sequencing

Edward Stephens, Dr Tian Mun Chee, Mr Vihanga Dharmasena, Professor Kwun Fong, Professor Ian Yang

PT01.36 Leveraging extracellular vesicle glycan signatures for prostate cancer detection

MS Trevor Enright, PhD Kai Tao, PhD Sinan Sabuncu, PhD Emek Demir, MD Mark Garzotto, BS Randall Armstrong, PhD Michelle Gomes

PT01.37 Lipidomic and proteomic approaches revealed glycerophospholipids as a signatures of hypoxic small extracellular vesicles from head and neck squamous cell carcinoma

Dr Alicja Głuszko, dr. hab. Mirosław Szczepański, dr. Andrzej Ciechanowicz, Prof. Theresa Whiteside, dr. Nils Ludwig

PT01.38 Lipidomic identification of novel small extracellular vesicle biomarkers for prostate cancer early diagnosis and risk progression stratification

PhD Meng Han, PhD Jie Gong, Professor Qi Wang, PhD Bairen Pang, PhD Cheng Zhou, PhD Zhihan Liu, Professor Junhui Jiang, Professor Yong Li

PT01.39 Multiplex profiling of endometriosis‐derived extracellular vesicles reveals novel potential biomarkers for endometriosis

MSc Karolina Soroczyńska, Tobias Tertel, Bernd Giebel, Małgorzata Czystowska‐Kuźmicz

PT01.40 Novel set of extracellular vesicle proteins as biomarkers for early detection of high grade serous ovarian cancer

Kalpana Deepa Priya Dorayappan, Dr. Michelle Lightfoot, Dr. Lianbo Yu, Dr. Colin Hisey, Dr. Takahiko Sakaue, Dr Muralidharan Anbalagan, Dr Casey Cosgrove, Dr Larry Maxwell, Dr Premal Thaker, Dr Beth Y. Karlan, Dr David O'Malley, Dr Raphael E. Pollock, Dr David E. Cohn, Dr Rajan Gogna, Dr Selvendiran Karuppaiyah

PT01.41 Proteomic analysis of cerebrospinal fluid in medulloblastoma and associated extracellular vesicle protein ‐ TKT as a potential biomarker

Research Professor Seung Ah Choi, Professor Seung‐Ki Kim, Professor Ji Hoon Phi

PT01.41 Plasma extracellular vesicle miR‐512‐3p modulates the GTPase activity and the angiogenic function of endothelial colony‐forming cells by targeting ARHGEF3 in pediatric Moyamoya disease

Research Professor Seung Ah Choi, Professor Eun Jung Koh, Professor Seung‐Ki Kim

PT01.42 Proteomic profiling of extracellular vesicles from lymphatic drainage fluid after optimized isolation reveals enriched tumor‐associated markers compared to plasma

Dr XINYU QU, Dr Leanne Leung, Dr Bojie Chen, Professor Zigui Chen, Professor Katie Meehan, Professor Jason Chan

PT01.45 Raman spectroscopy‐based profiling of plasma‐derived extracellular vesicles: a novel approach for differentiating cancerous diseases

Timea Boroczky, Matyas Bukva, Gabriella Dobra, Maria Harmati, Edina Sebestyen‐Gyukity, Yasmin Ranjous, Laszlo Szivos, Katalin Hideghety, Krisztina Budai, Judit Olah, Peter Horvath, Gyorgy Lazar, Zoltan Konya, Pal Barzo, Almos Klekner, Krisztina Buzas

PT01.46 Revealing urinary exosomal eiomarkers in progressive NAFLD: proteomic analysis in a rat model

Chao‐Yuan Chang, Visiting Staff Chun‐Jen Huang, Visiting Staff Syuan‐Hao Syu, Visiting Staff Tze‐Sian Chan

PT01.48 Small extracellular vesicle (sEV) proteins as a potential biomarker for endometriosis

Dr Hannah Nazri, Dr Raphael Heilig, Associate Professor Roman Fischer, Professor Benedikt Kessler, Dr Kavita S Subramaniam, Professor Christian Becker, Dr Thomas Tapmeier

PT01.49 Storage stability study of human urinary extracellular vesicles

MD Cahyani Gita Ambarsari, Professor MW Taal, MRCPCH MD(res) JJ Kim, Assistant Professor Dong‐Hyun Kim, Assistant Professor AM Piccinini

PT01.51 SWATH‐MS identified differentially expressed proteins in extracellular vesicles isolated from pleural effusions of Malignant Pleural Mesothelioma

Dr. Kelly Tian Mun Chee, Prof. Kwun M Fong, Prof. Ian A Yang, Assoc. Prof. Rayleen V Bowman

PT01.52 Systemic changes in Immune System‐Related Plasma Extracellular Vesicles During Healthy Aging

Dr. Xin Zhang, Dr. Sisi Ma, Syeda Iffat Naz, Janet Huebner, Dr. Erik Soderblom, Noor Alnemer, Dr. Constantin Aliferis, Dr. Virginia Kraus

PT01.53 The altered levels of urinary extracellular vesicles pre‐ and post‐surgery relative to proteomics change in breast cancer patients

Miss Nilobon Jeanmard, Dr. Rassanee Bissanum, Mr. Kittinun Leetanap*rn, Mr. Pongsakorn Choochuen, Assoc.Prof. Hutcha Sriplun, Miss Sawanya Charoenlappanit, Dr. Sittiruk Roytrakul, Assoc.Prof. Raphatphorn Navakanitworakul

PT01.54 The circulating extracellular vesicles in ovarian cancer study

Dr Andrew Lai, Dr Dominic Guanzon, Dr Shayna Sharma, Mrs Katherin Scholz‐Romero, Dr Yaowu He, Mr Weitong Huang, Dr Tanja Pejovic, Dr Carmen Winters, Professor Terry Morgan, Professor Jermaine Coward, Associate Professor Amy McCart Reed, Professor Sunil Lakhani, Professor Andreas Obermair, Professor Amanda Barnard, Professor Anna deFazio, Professor Lewis Perrin, Professor John Hooper, Professor Gregory Rice, Professor Carlos Salomon

PT01.58 Unique lipidomic profile sets Extracellular vesicles apart from other cellular fractions in ovarian cancer

Ms Shikha Rani, Dr Andrew Lai, Dr Dominic Guanzon, Mr Kaltin Ferguson, A/Prof Lewis C. Perrin, Prof John D. Hooper, Prof Carlos Salomon

PT01.59 Unlocking ovarian cancer detection: Long‐read sequencing reveals promising biomarkers using extracellular vesicle DNA methylation and mutation patterns

Dr Dominic Guanzon, Dr Subash Rai, Mr Rakesh Sankar, Ms Pragati Lodha, Ms Vidya Gummagatta, Dr Andrew Lai, Professor Lewis Perrin, Professor John Hooper, Professor Carlos Salomon

PT02.01 Altered protein nitrosylation patterns in extracellular vesicles isolated from activated microglia

Dr Natasha Vassileff, Dr Jereme Spiers, Miss Sarah Bamford, Dr Rohan Lowe, Dr Keshava Data, Professor Paul Pigram, Professor Andrew Hill

PT02.02 Antidepressant effects of aerobic exercise: are circulating EVs responsible?

Reine Khoury, Dr. Dariusz Zurawek, Gabriella Frosi, Assistant Professor Corina Nagy

PT02.03 BDNF/TrkB system dysregulation at the cell environment: extracellular vesicles as carriers of TrkB‐ICD in Alzheimer's disease

Mr. Tiago Costa‐Coelho, João Fonseca‐Gomes, Gonçalo Garcia, Mafalda Ferreira‐Manso, Catarina B. Ferreira, Carolina de Almeida‐Borlido, Juzoh Umemori, Mikko Hiltunen, Eero Castrén, Ana M. Sebastião, Alexandre de Mendonça, Dora Brites, Maria José Diógenes

PT02.04 Brain region‐specific changes in extracellular vesicles release and composition in tau R406W human organoid tauopathy model

Dr. Tina Bilousova, Nina Knitowski, Dr. Qing Cao, Shengkai Zhao, Swetha Atluri, Mikhail Melnik, Achyutha Kodavatikanti, Dr. Ranmal Samarasinghe, Dr. Jessica Rexach, Dr. Karen Gylys

PT02.05 Cerebrospinal fluid extracellular vesicle miRNAs identify synaptic transmission alterations in Alzheimer's disease

PhD Ursula Sandau, Trevor McFarland, PhD Sierra Smith, MD Douglas Galasko, MD Joseph Quinn, MD, PhD Randy Woltjer, PhD Julie Saugstad

PT02.06 Eukaryotic and microbiota‐derived extracellular vesicles in Parkinson's disease.

Tiana Koukoulis, Purnianto Adityas, David Finkelstein, Leah Beauchamp, Kevin Barnham, Dr Laura Vella

PT02.07 Exploitation of vitreous‐derived extracellular vesicles to study the central nervous system dynamics

Lien Cools, Dr. Cristiano Lucci, Sam Noppen, Dr. Charysse Vandendriessche, Drs. Kaat Verleye, Drs. Laura Raes, Elien Van Wonterghem, Prof. Inge Mertens, Prof. Dominique Schols, Prof. Roosmarijn E Vandenbroucke, Prof. Lies De Groef

PT02.09 How do tumour derived EVs interact with the maturing nervous system and lead to altered pain processing in cancer survivors?

Dr Hannah Jackson, Dr Anna Grabowska, Dr Victoria James, Dr Federico Dajas‐Bailador, Dr Beth Coyle, Dr Gareth Hathway

PT02.10 Inflammation‐associated microglial EVs exhibit morphological differences and enrichment for ribosomes

Mr William Phillips, Ms Irumi Amarasinghe, Dr Ebony Monson, Dr Nicholas Reynolds, Prof Karla Helbig, Dr Lesley Cheng, Prof Andrew F Hill

PT02.11 Isolation of spontaneously‐released brain extracellular vesicles: implications for stress‐driven brain pathologies

Dr Ioannis Sotiropoulos, Dr Patricia Gomes, Dr Cristian Bodo, Dr Carlos Noguera‐Ortiz, Dr Martina Samiotaki, Dr Minghao Chen, Dr Carina Soares‐Cunha, Dr Joana M. Silva, Dr Bárbara Coimbra, Dr George Stamatakis, Dr Liliana Santos, Dr George Panayotou, Professor Clarissa L. Waites, Proffessor Christos Gatsogiannis, Professor Nuno Sousa, Professor Dimitrios Kapogiannis, Dr Bruno Costa‐Silva

PT02.12 Metabolic analysis of extracellular vesicles isolated from human brain tissue in Alzheimer's disease context

PhD student Patricia Hernández‐López, Dr. Elisabeth Rackles, Dr. Oihane E. Albóniga, Dr. Juan Manuel Falcon‐Pérez

PT02.13 Mitochondrial proteins are exported from cells via sEVs in Parkinson's disease

Mr Adityas Purnianto, Ms Mitali Kulkarni, Professor Scott Ayton, Professor Catriona McLean, Professor Ashley Bush, Professor David Finkelstein, Professor Kevin Barnham, Dr Laura Vella

PT02.14 Plasma‐derived small extracellular vesicles in alzheimer's disease progression: insights into synaptic dysfunction and neuroinflammation

Mr Rishabh Singh, Ms Sanskriti Rai, Dr Prahalad Singh Bharti, Dr Prasun Chatterjee, Dr Saroj Kumar

PT02.15 Primary rat cortical tri‐culture to study cellular response to cancer EVs

Mrs. Rachel Rachel, Dr. Randy Carney, Hyehyun Kim, Dr. Erkin Seker

PT02.16 Protein nitrosothiol patterns altered in extracellular vesicles from Alzheimer's disease brain cortex

Dr Natasha Vassileff, Dr Rohan Lowe, Dr Keshava Datta, Professor Catriona McLean, Professor Andrew Hill, Dr Jereme Spiers

PT02.17 Proteomic analysis of brain‐dervied extracellular vesicles in Huntington's disease

Miss Mitali Manish Kulkarni, Mr. Adityas Purnianto, Miss Tiana Koukoulis, Miss Huaqi Su, Miss Geraldine Kong, Professor Anthony Hannan, Dr. Laura.J Vella

PT02.19 Uncovering the composition of extracellular vesicles (EVs) regulated by Translin‐Associated Factor X (TRAX) that modulates microglial identity

Dr. Yu‐Ting Weng, Ph.D. Yijuang Chern

PT02.22 Extracellular vesicles in mood disorders: a systematic review

Dr Cristian‐Daniel Llach¹, Ms Gia Han Le¹, Dr Gerard Anmella², Dr Joshua Rosenblat¹, Dr Anna Gimenez‐Palomo², Dr Isabella Pacchiarotti², Dr Eduard Vieta², Dr Roger McIntyre¹, Dr Rodrigo Mansur¹

¹University Of Toronto, Toronto, Canada, ²Bipolar and Depressive Disorders Unit, Hospital Clinic de Barcelona, Barcelona, Spain

PT03.01 Analysis of the phenotypical changes of plasma EVs over time in healthy donors

Rikke Bæk, Maiken Mellergaard, Rikke Wehner Rasmussen, Rikke Bülow Eschen, Evo Lindersson Søndergaard, Aase Handberg, Malene Møller Jørgensen

PT03.02 Comparison of primed mesenchymal stromal cells secretome following different methods of purification with a large panel of characterization tools

PHD Student Guillaume Valade, PHD Student Marine DE TADDEO, Mrs Muriel NIVET, Mrs Marion GROSBOT, Mrs Claire LANGLE, Mrs Sylvie GOULINET, PHD Philippe MAUDUIT, Mr Vincent JUNG JUNG, PHD Chiara GUERRERA, MD, PHD Sébastien BANZET, PHD Juliette PELTZER, PHD Marina TROUILLAS

PT03.03 Evaluation of the physical properties and pharmaco*kinetics of EVs purified by the microfiltration membrane with ion exchange function

Ms. Ayano Higaki, Mr. Keita Inoue, Ms. Mizuki Kobayashi, Ms. Makiko Hiraoka, Mr. Yosh*taka Kawakami, Ph.D. Naohiro Seo

PT03.04 Is it feasible to distinguish extracellular vesicles by their biophysical properties?

Mr Fredrik Stridfeldt, MSc Hanna Kylhammar, Dr Vipin Agrawal, MSc VIkash Pandey, Dr André Görgens, Professor Samir El Andaloussi, Professor Dhrubaditya Mitra, Professor Apurba Dev

PT03.06 Characterization of human MSC‐derived extracellular vesicle preparations using size‐exclusion HPLC and ion‐exchange HPLC coupled with multi‐angle light scattering detection

Dr. Hirotaka Nishimura, Dr. Tomofumi Yamamoto, Dr. Noritaka Hashii, Dr. Akiko Ishii‐Watabe

PT03.07 EVs during zebrafish larvae development

Dr.med. Linda‐marie Mulzer¹, Tim Felger¹, PD Dr. med. habil. Dr. rer. nat. Luis Muñoz², Gesa Engl¹, Prof. Dr. med. Heiko Reutter¹, Leila Pourtalebijahromi³, Prof. Dr. Gregor Fuhrmann³, Philipp Arnold⁴, Dr. med. Alina Hilger¹

¹Department of Pediatrics and Adolescent Medicine, Friedrich‐Alexander University of Erlangen‐Nürnberg, Erlangen, Germany, ²Department of Rheumatology and Immunology, Friedrich‐Alexander University Erlangen‐Nürnberg, Erlangen, Germany, ³Friedrich‐Alexander University Erlangen‐Nürnberg, Department of Biology, Chair of Pharmaceutical Biology, Erlangen, Germany, ⁴Friedrich‐Alexander University Erlangen‐Nürnberg, Institute for Functional and Clinical Anatomy, Erlangen, Germany

PT03.08 Activated human mast cells produce extracellular vesicles that change the metabolic function of target cells

Senior Investigator Marianna Kulka, Dr. Marcelo Marcet‐Palacios, Sabrina Rodrigues Meira

PT03.09 Advancing ultra‐low, ultra‐deep extracellular vesicle proteomics

Prof David Greening, Mr Alin Rai, Ms Haoyun Fang, Ms Bethany Claridge, Mr David Greening

PT03.10 Amniotic fluid EV proteome is a clear representation of gestational age‐dependent fetal development

Dr Ishara Atukorala, Dr Ching‐Seng Ang, Ms Sally Beard, Ms Bianca Fato, Dr Natasha de Alwis, Dr Hamish Brown, Professor Natalie Hannan, Professor Lisa Hui

PT03.11 Assessing the compartmentalisation of small non‐coding RNAs in the circulation

Dr I‐Jou Teng, Dr Kaloyan Takov, Dr Clemens Gutmann, Prof. Manuel Mayr

PT03.12 Astrocyte‐enriched extracellular vesicle protein concentrations after proteinase K treatment

Dr. Leandra Figueroa‐Hall, Dr. Kaiping Burrows, Dr. Ahlam Alarbi, Dr. Chibing Tan, Dr. Bethany Hannafon, Dr. Rajagopal Ramesh, Dr. Victoria Risbrougn, Dr. T. Kent Teague, Dr. Martin Paulus

PT03.13 Characterization of extracellular vesicles with capillary electrophoresis

Aleksandra Steć, Ph.D. Joanna Jońca, Ph.D. Agata Płoska, Prof. Leszek Kalinowski, Assoc. Prof. Bartosz Wielgomas, Ph.D. Krzysztof Waleron, Prof. Bogdan Lewczuk, Mr. Grzegorz Czyrski, Ph.D. Andrea Heinz, Ph.D. Szymon Dziomba

PT03.14 Circadian mass spectrometry‐based proteome profiling of salivary extracellular vesicles

Dr Carlos Andres Palma Henriquez, Ms Siena Barton, Dr Sara Nikseresht, Mr Sadman Bhuiyan, Dr Mozhgan Shojaee, Dr Kartini Asari, Dr Pingping Han, Dr Ramin Khanabdali, Dr Gregory Rice

PT03.15 FunRich enables enrichment analysis of extracellular vesicles OMICs datasets

Mr Sriram Gummadi

PT03.16 Modulating nonspecific uptake of engineered extracellular vesicles

Beth DiBiase^{Chemical and Biological Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}, Roxana Mitrut^{Chemical and Biological Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}, Taylor Gunnels^{Biomedical Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}, Dr. Neha Kamat^{Biomedical Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}, Dr. Joshua Leonard^{Chemical and Biological Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}

PT03.17 Multi‐omics characterization of highly enriched human plasma small extracellular vesicles

Ms Huaqi Su, Assoc. Prof. Kevin Barnham, Prof. Gavin Reid, Dr. Laura Vella

PT03.20 Proteomic analysis of extracellular vesicles secreted by human umbilical cord mesenchymal stem/stromal cells under stimulated conditions

Dr. Chaiyong Koaykul, Dr. Kunthika Mokdarta, Dr. Poorichaya Somparn, Dr. Jiradej Makjaroen, Dr. Chatikorn Boonkrai, Dr. Trairak Pisitkun

PT03.22 Sizing and visualization of single EVs using a super‐resolution based workflow to characterize EV populations

Dr James Rhodes, Dr Kathleen M Lennon, Dr Colbie Chinowsky, Dr Abigail Neininger‐Castro, Ms Chloe Celingant‐Copie, Dr Daniel Zollinger, Dr Grace DeSantis

PT03.23 The modulating effect of short, cationic peptides on EV's protein corona

PhD Imola Cs. Szigyarto, Priyanka Singh, Tasvilla Sonallya, PhD Aniko Gaal, PhD Lilla Turiak, PhD Laszlo Drahos, PhD Zoltan Varga, PhD Tamas Beke‐Somfai

PT03.24 Vesiclepedia and ExoCarta: A web‐based compendiums of extracellular vesicles cargo and extracellular particles

Mr Sriram Gummadi

PT03.26 Establishment of an immunocapture method for the separation of a rheumatoid arthritis‐related CD90+ subpopulation of extracellular vesicles

M.Sc. Stefanie Kurth, PhD André Tiaden, M.Sc. Edveena Hanser, Ute Heider, PhD Stefan Wild, Professor Diego Kyburz

PT03.27 ExoPAS: numerous and pure isolation of exosomes using cationic material and PEG

Wonjae Kim, Student Kangmin Lee

PT03.28 A biomimetic vortex tangential flow filtration (VTFF) system for efficient isolation and purification of extracellular vesicles

Ph.D. Candidate Yuxin Qu^{School of Biomedical Engineering, Tsinghua University, Beijing, China}, Assistant professor Han Wang^{School of Biomedical Engineering, Tsinghua University, Beijing, China}, Lan Xie^{School of Basic Medical Sciences, Tsinghua University, Beijing, China}

PT03.29 A protocol to differentiate the chondrogenic ATDC5 cell‐line for the collection of chondrocyte‐derived extracellular vesicles

Mr Jose Marchan‐Alvarez, Miss Loes Teeuwen, Mr Doste Mamand, Prof Susanne Gabrielsson, Prof Klas Blomgren, Dr Oscar Wiklander, Dr Phillip Newton

PT03.30 A quick, cost‐free, and user‐friendly cleanup protocol for dye and drug removal from small extracellular vesicle solution

Ioannis Isaioglou, Gloria Lopez‐Madrigal, Jasmeen Merzaban

PT03.31 A standardized multi‐stage purification process and comprehensive characterization of extracellular vesicles derived from HEK293F cells

Research associate Nhan Vo, Research associate Chau Tran, Research associate HB Nam Tran, Scientist T Nhat Nguyen, Research associate Thieu Nguyen, Scientist DN Diem Nguyen, Research associate Tran Pham, R&D lead Hoai‐Nghia Nguyen, R&D specialist Lan‐N Tu

PT03.32 A survey study on the status of extracellular vesicle (EV) research in malaysia: current updates

Ts. Dr. Norhayati Liaqat Ali Khan, Dr. Nadiah Abu, Dr. Wai Leng Lee, Dr. Muhammad Farid Nazer Muhammad Faruqu, Dr. Jia Xian Law, Associate Professor Dr. Norshariza Nordin, Dr. Maryam Azlan, Associate Professor Dr. Rajesh Ramasamy, Dr. Sik Loo Tan, Associate Professor Dr. Wan Nazatul Shima Shahidan, Mr. See Nguan Ng, Dr. Kok Lun Pang, Dr. Vijayendran Govindasamy, Mr. Benson Koh, Dr. Pan Pan Chong, Miss Yoong Yi Chong, Mrs. Nur Hidayah Hassan, Mr. Nazmul Huda Syed, Mrs. Maimonah Eissa Sheikh Al‐Masawa

PT03.33 Advancing scalable production of purified adipose‐derived stem cell extracellular vesicles

Jing Zhou, Ph.D candidate Jiajia Dai, Ph.D candidate Haonan Di, Ph.D candidate Yunyun Hu, Ph.D candidate Niangui Cai, professor Xiaomei Yan

PT03.35 Benchmarking surface functionalization strategies for marker independent EV capture and profiling

Mr. Hugues Martin, Dr. Andreas Wallucks, Dr. Andy Ng, Ms. Molly Shen, Dr. David Juncker

PT03.36 Beyond the boundaries of conventional isolation techniques: Functional self‐assembled coordination polymer nanoparticles for instant one‐step selective and efficient enrichment of exosomes – ExoFlocs™

Mr Mohamed Sallam, Mr Cong‐Minh Nguyen, Dr Amandeep Singh Pannu, Dr Indira Prasadam, Mr Yezhou Yu, Professor Serge Muyldermans, Dr Frank Sainsbury, Professor Nam‐Trung Nguyen, Professor Nobuo Kimizuka

PT03.38 Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy

Jie Gong, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Meng Han, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Bairen Pang, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Qi Wang, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Haotian Chen, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Zhihan Liu, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Cheng Zhou, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Yong Li, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Junhui Jiang

PT03.39 Comparison of asymmetric depth filtration and ultrafiltration combined with size‐exclusion chromatography for EV isolation from cell culture media

Dr. Vasiliy Chernyshev, Dr. Elena Svirshchevskaya, Mr. Mikhail Ivanov, Dr. Denis Silachev

PT03.40 Comprehensive evaluation of extracellular vesicle markers through diverse isolation strategies

Dr. Kaiping Burrows, Dr. Leandra Figueroa‐Hall, Dr. Ahlam Alarbi, Dr. Bethany Hannafon, Cole Hladik, Dr. Rajagopal Ramesh, Dr. Victoria Risbrough, Dr. T. Kent Teague, Dr. Martin Paulus

PT03.41 Confident isolation and proteomics of bacterial extracellular vesicles by size exclusion chromatography

Ms Haekang Yang, Ms Shinwon Chae, Mr Chul Won Seo, Ms Seoyeon Kim, Professor Yoon‐Jin Lee, Professor Dongsic Choi

PT03.42 Development of a method for large‐scale purification of extracellular vesicles using the PS affinity method

Dr. Afshin Iram, Shotaro Masuda, Hana Onizuka, PhD. Ryo Ukekawa, PhD. Takahiro Nishibu

PT03.43 Development of an applicable method for bacterial extracellular vesicle isolation from mouse stool supernatant

Shujin Wei, Professor Wanli Xing

PT03.44 Does EV purity affect downstream functionality?

Research Officer Janice Tan, Principal Investigator Ivy Ho

PT03.46 Evolution of an EV enrichment protocol: from minimal information to proteomics

Dr Felicity Dunlop, Dr Shaun Mason, Dr Taeyoung Kang, Professor Suresh Mathivanan, Professor Aaron Russell

PT03.47 ExoCAS‐2: rapid and pure isolation of exosomes by anionic exchange using magnetic beads

Student Jaeeun Lee

PT03.48 ExoFilter: large capacity extraction of EVs using a positive charge mesh filter in continuous flow

Student Yongwoo Kim

PT03.49 miRQuick: An innovative charge‐based EV isolation method for highly efficient extraction of EV‐miRNAs from liquid samples

Student Lee Kangmin

PT03.50 High‐throughput isolation and sorting of nanoparticle loaded exosomes

Dr. Hye Sun Park, Taewoong Son, Mi Young Cho, Hyunseung Lee, Eun Hee Han, Dr. Kwan Soo Hong

PT03.52 Impact of hyaluronidase on tetraspanin expression of extracellular vesicles (EVs) in synovial fluid from patients with rheumatoid arthritis and osteoarthritis using the Exoview platform.

Mrs. Edveena Hanser, Prof. Dr. Diego Kyburz

PT03.54 Isolation and enrichment of extracellular vesicles with double‐positive membrane protein for subsequent biological studies

Dr. Huixian Lin, Dr. Chunchen Liu, Prof. Bo Li, Prof. Lei Zheng

PT03.55 Molecular imprinted polymer‐based artificial peptide (MIPap) enables isolation of astrocyte‐specific extracellular vesicles (asEV) in serum

Yong Shin, Professor Eun Jae Lee

PT03.56 Novel strategy for affinity capture and release sEV

Professor Wei Duan, Mr Rajindra Napit, Dr Rocky Chowdhury, Mr Satendra Jaysawal

PT03.57 Optimization of separation methodologies for obtaining high yield‐high purity urinary extracellular vesicles

Beatriz Martín‐Gracia, Optimization of separation methodologies for obtaining high yield‐high purity urinary extracellular vesicles Håkon Flaten, Optimization of separation methodologies for obtaining high yield‐high purity urinary extracellular vesicles Krizia Sagini, Optimization of separation methodologies for obtaining high yield‐high purity urinary extracellular vesicles Alicia Llorente

PT03.58 Optimizing high‐throughput isolation of extracellular vesicles from primary cells in small to medium‐scale 3‐dementional bioreactors with serial purification methods

Dr. Zheng Zhao

PT03.59 Pillared interdigitated electrodes for small extracellular vesicle capture

Miss Emma Morris, Associate Professor Karl Hassan, Professor Craig Priest, Dr Bin Guan, Dr Renee Goreham

PT03.60 Protocol optimisation for extracellular vesicle isolation and characterisation: evaluation of ultracentrifugation, size exclusion chromatography and charged core bead chromatography methods

Dr Farha Ramzan, Hui Hui Phua, Dr Vidit Satokar, Dr Shikha Pundir, Dr Anastasia Artuyants, Dr Cherie Blenkiron, Dr Chris Pook, Prof Mark Vickers, Dr Ben Albert

PT03.62 Rapid and efficient isolation platform for plasma extracellular

vesicles: EV‐FISHER

Dr Weilun Pan, Prof Lei Zheng, Prof Jinxiang Chen, Prof Bo Li

PT03.63 Salivary extracellular vesicles isolation methods impact the robustness of biomarkers detection

Dr Jérémy Boulestreau^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4,France}, Dr Laurence Molina^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4,France}, Alimata Ouedraogo^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4,France}, Louen Laramy^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4,France}, Ines Grich^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4,France}, Dr Thi Nhu Ngoc Van^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4,France; SkillCell, Montpellier, France}, Dr Franck Molina^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182,34184, Montpellier, CEDEX 4, France}, Dr Malik Kahli^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182,34184, Montpellier, CEDEX 4, France}

PT03.64 Single‐particle multiplex analysis of EV‐biophysical properties of fractionated particle populations by ion exchange chromatography

Professsor Takanori Ichiki, Chiharu Mizoi, Kento Toyoda, Professor Naohiro Seo

PT03.65 Tailored cellulose nanofiber sheets capture and preserve small extracellular vesicles from micro‐volume body fluids and reveal the unknown profiles of extracellular vesicles

M.D., Ph.D.Akira Yokoi, M.D., Ph.D. Kosuke Yoshida, B.Sc. Masami Kitagawa, Ph.D. Takao Yasui, M.D., Ph.D. Hiroaki Kajiyama

PT03.66 Targeting EV enriched lipids for non‐biased capture and analysis

Dr Bradley Whitehead, PhD Litten S Sørensen, Anders T Boysen, Prof Peter Nejsum

PT03.68 Xeno‐free human platelet lysate depleted of exosomes for enhanced extracellular vesicle yield from stem cells, immune cells, and cancer cells

Mr. Yee‐Hsien Lin, Mr. Han‐Tse Lin, Mr. William Milligan, Dr. Min‐ChangHuang

PT03.70 Real‐time Label‐free platforms for size determination and cell interaction studies of extracellular vesicles

Msc In Medicinal Chemistry, doctoral researcher in Pharmacy Elena Scurti¹, PhD Martina Hànzlikova¹, MSc Johanna Puutio², PhD Fadak Howaili³, PhD Kai Härkönen⁴, Professor Pia Siljander², PhD Saara Laitinen⁴, Professor Tapani Viitala^1,3

¹Division of Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland, ²EVcore facility, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland, ³Åbo Akademy University, Turku, Finland, ⁴Finnish Red Cross Blood Service, Helsinki, Finland

PT03.71 How Centrifugation Can Improve Your EV Workflow

Ms. Amy Henrickson¹, Dr. Lutz Ehrhardt, Dr. Shawn sternisha

¹Beckman Coulter, Indianapolis, United States

PT03.72 Optimizing a workflow for the analysis of extracellular vesicles

Dr. Anis Larbi¹

¹Beckman Coulter Life Sciences, France

PT03.73 MISEV 2023: the Beckman Coulter Life Sciences approach for Extracellular Particles

Dr. Anis Larbi¹

¹Beckman Coulter Life Sciences, France

PT03.75 EV Quant: A quantitative web‐based compendium of extracellular vesicles cargo for studies in vesiclepedia

Mr SRIRAM GUMMADI¹

¹Latrobe University, Australia

PT03.76 Optimized protocol for isolation of extracellular vesicles (EV) ‐ carried microRNAs from platelet‐free plasma using size‐exclusion chromatography (SEC) and phenol – guanidine extraction.

MD Miłosz Majka¹, PhD Katarzyna Czarzasta², MD, PhD Małgorzata Wojciechowska², PhD Małgorzata Czystowska‐Kuźmicz¹

¹Medical University of Warsaw, Chair and Department of Biochemisrty, Warsaw, Poland, ²Medical University of Warsaw, Laboratory of Centre for Preclinical Research, Chair and Department of Experimental and Clinical Physiology, Warsaw, Poland

PT03.77 Comparative Analysis of Plasma and Serum Exosomal Small RNA Sequencing Profiles

Dr. Alex Chauhan¹, Hinal Zala¹, Simone Yamasaki¹, Enaam Merchant¹, Dr. Mohamed El‐Mogy¹, Dr. Songsong Geng¹, Dr. Taha Haj‐Ahmad¹, Dr. Yousef Haj‐Ahmad¹

¹Norgen Biotek Corp., Thorold, Canada

PT03.80 Innovative Ultrapure Exosome Extraction Using Hybrid Charge‐Based Filtration and Tangential Flow Filtration

Mr. Yoing‐woo Kim¹, Mr. Kang‐Min Lee¹, Professor Sehyun Shin¹

¹Korea University, Seoul, South Korea

PT03.81 Use of advanced aptamer technology in EV research

Mr. Rajindra Napit¹, Mr. Satendra Jyasawal¹, Ms. Jasmine Catague¹, Mr. Haben Melke¹, Dr. Rocky Chowdhury¹, Dr. Lingxue Kong¹, Dr. Wei Duan¹

¹Deakin University, Warun Ponds, Geelong, Australia

PT03.82 Purifying exosomes to meet manufacturing demand using a gentle, size‐based, and scalable purification solution

Dr Jagan Billakanti¹, Dr Jon Lundqvist, Dr Peter Guterstam

¹Cytiva, Brisbane, Australia

PT03.83 Isolation of extracellular vesicles in aqueous two‐phase systems for cancer diagnosis

Ph.D. Candidate Minyeob Lim¹

¹POSTECH, Pohang, South Korea

PT04.01 25HC depleted accessible cholesterol to restrict SFTSV infection and infectious‐EVs mediated tramsmission

Postdoctor Rui Zhang

PT04.04 Bacterial extracellular vesicles contain metabolites that could contribute to the pathological hallmarks of Alzheimer's disease

Samuel Wachamo^{Department of Neuroscience, Neuroscience Graduate Program, Center for Brain Immunology and Glia, Medical Scientist Training Program,University of Virginia, Charlottesville, VA, USA}, Alisha Thakur, Mallarie Broadway^{Department of Neuroscience, Neuroscience Graduate Program, Center forBrain Immunology and Glia, Medical Scientist Training Program, University of Virginia, Charlottesville, VA, USA}, Dr. Alban Gaultier^{Department of Neuroscience,Neuroscience Graduate Program, Center for Brain Immunology and Glia, Medical Scientist Training Program, University of Virginia, Charlottesville, VA, USA}

PT04.05 Bacterial outer membrane vesicles trigger mitochondrial stress in macrophages

Ms Chantelle Blyth, Dr Michael Lazarou, Dr Thomas Naderer

PT04.06 Comparative analysis of intestinal microbiota‐derived extracellular vesicles in newborns from vagin*l and cesarean section delivery: implications for modulation of immune system cells

Ms. Catalina Adasme‐Vidal, Mr. Aliosha I. Figueroa‐Valdés, Ms. Camila Fuentes, Ms. Patricia Valdebenito, Mr. Sebastián Illanes, Ms. Francisca Alcayaga‐Miranda

PT04.08 Exosomal miRNAs as markers of the biological effects of plant extracts

Doctor Alisa Petkevich, Doctor AleksandrAbramov, Professor Vadim Pospelov

PT04.09 Exploration of Purified Extracellular Vesicles (EVs) from Trypanosoma cruzi Y and G Strains on Host Cell Interaction

PhD student Paula Meneghetti, Ana Claudia Torrecilhas

PT04.10 Explore food microbes with preservative tolerance to spread of antimicrobial resistance from perspective of extracellular vesicles

Dr. Bao‐Hong Lee, MS. Yi‐Tsen Chang, Mr. You‐Zuo Chen, Mr. Hui‐Chun Lin, Dr. Wei‐hsuan Hsu

PT04.11 Exploring the extracellular vesicles derived from food spoilage microorganisms in the transmission of antibiotic resistance and potential impact on the gut environment

Dr. Bao‐Hong Lee, MS. Yi‐Tsen Chang, Mr. Hui‐Chun Lin, Mr. You‐Zuo Chen, Dr. Tang‐Long Shen, Dr. Wei‐hsuan Hsu

PT04.13 Extracellular vesicles derived from Akkermansia muciniphila

Promote placentation and mitigate preeclampsia

Ph.d Zihao Ou

PT04.14 Extracellular vesicles derived from Candida albicans promote lung injury through inducing ferroptosis of macrophages

Miss Yiyi Huang, Doctor Kening Zhao, Miss Yuneng Hua, Miss Mei Huang, Doctor Ruyi Zhang, Doctor Jingyu Wang, Mr Fan Bu, Miss Junhui Wang, Professor Lei Zheng, Professor Qian Wang, Professor Xiumei Hu

PT04.15 Extracellular vesicles derived from Naegleria fowleri stimulate cytokine production by innate immune cells

Asst. Prof. Sakaorat Lertjuthap*rn, Ms Narinee Srimark, Mrs Hathai Sawasdipokin, Ms Kasama Sukapirom, Ms Jinjuta Somkird, Prof. Kovit Pattanapanyasat, Ladawan Khowawisetsut

PT04.16 Extracellular Vesicles isolated from Virulent and Non‐Virulent trypomastigotes forms from Trypanosoma cruzi in Host Cell Modulation

Ana Claudia Torrecilhas, Master Nicholy Lozano, Full Professor Sergio Schenkman

PT04.17 Extracellular vesicles release from Aeromonas hydrophila: proteomic analysis and immunomodulatory activity

Professor MAHANAMA DE ZOYSA, Mr. Mawalle Kankanamge Hasitha Madhawa Dias

PT04.18 Gram‐positive bacterial extracellular vesicles released by Streptococcus parauberis: Proteomic profiling and anti‐inflammatory activity

Professor MAHANAMA DE ZOYSA, Mr. E.H.T. Thuslahn E.H.T. Thuslahn Jayathilaka, Mr. Mawalle Kankanamge Hasitha Madhawa Dias, Dr. Chamilani Nikapitiya Nikapitiya

PT04.20 Helicobacter pylori cytotoxin, VacA, hijacks dendritic cell extracellular vesicles

Miss Ruby Gorman‐batt¹, Associate Professor Meredith O'Keeffe, Doctor Terry Kwok‐Schuelein

¹Monash University, Clayton, Australia

PT04.20 Helicobacter pylori cytotoxin, VacA, hijacks dendritic cell extracellular vesicles to dysregulate immune cell functions

Miss Ruby Gorman‐batt, Meredith O'Keeffe, Terry Kwok

PT04.21 Helicobacter pylori extracellular vesicles contain functionally active enzymes promoting bacterial survival

Miss Nina Colon, Mr Liam Gubbels, Professor Richard L. Ferrero

PT04.23 Infective forms of Leishmania show different biological aspects and differ in the extracellular vesicle target mechanisms: Looking at the fundamental bases to understand differences

Dr Mauro Javier Cortez Veliz, Miss Deborah Brandt‐Almeida, Mrs Jenicer Kazumi Umada Yokoyama Yasunaka, Dr Simon Ngao Mule, Dr Giuseppe Palmisano, Dr Ana Claudia Torrecilhas

PT04.23 Host‐Parasite Interface: Exploring the Interaction of Trypanosoma cruzi trypomastigotes forms Y strain Extracellular Vesicles (EVs) with Human Monocytes and Macrophage

PhD student Juliana Fortes, Master student Nathani Negreiros, Ana Claudia Torrecilhas

PT04.24 Investigating the impact of Pseudomonas aeruginosa outer membrane vesicles on alveolar macrophage responses

Miss Isabella Stuart, Mr Joshua Nickson, Dr Seong Hoong‐Chow, Associate professor Thomas Naderer

PT04.25 Isolation and physiological characterisation of Ascochyta rabiei small extracellular vesicles

Ms Matin Ghaheri, Dr Ido Bar, Dr Prabhakaran T. Sambasivam, Dr Muhammad J. A. Shiddiky, Mr Abolfazl Jangholi, Prof Chamindie Punyadeera, Prof Rebecca Ford

PT04.26 Leishmania extracellular vesicles genomic cargo: sharing is caring

Associate Professor David Langlais, MSc Atia Amin, PhD Ana Victoria Ibarra Meneses, Associate Professor Christopher Fernandez‐Prada

PT04.27 Microbiome derived EVs regenerate intestinal stem cells against radiation injury

Dr. Payel Bhanja, Dr. Rishi Man Chugh, Dr. Kafayat Yusuf, Dr. Badal Roy, Dr. Shahid Umar, Dr. Subhrajit Saha

PT04.29 Microbiota‐derived extracellular vesicles regulate host liver gluconeogenesis

Dr Jian Tan, Ms Jemma Taitz, Dr Duan Ni, Ms Camille Potier, Prof Ralph Nanan, Prof Laurence Macia

PT04.30 Presence of viral particles and origin of extracelluar vesicles isolated from patients with COVID‐19 and their association with clinical outcome

Msc Jaques Franco Novaes De Carvalho, Msc. Gabriela Rodrigues Barbosa, Msc Marina Malheiros Araújo Silvestrini, Dr. Sidneia Sousa Santos, Dr. Flávio Freitas, Dr. Nancy Cristina Junqueira Bellei, Dr. Andréa Teixeira de Carvalho, Dr. Ana Claudia Torrecilhas, Dr. Reinaldo Salomão

PT04.31 Probiotic extracellular vesicles: Characterisation and unravelling the proteomic cargo of extracellular vesicles derived from Lactobacillus delbrueckii

Mr Kyle Bramich, Dr Rahul Sanwlani, Prof Suresh Mathivanan

PT04.32 Proteomic analysis of Olive flounder (Paralichthys olivaceus) plasma derived exosomes responses to Edwardsiella piscicida infection

Professor MAHANAMA DE ZOYSA, Mr. E.H.T. Thuslahn Jayathilaka, Mr. Mawalle Kankanamge Hasitha Madhawa Dias, Dr. Chamilani Nikapitiya Nikapitiya

PT04.33 Role of HIV‐associated extracellular vesicles in human papillomavirus (HPV) infection

Professor Ge Jin, Dr. Zhimin Feng

PT04.33 Role of extracellular vesicles in the pathogensis of Citrus exocortis viroid infection tomato plants

Professor Tang‐long Shen, Hao‐Yuan Chien, Ta‐Hsin Ku

PT04.35 TcVPS23: A component of ESCRT‐I complex is a key factor in secretion of extracellular vesicles, endocytosis of Transferrin and act as important virulence factor in Trypanosoma cruzi experimental infection

Pos‐doctoral Nadjania Saraiva de Lira Silva, Ana Claudia Torrecilhas, Full Professor Sergio Schenkman

PT04.37 The Helicobacter pylori autotransporter ImaA associates with extracellular vesicles to promote host inflammatory responses in gastric epithelial cells

Mr Angus Cramond, Ms Nina Colon, Mr Jack Emery, Dr Dongmei Tong, Dr Caroline Skene, Professor Richard L. Ferrero

PT04.38 The Helicobacter pylori virulence factor, Tipa, is carried by bacterial extracellular vesicles to the nuclear compartment of host cells

Mr Jack Emery, Doctor Variya (Way) Nemidkanam, Ms Nina Colon, Ms Kate Friesen, Ms Georgie‐Wray McCann, Associate Professor David McGee, Doctor Natalia Castaño‐Rodríguez, Doctor Dongmei Tong, Doctor Caroline Skene, Doctor Laurent Terradot, Professor Richard L. Ferrero

PT04.39 The impact of maternally‐derived gut bacterial extracellular vesicles on the offspring's developing immune system

Miss Jemma Taitz, Mr Jian Tan, Mr Duan Ni, Mr Georges Grau, Mr Nicholas King, Mr Ralph Nanan, Ms Laurence Macia

PT04.40 The role of Neisseria gonorrhoeae outer membrane vesicles in inducing trained immunity in Macrophages

Dr Jiaru Yang, Dr. Seong Hoong Chow, Dr. Pankaj Deo, Associate Professor Thomas Naderer

PT04.43 Using circulating bacterial outer membrane vesicles to diagnose bacterial infections

Phd Qianbei Li, Professor Lei Zheng

PT04.45 Foam cell‐derived extracellular vesicles regulate the environment surrounding atherosclerotic plaques

Foam cell‐derived extracellular vesicles regulate the environment surrounding atherosclerotic plaques Akihiko Okamura¹, Dr. Yusuke Yoshioka¹, Shungo Hikoso², Takahiro Ochiya¹

¹Department of Molecular and Cellular Medicine, Tokyo Medical University, 6‐7‐1 Nishishinjuku, Shinjuku‐ku, Japan, ²Department of Cardiovascular Medicine, Nara Medical University, 840 Shijo‐cho, Kashihara, Japan

PT04.46 Hypericum perforatum‐derived exosomes‐like nanovesicles‐based photosensitizer for photodynamic therapy in adipose tissues

Professor Jianbo Wu¹, Dr. Ziyu Li

¹Southwest Medical University, Luzhou, China

PT05.03 Characterising soluble TREM2 in extracellular vesicles derived from cultured human macrophages

Miss Drishya Mainali, Mr Anjie Ge, Dr Monokesh Sen, Miss Yvonne A. Candia, Dr. Claire Goldsbury, A/Prof. Laura Piccio

PT05.04 CRISPR/Cas9, genome editing and EVs: Exogenous bacterial Cas9 expression alters small EV secretion and their protein cargo in p53 dependent manner

Professor Suresh Mathivanan

PT05.05 Cytoplasmic DNA accumulation upon the inhibition of small extracellular vesicles secretion induces cGAS and ULBP1 activation in acute myeloid leukemia blasts

Dr. Jamal Ghanam, Dr. Venkatesh Kumar Chetty, Prof. Dr. Dirk Reinhardt, PD. Dr. Basant Kumar Thakur

PT05.06 Defining the parameters for sorting of RNA cargo into extracellular vesicles

Associate Professor Mona Batish, Graduate Student Ahmed Abdelgawad, Assistant Professor Vijay Parashar

PT05.07 Effects of electromagnetic fields on the release and content of extracellular vesicles

Hailong Wang, Research Associate YiHua Wang, Professor Gregory Worrell

PT05.10 Establishment of a method for mass production of extracellular vesicles based on microRNA regulation

Dr. Tomofumi Yamamoto, Dr. Hirotaka NIshimura, Dr. Noritaka Hashii, Dr. Akiko Ishii‐Watabe, Dr. Yusuke Yamamoto, Prof. Takahiro Ochiya

PT05.12 Exploiting the human peptidome for discovery of EV release‐modulating agents

Dr. Ruediger Gross, Ms. Hanna Reßin, Mr. Nico Preißing, Dr. Ludger Ständker, Prof. Dr. Jan Münch

PT05.13 Gene copy numbers dictate extracellular vesicle cargo

Sumeet Poudel, Jerilyn Izac, Zhiyong He, Lili Wang

PT05.16 Identification of cis‐regulatory elements involved in exosomal cargo sorting

Phd Student Gizaw Gebeyehu, Research Professor Tibor Rauch, Associate Professor Marianna Pap, Dr Geza Makkai, Dr Tibor Janosi

PT05.17 Lipid nanoparticles (LNPs) alter transcriptomic contents of extracellular vesicles (EVs) leading to functional LNP‐mRNA repackaging into EVs

Benyapa Tangruksa^{1. Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg,Gothenburg 41346, Sweden 2. Systems Biology Research Center, School of Bioscience, University of Skövde, Skövde SE‐54128, Sweden}, Doctor Muhammad Nawaz^{1. Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg 41346,Sweden}, Adjunct Professor Sepideh Heydarkhan‐Hagvall^{2. Systems Biology Research Center, School of Bioscience, University of Skövde, SkövdeSE‐54128, Sweden}, Professor Jane Synnergren^{2. Systems Biology Research Center, School of Bioscience, University of Skövde, Skövde SE‐54128, Sweden3. Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg 41345, Sweden}, Associate Professor Hadi Valadi^{1. Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg,Gothenburg 41346, Sweden}

PT05.18 Metalloprotease ADAM10 in secretion, composition, and targeting of extracellular vesicles

MSc Christopher C. Reimann, MSc Feizhi Song, Dr. rer. nat. Dipl. Hum.‐Biol. Hermann C. Altmeppen, Dr. Lesley Cheng, Prof. Markus Glatzel, Prof. Marina Mikhaylova, Prof. Andrew F. Hill

PT05.20 The “torn bag mechanism” of small extracellular vesicle release through rupture of the limiting membrane of en bloc released large EVs

Dr. Tamás Visnovitz, Ms Dorina Lenzinger, Ms Anna Koncz, Ms Tünde Bárkai, Dr. Krisztina V Vukman, Ms Alicia Galinsoga, Dr. Krisztina Németh, Ms Kelsey Fletcher, Dr. Péter Lőrincz, Dr. Gábor Valcz, Prof. Edit I Buzás

PT05.23 β‐catenin regulates the biogenesis and secretion of small extracellular vesicles by modulating senescence

Dr Taeyoung Kang

PT05.24 The RNA‐Binding Protein NPM1 Is a Component of Exosomal Machinery Controlling mRNA Sorting through Binding to Specific RNA Motif

Student Kaixiang Zhang^1,2, Ying Zhang¹, Hang Yin^1,2

¹School of Pharmaceutical Sciences, Tsinghua University, Beijing, China, ²Tsinghua‐Peking Center for Life Sciences, Tsinghua University, Beijing, China

PT05.25 Glycosylphosphatidylinositol‐anchored Proteins Promote Cellular Membrane Trafficking and Enhance Extracellular Vesicles Production

Scientist Tong Zhao¹, Associate Scientist Wei Zhao¹, Associate Researcher Shengya Xu¹, Associate Researcher Moxuan Yang¹

¹TheraXyte Bioscience, Beijing, China

PT05.26 Immunomodulatory effect of AFSC conditioned media generated using Micregen's cell‐free secretome technology platform linked with neural protection

Dr Robert Mitchell¹, Mr Andrew Parnell¹, Dr Ben Mellows¹, Professor Ketan Patel¹, Dr Steve Ray¹

¹Micregen Limited, Reading, United Kingdom

Extracellular vesicles in human body fluids compete with virus particles for binding of phosphatidylserine receptors to prevent infection and transmission

Dr. Ruediger Gross, Hanna Reßin, Pascal von Maltitz, Dan Albers, Laura Schneider, Hanna Bley, Markus Hoffmann, Mirco Cortese, Dhanu Gupta, Miriam Deniz, Jae‐Yeon Choi, Jenny Jansen, Christian Preußer, Kai Seehafer, Stefan Pöhlmann, Dennis R Voelker, Christiane Goffiniet, Elke Pogge‐von Strandmann, Uwe Bunz, Ralf Bartenschlager, Samir El Andaloussi, Konstantin MJ Sparrer, Eva Herker, Stephan Becker, Frank Kirchhoff, Jan Münch, Janis A Müller

Plenary Session 1 and Featured Abstract, Plenary 1, May 9, 2024, 9:00 AM ‐ 10:15 AM

Introduction

Several human viral pathogens, including flaviviruses Zika and Dengue virus, are present in human body fluids, yet rarely transmitted sexually or orally. A possible explanation are extracellular vesicles (EVs) in sem*n and saliva which have been shown to inhibit flavivirus infection. The exact mechanism, whether this activity occurs in EVs from different sources, and which other viruses are affected are unclear.

We here propose that the antiviral activity of these EVs involves competitive binding of phosphatidylserine (PS) receptors. PS‐receptors are commonly used as EV attachment factors and are sensors of apoptosis, a phenomenon exploited by several virus families in a process termed “viral apoptotic mimicry”, leading to uptake of virions and subsequent infection.

Methods

To study the molecular mechanism of antiviral EVs, we purified EVs from human sem*n, saliva, urine, breast milk, and blood using tangential flow filtration and size‐exclusion chromatography. We characterized them biophysically by NTA and quantified the abundance (shotgun lipidomics) and exposure (bead‐assisted and nano‐flow cytometry) of PS. The antiviral activity was tested against a diverse panel of apoptotic mimicry viruses and viruses known to use other modes of attachment. To elucidate the specificity of PS on antiviral activity, we modified the exposed lipid leaflet enzymatically and imaged their interaction with PS receptors using confocal microscopy.

Results

Using Zika virus as a model for viral apoptotic mimicry, we show that EVs from all used sources inhibit infection of cell lines and primary tissues, while synthetic control vesicles lacking PS are inactive. Lipidomics and flow cytometry using lactadherin revealed that antiviral EVs are rich in surface‐exposed PS. PS‐exposing EVs prevented virion attachment and infection by competing with virions for cellular PS‐receptors. This antiviral activity was reduced upon enzymatic removal of lipid headgroups and restored by cyclodextrin‐mediated PS‐donation. The antiviral activity extended to other viruses known to use PS receptors, including Dengue, West‐Nile‐, Chikungunya‐, Ebola and vesicular stomatitis viruses, but not viruses using other receptors such as SARS‐CoV‐2, HIV‐1 or herpesviruses.

Conclusions

Overall, our results identify PS‐exposure as an EV‐based innate defense mechanism that may play a key role in restricting viral transmission via body fluids.

Machine learning models detect blood ‘fingerprints’ for accurate glioblastoma tumour surveillance

Dr Susannah Hallal, Dr Ágota Tűzesi, Dr Abhishek Vijayan, Dr Laveniya Satgunaseelan, Associate Professor Hao‐Wen Sim, Associate Professor Brindha Shivalingam, Associate Professor Michael Buckland, Associate Professor Fatemeh Vafaee, Dr Kimberley Alexander

Plenary Session 2 and Featured Abstract, Plenary 1, May 10, 2024, 9:00 AM ‐ 10:10 AM

Introduction: Glioblastoma (GBM) is the most common and aggressive adult primary brain tumour, and patients face distressingly short survival outcomes of only 14 months. GBM tumours often recur quickly, acquiring more aggressive and treatment‐resistant features that cannot be accurately detected with currently available monitoring methods. The development of circulating biomarkers that offer early and precise indications of GBM recurrence holds immense potential for enhancing patient care. To address this critical need, we have developed a novel, extracellular vesicle (EV)‐based multianalyte liquid biopsy strategy that detects sensitive biomarker signatures or ‘blood fingerprints’ for routine assessment of GBM tumour activity and treatment response.

Methods: Using size‐exclusion chromatography, EVs were isolated from 104 serial plasma specimens (1 mL) from 50 patients diagnosed with GBM IDH‐wildtype. The serial plasma samples were collected at three clinical timepoints: before (Pre‐OP, n=27) and after (Post‐OP, n=49) first surgeries, and upon pathologically‐confirmed recurrence (REC, n=28). Captured plasma‐EV populations were characterised by nanoparticle tracking analysis, cryo‐transmission electron microscopy and mass spectrometry. The plasma‐EVs were analysed using established, complementary EV proteomics and small‐RNA (sRNA) sequencing platforms. Filtered and normalised proteomics and sRNA datasets were passed through a cross‐validation pipeline (30 iterations; 80%‐train:20%‐test) using multiple feature selection methods and classification models. The best‐performing protein and sRNA candidate biomarkers were identified separately. Multianalyte models were generated using an ensemble stacking machine learning method that combined the performance power of the best performing protein and sRNA biomarkers.

Results: Our pipeline generated 4117 proteins and 272 sRNA transcripts common to all plasma specimens. We described three multianalyte (protein and sRNA) blood ‘fingerprints’ that comprised the best‐performing biomarkers for classifying patients according to GBM tumour burden (Pre‐OP vs Post‐OP), recurrence (Post‐OP vs REC) and treatment resistance (Pre‐OP vs REC). All models had a training performance of 100% and cross‐validation test accuracy rates that ranged from 89.9‐93.4%.

Conclusions: Our EV‐based liquid biopsy strategy holds promise for accurately monitoring GBM patients and distinguishing recurrence from treatment‐effects. Independent validation of our blood fingerprints is underway utilising plasma specimens and clinicopathologic data captured from two external GBM cohorts, the VERTU study (trial ACTRN12615000407594) and GlioNET observational study.

Barcoding of small extracellular vesicles with CRISPR‐gRNA enables high‐throughput, subpopulation‐specific analysis of their release regulators

Prof. Dr. Ryosuke Kojima, Mr. Koki Kunitake, Professor Tadahaya Mizuno, Professor Yasuteru Urano

Plenary Session 3 and Featured Abstract, Plenary 1, May 11, 2024, 9:00 AM ‐ 10:05 AM

Small Extracellular Vesicles (sEVs) are important mediators of cell‐to‐cell communication in both physiological and pathological contexts, including cancer metastasis. These facts highlight the potential of sEV biogenesis and release processes (“release” processes hereafter) as novel therapeutic targets. Furthermore, sEVs are also attracting attention as highly biocompatible delivery vesicles, and therefore methods to control/enhance their production are of great interest for biotechnological applications. Despite the importance of sEV release processes, a comprehensive understanding of their regulation has been difficult with conventional low‐throughput assays using small‐molecule inhibitors or siRNAs in separate wells.

Here we report a novel high‐throughput pooled screening system to identify key players of sEV release processes. We actively incorporated guide RNA (gRNA) for Cas9 into sEVs through the interaction of gRNA and dead Cas9 (dCas9) fused with an sEV marker in a pooled CRISPR screening format. This allows sEV‐loaded gRNA to work as a “barcode” linking each sEV to the perturbation of gene expression in its originating cell. Quantification of the composition of barcode gRNA in both sEVs and cells allows high‐throughput, genome‐wide exploration of genes involved in sEV release while canceling out the effects on cellular activities (e.g., proliferation, barcode transcription). We call this assay platform CRISPR‐assisted individually barcoded sEV‐based release regulator (CIBER) screening. CIBER screening using multiple sEV markers in combination with bioinformatic analyses revealed both known and previously unknown factors controlling sEV release processes, uncovering different effects of V‐type ATPases, mitochondrial electron transport, and the cell cycle on the release of CD63+ and CD9+ sEVs. We believe this work provides a basis for detailed studies on the biogenesis, release, and heterogeneity of sEVs. (Kunitake, Kojima* et al, bioRxiv 2023, doi.org/10.1101/2023.09.28.559700)

In vivo visualization of endothelial cell‐derived extracellular vesicle formation in steady state and malignant conditions

Dr Georgia Atkin‐Smith, Jascinta Santavanond, Amanda Light, Joel Rimes, Andre Samson, Jeremy Er, Joy Liu, Darryl Johnson, Melanie Le Page, Pradeep Rajasekhar, Raymond Yip, Niall Geoghegan, Kelly Rogers, Catherine Chang, Vanessa Bryant, Mai Margetts, Cristina Keightley, Trevor Kilpatrick, Michele Binder, Sharon Tran, Erinna Lee, Doug Fairlie, Dilara Ozkocak, Andrew Wei, Edwin Hawkins, Ivan Poon

Plenary Session 4 and Featured Abstract, Plenary 1, May 12, 2024, 9:00 AM ‐ 10:00 AM

Introduction:

Endothelial cells are integral components of all vasculature within complex organisms and are vital in diverse processes such as embryonic development, angiogenesis and wound healing. Despite their low turnover, endothelial cells undergo rapid growth and remodelling under both homeostatic and stressed conditions. Understanding how these processes are regulated, and how endothelial cells mediate communication throughout the organism remains a fundamental question in cell biology. The generation of extracellular vesicles (EVs) has emerged as a key mechanism for cell‐to‐cell communication, as well as for the removal of cellular waste. However, the formation, function and clearance of large, endothelial cell‐derived EVs in vivo is yet to be fully defined.

Methods:

We used a series of world class imaging techniques to study the formation of large endothelial cell‐derived EVs in model organisms. This includes 4D intravital microscopy of the bone marrow calvarium of live, endothelial cell reporter mice, as well as dual confocal multiphoton microscopy of cleared tissue. To complement our mouse studies, we also performed extensive time lapse microscopy on endothelial reporter zebrafish. We paired our elegant imaging approaches with comprehensive traditional and imaging flow cytometry analysis to examine the formation, contents and clearance of EVs under homeostatic settings and under the stress of various blood cancer models.

Results:

For the first time, we captured the formation of large endothelial cell‐derived EVs in vivo, and acquired 4D information of this process including spatial and temporal insights. We identified these EVs to be a population of large, mitochondria‐rich EVs present under homeostatic settings, and identified in murine, zebrafish and human samples. We demonstrate that these EVs can interact with and be efferocytosed by various immune cell populations as a potential clearance mechanism. Excitingly, we show that elevated levels of circulating large, endothelial cell‐derived EVs correlated with the degradation of the bone marrow vasculature caused by acute myeloid leukemia (AML).

Summary/Conclusions:

Collectively, our study provides in vivo spatio‐temporal characterization of EV formation in the murine vasculature, and suggests that circulating, large endothelial cell‐derived EVs may provide a snapshot of endothelial cell health that reflects the extent of tissue damage at distal sites.

OT01.O02. miR‐151a‐5p cargo in neuron‐derived extracellular vesicles is a biomarker and mediator of antidepressant treatment response

PhD Dariusz Żurawek, PhD Alice Morgunova, PhD Laura Fiori, M.S. Jennie Yang, PhD Claudia Belliveau, M.S. Pascale Ibrahim, M.S. Jean Francois Théroux, M.S. Ryan Denniston, Prof. Sidney H. Kennedy, Prof. Raymond W. Lam, Prof. Roumen Milev, PhD Susan Rotzinger, MD Claudio N. Soares, MD Valerie H. Taylor, MD Rudolf Uher, PhD Jane A. Foster, MD Benicio N. Frey, PhD Cecilia Flores, PhD Corina Nagy, MD Gustavo Turecki

Introductory Talk and Oral Session: OT01 Towards the Clinic, Plenary 1, May 9, 2024, 10:40 AM ‐ 12:00 PM

Introduction

The difficulty of studying the biochemical processes of the living brain has been a limitation in advancing our understanding of the molecular mechanisms of antidepressant treatment response. Recently, it has been reported that various brain cell types, including neurons, release extracellular vesicles with specific molecular cargo that can cross the blood‐brain barrier. Detecting these vesicles in the periphery may serve as non‐invasive biomarkers, providing insights into the true molecular state of the brain and aiding our understanding of antidepressant treatment mediators.

Methods

We analyzed 430 human plasma samples from the CAN‐BIND‐1 clinical trial (clinicaltrials.gov, NCT01655706). Informed consent was obtained from participants and the trial has an ethical approval. The samples were collected before and after an 8‐week escitalopram treatment from depressed patients, distinguishing between responders and non‐responders to the treatment, and untreated healthy controls. Neuron‐derived extracellular vesicles (NEVs) were isolated from plasma using size exclusion chromatography following by immunoprecipitation against SNAP25, a protein exclusively expressed in neurons and found on the surface of NEVs. Presence of EV markers and potential contamination in NEV samples were tested by Western blot. Transmission electron microscopy and nanoparticle tracking analysis were used to determine the morphology and size distribution of NEVs. Small RNA‐seq profiling followed by RT‐qPCR validation were used to determine miRNA cargo in NEVs.

Results

Before treatment, depressed patients had lower miR‐151a‐5p levels in NEVs than controls. However, these levels significantly increased over time only in the group of depressed patients who responded to antidepressant treatment. miR‐151a‐5p levels in NEVs demonstrated good predictive value in discriminating between responders and non‐responders. In vitro and in silico experiments revealed that miR‐151a‐5p negatively regulates a gene set enriched in the prefrontal cortex, responsible for regulating glutamatergic signaling. Engineered NEVs enriched with miR‐151a‐5p and delivered to prefrontal cortex displayed antidepressant properties in vivo and effectively ameliorated the behavioral deficits induced by chronic social defeat stress in mice. Animal studies were approved by an appropriate ethical committee.

Conclusion

miR‐151a‐5p cargo in NEVs may be a molecular mediator of effective antidepressant treatment response.

OT01.O03. First‐in‐human clinical trial of allogeneic platelet extracellular vesicles as a potential therapeutic for chronic wound healing

Dr. Jancy Johnson, Dr. Gregor Lichtfuss

Introductory Talk and Oral Session: OT01 Towards the Clinic, Plenary 1, May 9, 2024, 10:40 AM ‐ 12:00 PM

Introduction:

Chronic wounds are damaged tissue that stay unhealed for longer than 4 weeks and are currently lacking effective treatment methods. In vitro and animal studies have shown that the release of growth factors and nucleic acids by activated platelets through extracellular vesicles (EVs) are pivotal for healthy wound healing. Hence, platelet‐derived EVs (pEVs) could represent a next‐generation therapeutic for the treatment of chronic wounds.

However clinical assessment of pEV‐based therapeutics has been hampered to‐date by challenges in scalable and reproducible EV manufacturing. This study aimed to address these gaps by isolating clinical‐grade pEVs using a scalable, proprietary chromatography‐based method, evaluating their functional efficacy in cell‐based assays and lastly, assessing their safety upon administration in humans in a Phase I clinical trial.

Methods:

pEVs were isolated using Ligand Exosome Affinity Purification (Exopharm) and characterized for particle size, count and morphology. In vitro assays were performed to assess the effect of pEVs on cellular processes important for wound healing. Lastly, a Phase I Clinical trial approved by the Australian Red Cross Lifeblood was conducted in healthy volunteer adults to assess safety of pEV administration following a skin punch biopsy (n = 11).

Results:

Clinical‐grade pEVs were found to adhere to MISEV guidelines, ranging between 65‐200nm in size, possessing a lipid bilayer and positive for EV proteins such as ALIX and Syntenin. pEVs significantly improved cellular functions such as dermal fibroblast proliferation and migration in vitro suggesting a potential regenerative function of pEVs after injury. Sterility and endotoxin testing revealed that the pEVs were suitable for administration for clinical trials.

In humans, a single dose of pEVs (300 µg mL‐1) was injected, following a skin punch biopsy. A mean healing time of 22.8 ± 8.7 days was recorded for pEV and placebo‐treated groups. Importantly, pEV treatment was found to be safe and well‐tolerated with no adverse events recorded.

Conclusion:

These results demonstrated for the first time, that allogeneic pEVs can be manufactured under clean‐room conditions, at clinical scale, and are safe when administered to humans. These findings also support future studies that assess efficacy of pEV‐therapeutics in patients with disrupted wound healing.

OT01.O04. Navigating the regulatory and commercial challenges of translating extracellular vesicle‐based biomarkers into clinical practice

Dr Olasehinde Olusanya

Introductory Talk and Oral Session: OT01 Towards the Clinic, Plenary 1, May 9, 2024, 10:40 AM ‐ 12:00 PM

Method: This project involved the formation of a team including academic experts with varied backgrounds in the field of extracellular vesicles (EVs). The primary objective of the team was to investigate the potential of biomarkers derived from EVs for the purpose of medicine development. The research team engaged in a comprehensive investigation of the available literature and executed a series of tests to ascertain and assess the prevailing obstacles and prospects associated with the regulation and commercialization of biomarkers derived from extracellular vesicles (EVs).

Results: Based on the results of our research, we suggest that the extracellular vesicle (EV) community should prioritize the advancement of therapeutic applications grounded on empirical evidence. Additionally, efforts should be made to modify current regulatory criteria to encompass biomarkers derived from EVs. Furthermore, it is imperative to make endeavours in the identification of commercially feasible uses of extracellular vesicle (EV)‐based biomarkers and to produce comprehensive, integrated data at the systems level to enhance their utilization in clinical decision‐making processes. To comprehensively comprehend the practical consequences of utilizing EV‐based biomarkers in the realms of diagnosis and therapy, it is imperative to engage in collaborative endeavours with clinical organizations. These measures have the potential to facilitate the integration of EV‐based biomarker development with their application in clinical settings.

Summary/Conclusion:

In summary, by the resolution of regulatory and commercialization obstacles, as well as the promotion of cooperation between the extracellular vesicle (EV) community and clinical institutions, we can facilitate the effective incorporation of EV‐derived biomarkers into clinical settings. This would not only improve disease diagnosis and therapeutic evaluation, but also expedite the advancement of novel treatment modalities. Further investigation is warranted in this domain to effectively harness the potential of extracellular vesicle (EV)‐based biomarkers in the context of pharmaceutical development, with the ultimate goal of enhancing patient outcomes.

Keywords: Extracellular vesicles (EVs), EV‐based biomarkers, Regulation, Clinical applications Biomarker development, Diagnostic tools

OT01.OWP01. Serum extracellular vesicle profiling to determine extracorporeal photopheresis response in graft versus host disease

Miss Kimberly Schell, Doctor Aisling Flinn, Professor Matthew Collin, Professor Andrew Gennery, Doctor Rachel Crossland

Introductory Talk and Oral Session: OT01 Towards the Clinic, Plenary 1, May 9, 2024, 10:40 AM ‐ 12:00 PM

Introduction

Graft versus Host disease (GvHD) is a serious complication of allogeneic haematopoietic stem cell transplantation (HSCT), affecting 40‐70% of patients and leading to mortality in 40‐60%. Standard steroid treatment can cause adverse effects, and lead to steroid refractory disease. Extracorporeal photopheresis (ECP) therapy is a safe alternative, though the mechanism of action is unknown, and biomarkers to determine ECP response are lacking. This study aims to investigate the role of serum extracellular vesicles (EVs) during ECP therapy for GvHD.

Methods

Serum and peripheral blood mononuclear cell (PBMC) samples were acquired from n=12 paediatric ECP patients before each treatment cycle with appropriate ethical approval and informed consent. EVs were isolated and characterised from four sequential timepoints (TP) by precipitation for n=6 complete (CR) and n=6 partial/non‐responders (PR). EV microRNA cargo and surface marker expression profiling was performed using NanoString, MACSPlex and ImageStream, respectively. T Cell Receptors (TCRs) will be assessed via the TCR diversity nCounter panel, and serum cytokines via MSD assays in PBMC and serum, respectively.

Results

EV modal size was significantly larger for patients who reached CR vs. PR, and this was most pronounced after commencement of ECP. Analysing EV microRNA composition showed 3 principal component analysis (PCA) clusters; PR and two separate clusters of mostly CR. There was no treatment cycle‐based clustering. Eight microRNAs were significantly differentially expressed in CR vs. PR patients; hsa‐miR‐23a‐3p was downregulated while hsa‐miR‐144‐3p, hsa‐miR‐548ar‐3p, hsa‐miR‐378e, hsa‐miR‐21‐5p, hsa‐let‐7i‐5p, hsa‐miR‐579‐3p, and hsa‐miR‐1283 were upregulated (p<0.05). MicroRNA target analysis showed enrichment of GvHD and/or ECP‐related pathways, such as allograft rejection, interleukin, and TLR‐signalling. The most highly expressed EV‐microRNA post‐ECP in all patients was anti‐inflammatory miR‐451a. Focusing on EV‐markers, there was a significant interaction between ECP cycle and response for CD40, CD42a, CD56, CD41b, CD31, and CD49e, while HLA‐DPDQDR, CD14, CD20 and CD44 were significantly downregulated post‐ECP, and CD8, CD44, CD142 and CD146 were expressed at a significantly higher level in PR vs. CR.

Conclusions

EVs show potential for biomarkers of ECP response, and their molecular cargo and surface markers may help elucidate the mechanisms of ECP action.

OT01.OWP02. Cracking the code: Understanding oncogenic small EVs in pancreatic cancer diagnostic landscape

Ms Arunima Panda, Mrs Ilaria Casari, Dr Abir Halder, Dr Walid Abu Shawish, Dr Danielle Dye, Prof Krish Ragunath, A/Prof David Greening, Prof Marco Falasca

Introductory Talk and Oral Session: OT01 Towards the Clinic, Plenary 1, May 9, 2024, 10:40 AM ‐ 12:00 PM

Introduction

Pancreatic cancer (PC) is one the most aggressive malignancies marked by rising incidence and high fatality rates. PC is characterised by a lack of early symptoms, delayed diagnosis, and resistance to conventional chemotherapy, making it challenging. The PC marker, Ca19.9, lacks efficiency and efficacy. Consequently, early detection and effective treatment options are crucial in confronting this challenge. Small extracellular vesicles (sEVs) are employed by cancer cells for intracellular communication and to promote cancer progression. Identification and molecular analysis of the distinctive molecules transported by sEVs associated with PC hold the potential to serve as biomarkers, offering molecular signatures for PC diagnosis.

Methods

We performed characterisation of sEVs from PC and patient‐derived xenograft (PDX) cell lines based on phenotypical, physiological, and functional characteristics. To support our functional results, we performed a CRISPR knockdown. Plasma samples were collected from 30 suspected PC patients and 14 disease‐free participants. Ethical approval was obtained for the use of human blood samples from PC patients and age‐matched healthy participants (Royal Perth Hospital RGS4208).

Results

Our research has identified specific oncoproteins only found in sEVs derived from PC cells. Interestingly, sEVs derived from human PC cell lines and PDX cells contain unique cargo, including metastatic regulatory factors and pivotal signalling molecules crucial for PC progression. Furthermore, our proteomic analysis of sEVs from human PC cell lines and PDX cell lines has shown higher expression of critical enzymes involved in inositol synthesis and metabolism, including the transporter SLC5A3. SLC5A3 plays a role in inositol transport and the generation of essential signalling molecules like phosphatidylinositol 4,5‐bisphosphate. SLC5A3 can play the role of a prognostic factor for PC. Our findings have been validated in sEVs from PC, PDX cell lines, knockdown of PDX cell lines and plasma samples from PC and healthy participants.

Conclusion

Oncoproteins such as SLC5A3 can be potential PC biomarkers and improve the diagnostic landscape of PC diagnosis.

Funding:

This research is funded by PanKind, The Australian Pancreatic Cancer Foundation (www.pankind.org.au) and the Royal Perth Springboard Plus Grant.

OT01.OWP03. Hydrogel loaded with microalgae‐derived extracellular vesicles for preventing skin ultraviolet damage

Miss Jiarong Cui, Prof. Min Zhou

Introductory Talk and Oral Session: OT01 Towards the Clinic, Plenary 1, May 9, 2024, 10:40 AM ‐ 12:00 PM

Introduction

Spirulina platensis (S. platensis, SP), a naturally occurring edible microalgae, has been noticed for its numerous beneficial properties, including antioxidant, anti‐inflammation, and immunomodulatory effects. However, the challenges associated with its micron‐scale size resulting in difficult skin absorption, and potential for skin allergies hinder its application in the treatment of skin diseases. As nanoscale phospholipid bilayer vesicles, SP extracellular vesicles (SP‐EVs) contain components from the original cells, which is expected to inherit the application value of SP and be used in the skin diseases. Consequently, this research aims to isolate extracellular vesicles from the culture supernatants of SP and examine their potential to prevent skin damage caused by ultraviolet radiation.

Methods

In this study, SP‐EVs were isolated using differential ultracentrifugation, and their structure and size were determined through the utilization of a transmission electron microscope, atomic force microscope, and dynamic light scattering. To enhance the adhesion of this system, SP‐EVs were incorporated into a mixture of carboxymethyl chitosan (CMCS) and sodium alginate (SA), and subsequently cross‐linked with genipin to form a hydrogel complex. To ensure optimal biosafety and adhesion properties, hydrogels with a final concentration of 0.04% genipin were selected from various concentration ratios for subsequent cell and animal experiments.

Results

The results of our study indicate that treatment with SP‐EVs effectively mitigated the generation of reactive oxygen species, DNA damage, and mitochondrial alterations in keratinocytes stimulated with H2O2 or exposed to UV irradiation. Furthermore, SP‐EVs therapy demonstrated enhanced antioxidant capacities, as evidenced by increased activity of glutathione peroxidase and superoxide dismutase, along with a decrease in malondialdehyde concentration, thus protecting against oxidative stress‐induced skin damage. Additionally, in both cellular and animal models, SP‐EVs treatment exhibited anti‐inflammatory properties in cells and tissues.

Conclusion

In brief, we have successfully fabricated a hydrogel with exceptional adhesion properties by incorporating SP‐EVs into the biocompatible CMCS/SA substrates and subsequently cross‐linking them with genipin. Considering the remarkable stability, absence of adverse toxicity, and absence of anaphylactic reactions, SP‐EVs exhibit promising potential as a dermatological nanotherapeutic agent for mitigating skin UV damage via their antioxidant, anti‐inflammatory, and other therapeutic effects.

OT02.O02. Multi‐omic insights into extracellular vesicles mediating drug resistance in leishmania parasites

Associate Professor Christopher Fernandez‐Prada

Introductory Talk and Oral Session: OT02 Pathogen Host Response, Eureka, May 9, 2024, 10:40 AM ‐ 12:00 PM

Introduction: Leishmaniasis, a significant tropical disease caused by Leishmania protozoan parasites, presents challenges due to emerging drug‐resistant strains. Understanding the role of extracellular vesicles (EVs) in propagating drug resistance is crucial for the development of effective treatments and potential vaccines.

Methods: Our comprehensive study combined proteomics, lipidomics, and genomics to analyze both Leishmania parasites and their EVs. The EVs were isolated from Leishmania infantum promastigotes using a protocol applicable to various Leishmania spp. strains. This involved multiple centrifugation steps, filtering, and ultracentrifugation to retrieve and purify small EVs. DNA was extracted from these EVs using the DNeasy Blood and Tissue Kit and sequenced using an Illumina sequencer. Proteomic analysis involved mass spectrometry to identify and quantify proteins in both parasites and EVs. Lipidomic profiling was conducted using high‐performance liquid chromatography coupled with mass spectrometry (HPLC‐MS), focusing on the lipid composition of EVs from drug‐resistant strains. Genomic analysis included Next‐Generation Sequencing (NGS) and PCR assays to examine the DNA content of EVs and assess their role in horizontal gene transfer (HGT).

Results: Our findings revealed significant changes in the EVs of drug‐resistant parasites, including variations in morphology, size, and distribution. The proteomic analysis identified a diverse array of proteins, including virulence factors and proteins encoded by drug‐resistance genes. Lipidomics revealed a notable shift in the lipid composition of EVs from drug‐resistant strains. Genomic studies confirmed the enrichment of circular amplicons carrying drug‐resistance genes in EVs. These EVs were shown to transfer drug‐resistance traits to naïve parasites, altering their drug sensitivity, enhancing their growth, and improving their control of reactive oxygen species.

Summary/Conclusion: This research provides the first evidence of EVs as an efficient platform for HGT in eukaryotic parasites, facilitating the rapid transmission of drug‐resistance genes and enhancing the global fitness of recipient parasites. Our findings highlight the critical role of EVs in the dissemination of drug resistance in Leishmania, offering novel insights for therapeutic strategies targeting these vesicles to combat drug‐resistant strains.

OT02.O03. Common mechanisms of protection against pathogenic gram‐negative bacteria by host‐derived sEV

Dr. Adam Fleming, Mr. Graham Matulis, Ms. Heather Hobbs, Dr. Valentin Giroux, Mr. Hunter Mason, Dr. Weidong Zhou, Dr. Valerie Calvert, Dr. Nitin Agrawal, Professor Emanuel Petricoin, Dr. Rekha Panchal, Professor Igor Almeida, Dr. Sina Bavari, Professor Ramin Hakami

Introductory Talk and Oral Session: OT02 Pathogen Host Response, Eureka, May 9, 2024, 10:40 AM ‐ 12:00 PM

Introduction:

Gram‐negative bacteria (GNB) have significant clinical importance in hospitals and are among the world's most significant public health problems due to their high resistance to antibiotics. We have used two well‐established model human pathogens, Yersinia pestis (Yp), and Burkholderia pseudomallei (Bp), to address the strong gap in knowledge regarding the molecular mechanisms by which host‐derived sEV help protect against pathogenic gram‐negative bacteria.

Methods:

sEV were purified from naïve U937 monocytes (EXu) and infected U937 (EXi) by differential centrifugation followed by density gradient purification, and characterized by SEM, confocal microscopy, NTA, marker analysis (CD63, TSG101, Flotillin‐1), and LC‐MS/MS analysis to profile sEV content and check for presence of LPS. Immune responses of naïve U937 cells and response mechanisms were analyzed following treatment with equivalent amounts of EXi or EXu (as control). These included macrophage differentiation assays, multiplex measurements of inflammatory cytokines both in vitro and in vivo, bacterial clearance assays, quantitative reverse protein microarray (RPMA) analysis of 173 host signaling proteins, and siRNA knockdown of signaling proteins identified by RPMA and of the EXi‐induced cytokines in recipient cells. For all assays, at least four biological replicates were performed.

Results: Our results demonstrate that EXi induce activation of p38 kinase in recipient naïve monocytes that leads to their differentiation to macrophages and significant release of IL‐6, IL‐8 and IL‐10 cytokines, phenotypes that are intriguingly similar to when the cells are infected with the bacteria. The IL‐6 cytokine release in turn primes the recipient immune cells, leading to a dramatic increase in their capacity to clear the bacteria if they become infected. MS analysis showed lack of LPS in EXi, and also demonstrated the presence of specific bacterial proteins that have antigenic properties.

Summary/Conclusion: We have identified some of the main molecular mechanisms by which host‐derived EXi assist the host in clearing infection caused by gram‐negative pathogens. EXi prime distant naïve monocytes through modulation of distinct pathways such as p38 to mount IL‐6 dependent immune responses, protecting the cells from a possible subsequent infection. These results provide a basis for development of novel prevention strategies against infection with gram‐negative bacterial pathogens.

OT02.O04. Parasite extracellular vesicles selectively target human monocytes to induce T‐cell anergy and amelioration of DSS‐induced colitis in mice

Dr Anne Borup, Dr Farouq Mohammad Sharifpour, Dr Litten Sørensen Rossen, Dr Bradley Whitehead, MSc Anders Toftegaard Boysen, Dr Paul Giacomin, Mrs Kim Miles, Ms Maggie Veitch, Dr Andrea Ridolfi, Dr Marco Brucale, Dr Francesco Valle, Dr Lucia Paolini, Dr Paolo Bergese, Dr Alex Loukas, Professor Peter Nejsum

Introductory Talk and Oral Session: OT02 Pathogen Host Response, Eureka, May 9, 2024, 10:40 AM ‐ 12:00 PM

Introduction: Helminths cause chronic infection in the host through modulation of the host immune response. They suppress the pro‐inflammatory type 1 response in favour of a modulated type 2 response by releasing excretory/secretory products (ESPs). Extracellular vesicles (EVs) have been shown to be released with the ESPs; however, their immunomodulatory mechanism is not fully understood.

Methods: We isolated EVs from Ascaris ESPs using size exclusion chromatography (SEC) and characterized them using nanoparticle tracking analysis (NTA) and atomic force microscopy (AFM). Labelled EVs were generated in vivo, via incorporation of fluorescent lipid analogues during EV biogenesis, followed by flow cytometry and imaging flow cytometry to evaluate EV uptake in human PBMCs. Protease inhibition and PNGase were used further to understand the interaction between EVs and immune cells, while co‐cultures and cytokine release were used to evaluate T‐cell activation. A mouse model of inducible chemical colitis (dextran sodium sulfate) was used to assess the in vivo suppressive effects of Ascaris EVs.

Results: Using flow cytometry, we demonstrate that Ascaris EVs are primarily internalized in monocytes of human PBMCs. EVs induced a unique phenotype in the monocyte which was partly due to enzymatic activity by metalloproteinases. Enzymatic removal of N‐linked glycans from the surface of EVs did not alter their effect on the monocytes' expression profile. Ascaris EVs attenuated T‐cell activation in a monocyte dependent manner, and we demonstrate for the first time that these immunomodulatory effects are predominantly caused by EVs and not the EV‐depleted fractions. Lastly, we find that Ascaris EVs partly recover DSS induced colitis in mice as measured by weight gain/loss and clinical score.

Summary/Conclusion: We show that EVs from intestinal helminths can suppress the immune response in circulating human immune cells and ameliorate colitis in mice.

OT02.O05. Legionella pneumophila outer membrane vesicles promote macrophage survival while Legionella pneumophila induce inflammatory cell death pathways

Ms. ‐ Ayesha, Dr Franklin Wang Ngai Chow, Prof. Polly Hang‐mei LEUNG

Introductory Talk and Oral Session: OT02 Pathogen Host Response, Eureka, May 9, 2024, 10:40 AM ‐ 12:00 PM

Introduction: Legionella pneumophila is a gram‐negative bacterium and a respiratory pathogen associated with Legionellosis. Recent research has revealed that these bacteria release extracellular vesicles called outer membrane vesicles (OMVs) that contain effector molecules capable of triggering inflammatory responses in the host. However, there are insufficient comprehensive studies on the immunomodulatory capabilities of L. pneumophila OMVs, particularly when compared to the effect of L. pneumophila infection. To address this gap, we purified and characterized the L. pneumophila OMVs and explored their immunostimulatory effect on THP‐1‐derived macrophages in comparison with L. pneumophila infection

Methods: Highly purified OMVs were obtained from L. pneumophila using ultracentrifugation followed by density gradient ultracentrifugation. Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) were employed to prove the purity of vesicles. A human proteome profiler kit which contains 105 cytokines, growth factors, and chemokines was used for the analysis. The intensities of the resulting blots were measured using quick spot software and the resulting protein expression data was further analysed by IPA (Ingenuity pathway analysis).

Results: NTA and TEM revealed that L. pneumophila OMVs have diameter ranging between 100 and 250 nm. Human cytokine array analysis revealed that these OMVs greatly induced the expression of various cytokines, growth factors, and enzymes, such as IL‐1β, GM‐CSF, IL‐5, IL‐4, IL‐10, Growth hormone, FGF, DPPIV, and VCAM‐1 in THP‐1 derived macrophages when compared to L. pneumophila infection. Further IPA revealed that the immunostimulatory effects of L. pneumophila OMVs promote cell survival pathways while L. pneumophila infection induces macrophage cell death and inflammation pathways.

Conclusion: Our finding provides valuable insights into the role of OMVs as the key modulators of the immune system however, further research is necessary to understand their exact function in the pathogenesis of Legionnaires disease.

K E Y W O R D S

Cytokines, gram‐negative bacteria, inflammation, Legionella pneumophila.

OT03.O02. Extracellular vesicles as mediators of retinal homeostasis and immune modulation

Dr Yvette Wooff, Dr Adrian Cioanca, Miss Rakshanya Sekar, Associate Professor Riccardo Natoli

Introductory Talk and Oral Session: OT03 EVs in Tissue Function, Room 105‐106, May 9, 2024, 10:40 AM ‐ 12:00 PM

Introduction: Extracellular vesicles (EV) are membrane‐enclosed delivery vehicles, that function in cellular communication through the selective transfer of molecular cargo between cells. Since cell‐to‐cell communication is critical for tissue survival, dysregulation in EV communication is linked to the development of inflammatory and neurodegenerative diseases. Currently, little is known about retinal EV, due to technical difficulties in EV isolation. We therefore developed a novel protocol for the isolation of retinal EV, and quantified, characterised, and profiled the molecular contents of retinal EV in health and across degeneration.

Methods: Retinal EV were isolated from mouse and human retinas using papain digestion and ultracentrifugation. Retinal EV were characterised using nanotracking analysis, electron microscopy and western blot. Small RNA‐seq and tandem mass spectrometry was performed on retinal EV. To investigate the role of EV or EV‐miRNA on retinal homestasis, EV from healthy mouse retinas or EV‐abundant microRNA were supplemented into the degenerating retina via intravitreal injection. Electroretinography and optical coherence tomography were used to measure retinal function and morphology respectively following EV/miRNA administration, while TUNEL and IBA‐1+ immunohistochemistry was conducted on retinal cryosections to determine levels of cell death and inflammation.

Results: EV were found to alter in their concentration, size and molecular contents in degeneration compared to healthy controls. The top ten EV‐miRNA were found to make up ∼67% of the total retinal miRNA concentration, and along with upregulated differentially expressed EV proteins were identified to control inflammatory and cell survival pathways known to be heavily involved in retinal degenerations. Compared to controls, mice injected with healthy retinal EV had significantly higher retinal function, reduced inflammation and decreased photoreceptor cell death. Further, intravitreal administration of key EV miRNA protected the retina against photo‐oxidative damage‐induced retinal degeneration, ameliorating inflammation and cell death, and preserving retinal function.

Conclusions: Taken together, our data suggests that a loss of EV‐miRNA bioavailability is correlated to progressive retinal degeneration, while supplementation of healthy retinal EV or highly abundant EV‐miRNA can restore homeostatic communication pathways and slow the progression of retinal degeneration.

OT03.O03. Matrimeres: Cell‐secreted nanoscale mediators enabling systemic maintenance of tissue integrity and function

Dr. Koushik Debnath, Dr. Irfan Qayoom, Mr. Steven O'Donnell, Ms. Julia Ekiert, Ms. Can Wang, Mr. Mark Sanborn, Mr. Chang Liu, Ms. Ambar Rivera, Dr. Ik Sung Cho, Ms. Saiumamaheswari Saichellappa, Dr. Peter Toth, Prof. Dolly Mehta, Prof. Jalees Rehman, Prof. Xiaoping Du, Prof. Yu Gao, Jae‐Won Shin

Introductory Talk and Oral Session: OT03 EVs in Tissue Function, Room 105‐106, May 9, 2024, 10:40 AM ‐ 12:00 PM

Introduction: Rapid restoration of tissue barriers is essential for promoting regeneration post‐injury. However, the mechanism involved, especially when the extracellular matrix (ECM) remains compromised, is not well understood. Recent studies propose that nanoscale mediators secreted by cells play a role in transporting ECM molecules. However, these mediators consist of diverse subpopulations, including lipid membrane‐bound extracellular vesicles (EVs) and non‐vesicular extracellular particles (NVEPs). The specific subpopulation responsible for competently activating ECM signaling over a distance remains unclear. Here, we present the discovery of matrimeres as constitutive nanoscale mediators of tissue integrity and function.

Methods: We utilized the standard differential ultracentrifugation technique (EV‐TRACK ID EV150007) to isolate the crude nanoscale fraction secreted from mesenchymal stromal cells (MSCs). Subsequently, we employed immunoaffinity‐based approaches, nanoparticle tracking analysis, and conditional probability calculations in order to quantify nanoscale subpopulations based on CD63, fibronectin (FN), and Triton‐X sensitivity. Characterization of different nanoscale subpopulations involved various assays, including transmission electron microscopy and mass spec proteomics analysis. Following the isolation and characterization, we administered the fractionated or reconstituted nanoscale fractions to mice after inducing infectious or sterile inflammatory injury. Subsequent evaluations included the assessment of edema, vascular permeability, and tissue function.

Results: We define matrimeres as non‐vesicular nanoparticles released by cells, identifiable by a primary composition consisting of at least one matrix protein and DNA molecules serving as scaffolds. MSCs form matrimeres from FN and DNA within acidic intracellular compartments. Drawing inspiration from this natural process, we successfully reconstituted matrimeres using purified matrix proteins and DNA molecules. Matrimeres containing plasma FN circulate in the blood under normal conditions, but their levels significantly decrease during systemic inflammation in vivo. Administering exogenous matrimeres leads to a rapid restoration of vascular integrity and tissue function by actively reannealing endothelial cells post‐injury, and these matrimeres persist in the host tissue matrix.

Conclusions: We show that cells utilize a mechanism involving the assembly and systemic circulation of matrimeres to constitutively maintain tissue integrity. The ability to produce matrimeres at scale shows potential as a biologically inspired platform for tissue regeneration.

OT03.O04. Elucidation of the mechanisms of participation of mesenchymal stromal cells extracellular vesicles in the regulation of myofibroblasts differentiation on 2D and 3D models of fibrosis

Ms Anastasiya Tolstoluzhinskaya, Ms Natalia Basalova, Ms Anastasiya Efimenko

Introductory Talk and Oral Session: OT03 EVs in Tissue Function, Room 105‐106, May 9, 2024, 10:40 AM ‐ 12:00 PM

Introduction. The main effectors cells of fibrogenesis are myofibroblasts, that participate in formation and remodeling of the fibrotic unit named fibroblastic focus, consisting of specific extracellular matrix (ECM). Accumulating evidence indicates that mesenchymal stromal cells could inhibit the fibrosis development mainly by regulating the myofibroblast pool through secreted extracellular vesicles (EV‐MSCs) and specific microRNAs within EV‐MSCs. We explored the molecular mechanisms of the effect of EV‐MSCs and selected microRNAs as their cargo on myofibroblast transdifferentiation during fibrogenesis.

Methods. EV‐MSC fraction were isolated by ultrafiltration from MSC conditioned medium and characterized according to MISEV‐2018 recommendations. We added EV‐MSCs to the previously developed 2D and 3D in vitro models of fibrosis. A 2D model was a culture of myofibroblasts differentiated under the influence of the transforming growth factor β (TGFb). 3D model was created using a decellularized spheroid ECM surrounded by myofibroblasts, which imitated the fibroblastic focus. We studied the contribution of selected microRNAs transferred within EV‐MSCs and associated with fibrosis by transfection of EV‐MSCs with synthetic microRNA inhibitors (Qiagen).

Results. The addition of EV‐MSCs to 2D fibrosis model decreased the acquisition of myofibroblasts’ features and fibrotic ECM markers such as collagen type I and EDA‐fibronectin. Using 3D model, we showed that addition of EV‐MSCs resulted in a reduction of myofibroblasts features and the destruction of the structure of fibroblastic focus. We conducted an inhibitory analysis and showed that the antifibrotic effect of EV‐MSCs was associated with the presence of microRNA‐129, ‐29c and ‐92a in its composition.

Conclusions. Using 2D and 3D in vitro model simulating fibroblastic focus, we revealed that microRNAs‐129, ‐29c and ‐92a secreted by MSC within EV‐MSCs could significantly contribute to the regulation of myofibroblast transdifferentiation. These data further can be used to consider EV‐MSCs and specific antifibrotic microRNAs as promising candidates for the treatment of fibrosis. The study was supported by the State Assignment of Lomonosov MSU.

OT03.O05. Mechanical overload‐induced extracellular mitochondria and particles release from tendon cells leads to inflammation in tendinopathy

Dr. Ziming Chen, Mengyuan Li, Peilin Chen, Andrew Tai, Jiayue Li, Euphemie Bassonga, Junjie Gao, Delin Liu, David Wood, Brendan Kennedy, Qiujian Zheng, Professor Minghao Zheng

Introductory Talk and Oral Session: OT03 EVs in Tissue Function, Room 105‐106, May 9, 2024, 10:40 AM ‐ 12:00 PM

INTRODUCTION: Tendinopathy, the most prevalent musculoskeletal disease, is typically caused by mechanical overload. While its underlying pathology is associated with inflammation, it is not clear how overload induces the pathological process. The aim of the present study is to explore the link between the overload and inflammatory reactions in tendon.

METHODS: We performed RNA sequencing on human tendinopathic tissues to explore the cellular response in tendinopathy. We then generated mouse tendon organoid by performing three‐dimensional uniaxial stretching in bioreactors. Cyclic strain of uniaxial loadings included underload with 0% or 3% strain, normal load with 6% strain, and overload with 9% strain. Functional tests were performed by RT‐qPCR. Medium extracellular particles (mEPs) were isolated from culture medium by differential centrifugation. Flow cytometry, dynamic light scattering, electron microscopy, and immunoblotting were performed to characterize mEPs. MitoTracker‐labelled intracellular mitochondria of tendon organoid after loaded by different strain were observed through confocal live‐cell imaging. Raw 264.7 mouse macrophage cell line was used for chemotaxis assay in a Boyden Chamber System with Magnetic‐Activated Cell Sorting Technology to elucidate the role of ExtraMito in macrophage chemotaxis. Cytokines secretion by macrophages was analyzed by a bead‐based multiplex assay panel. N‐Acetyl‐L‐cysteine (NAC) was used as the antioxidant to tendon organoid to regulate mitochondrial fitness.

RESULTS: RNA sequencing showed that cellular activities including oxygen‐related reactions, extracellular particles, and inflammation were activated in human tendinopathic tissues. Compared to underload, normal load induced better formation of mitochondrial network, while overload fragmented mitochondrial network and induced tendon organoid to release mEPs including ExtraMito. High‐resolution confocal microscopy identified two forms of ExtraMito, including mitochondria‐encapsulated mEPs and free extracellular mitochondria. Overload led to the degeneration of the organoid and induced mEPs release containing ExtraMito. Chemotaxis assay showed that ExtraMito from overloaded tendon organoid induced macrophages chemotaxis. In addition, mEPs from overloaded tendon organoid induced the production of proinflammatory cytokines including IL‐6, CXCL1 and IL‐18. NAC treatment attenuated overload‐induced macrophage chemotaxis.

Conclusion: We identify for the first time that tendon cells release ExtraMito as a subtype of mEPs to the extracellular environment and mediate the inflammation, a process regulated by mechanical loading.

0T04.O02. Cellular interaction and uptake of human endogenous retrovirus (HERV) envelope‐displaying EVs

Dr. Zach Troyer, Sarah Marquez, PhD Olesia Gololobova, PhD Kenneth Witwer

Introductory Talk and Oral Session: OT04 EV Communication and Uptake, Room 109‐110, May 9, 2024, 10:40 AM ‐ 12:00 PM

Introduction: Extracellular vesicles (EVs) have been reported to fuse with cellular membranes pre‐ or post‐endocytosis and thereby to deliver cargo to the recipient cell cytosol; however, the efficiency of this process appears to be low and may depend on producer:recipient cell identities. One approach to boosting fusion‐mediated cargo delivery is to engineer EVs to display viral fusion proteins such as the vesicular stomatitis virus glycoprotein (VSV‐G) envelope. While potentiating delivery, proteins of pathogenic viruses may also be immunogenic, limiting clinical applications. Human endogenous retroviruses (HERVs) are found in the human genome and, although no longer infectious, encode proteins that may be less immunogenic than foreign viral proteins. Here, we engineered EV display of several HERV envelope (Env) proteins (Syncytin‐1, Syncytin‐2, and HERV‐K‐108‐Env) and assessed how they affect EV/cell interaction for two cell types (HEK293T and HeLa) that are commonly used for in vitro experiments.

Methods: Expi293F cells were used to produce EVs loaded with PalmGRET (an EV‐labelling EGFP‐Nanoluciferase (NLuc) fusion) and displaying one of several selected HERV Env proteins. EVs were separated by combined ultrafiltration and size exclusion chromatography. EVs were characterized per MISEV by electron microscopy, Western blot, and nanoflow cytometry and assessed for incorporation of PalmGRET and HERV Env. EVs were incubated with HEK293T or HeLa cells, and cell/EV association was measured by measuring cellular NLuc activity after washing away unbound EVs and treating cells with trypsin to remove surface‐bound EVs.

Results: EV enrichment of HERV Envs and PalmGRET was confirmed by immunoblot and nanoflow. For recipient HEK293T cells, EVs displaying Syncytin‐1 (HERV‐W Env) had increased cellular association (2.027‐fold/p<0.0001/n=6), but not uptake (1.121‐fold/p=0.838/n=3), compared with non‐surface‐modified control EVs. For HeLa cells, Syncytin‐1+ EVs had both increased association (5.701‐fold/p=0.0038/n=3) and uptake (2.857‐fold/p=0.01/n=3). Syncytin‐2+ and HERV‐K‐108‐Env+ EVs did not have increased HEK293T/HeLa interactions.

Conclusions: HERV Envs that are expressed in EV‐producing cells are also displayed on EVs. Certain HERV Env+ EVs have enhanced levels of interaction with target cells. However, the extent of this enhancement may differ by recipient cell type. These findings emphasize the importance of understanding the interactions of native and engineered EVs with different recipient cells.

0T04.O03. Functionalized engineered extracellular vesicles for targeted delivery to intervertebral disc cells

Ms Mia Kordowski, Dr Ana Salazar‐Puerta, Ms María Rincon‐Benavides, Mr Justin Richards, Dr Nina Tang, Dr Safdar Khan, Dr Elizabeth Yu, Dr Judith Hoyland, Dr Devina Purmessur, Dr Natalia Higuita‐Castro

Introductory Talk and Oral Session: OT04 EV Communication and Uptake, Room 109‐110, May 9, 2024, 10:40 AM ‐ 12:00 PM

Introduction

Chronic low back pain is a leading cause of disability worldwide and often results from intervertebral disc (IVD) degeneration¹. Our innovative approach involves using engineered extracellular vesicles (eEVs) loaded with transcription factors (TFs) and functionalized with cell specific transmembrane ligands to target nucleus pulposus (NP) and annulus fibrosus (AF) cells within the diseased IVD. This strategy offers a potential treatment for chronic low back pain.

Materials and Methods

In vitro, eEVs loaded with key developmental TFs FOXF1 and T to target NP and MKX and SCX to target AF, were derived from adult human dermal fibroblasts after nanoelectroporation with expression plasmids for each factor²‐⁴. Characterization involved nanoparticle tracking analysis, Western Blot, ELISA, and qRT‐PCR to confirm therapeutic TF packing and ligand presence. Fluorescently labeled eEVs were used to assess preferential uptake by human degenerate IVD cells (IRB 2015H038). Therapeutic efficacy of the functionalized TF loaded eEVs was evaluated via qRT‐PCR analysis of catabolic, inflammatory, and pain markers in degenerate cells before and after exposure to eEVs. Excitingly, we developed a method of high‐resolution microscopy to visualize efficient loading of protein cargo inside the eEVs and ligand presence on the membrane.

Results

Our research emphasizes the potential of using eEVs loaded with developmental TFs and functionalized with ligands for targeted delivery to NP or AF. Characterization validated successful loading of developmental TFs and ligands. Functionalized eEVs exhibited preferential internalization by NP or AF, leading to robust upregulation of TF expression. In contrast, non‐functionalized EVs were captured nonspecifically. Additionally, our findings demonstrated successful reprogramming of NP and AF cells towards a healthier, pro‐anabolic phenotype following exposure to eEVs, as confirmed by qRT‐PCR and enhanced collagen production.

Conclusions

Our research demonstrates the potential of functionalized eEVs for efficient and selective delivery of developmental TFs to degenerate cells within the IVD, resulting in reprogramming of both cell types towards a healthier phenotype. These findings underscore the importance of further exploring eEV‐based nanocarriers for targeting specific cell types and employing various techniques to confirm proper protein loading inside or on the membrane of eEVs. This research opens possibilities for tailored interventions in regenerative medicine.

0T04.O04. Phospholipid scrambling: a novel regulator of extracellular vesicle cargo packaging and function

Ms Akbar Marzan, Ms Monika Petrovska, Professor Suresh Mathivanan, Sarah Stewart

Introductory Talk and Oral Session: OT04 EV Communication and Uptake, Room 109‐110, May 9, 2024, 10:40 AM ‐ 12:00 PM

Introduction

Most mammalian proteins are secreted through the conventional secretory pathway, however a subset of cytosolic proteins are also secreted through unconventional protein secretion (UPS) pathways. We previously described a novel mechanism for UPS of non‐vesicular protein cargo, involving bi‐directional phospholipid scrambling at the plasma membrane. Extracellular vesicles (EVs) embody a major pathway for UPS. Therefore, we are now investigating whether phospholipid scrambling is also important for vesicular UPS, and its impact on EV biogenesis, packaging and function.

Methods

To investigate a potential role for phospholipid scrambling in EV biology, we investigated whether scramblases were expressed in EVs. EVs were isolated using ultracentrifugation and analysed using proteomics. Candidates were then investigated using CRISPR/Cas9 to generate scramblase knockout mammalian cell lines. EV biogenesis, secretion and function were assessed using biochemical approaches, confocal microscopy and proliferation assays.

Results

Proteomic analyses showed that EVs isolated from multiple mammalian cell lines contain phospholipid scramblases. Upon knockout of at least one scramblase, the protein content of EVs is altered. Loss of scramblase activity also reduces the number of EVs released per cell without affecting EV morphology. Confocal microscopy revealed that there may be perturbations both in the plasma membrane and endocytic system, leading to altered EV biogenesis and secretion. Finally, we showed that the pro‐proliferative function of cancer‐cell derived EVs is abolished when purified from scramblase knockout parent cells.

Summary/Conclusion

Together these results suggest that phospholipid scrambling and membrane homeostasis is important for the UPS of both soluble and vesicular cargo. EV biogenesis/secretion is decreased when scrambling activity is abolished. Equally, the protein cargo carried by scramblase‐deficient EVs is altered, and this leads to functional impairments. Together this data demonstrates phospholipid scramblase activity is an important regulator of EV biogenesis and function.

0T04.O05. Quantitative features of extracellular vesicle‐mediated crosstalk in multi‐cellular 3D tumor models

Dr. Maria Harmati, Akos Diosdi, Ferenc Kovács, Ede Migh, Gabriella Dobra, Timea Boroczky, Matyas Bukva, Edina Gyukity‐Sebestyen, Peter Horvath, Krisztina Buzas

Introductory Talk and Oral Session: OT04 EV Communication and Uptake, Room 109‐110, May 9, 2024, 10:40 AM ‐ 12:00 PM

Introduction. There are strong evidences that extracellular vesicle (EV) ‐mediated crosstalk between tumor and stroma cells has an essential role in tumor evolution and determines the outcome of the disease. However, there is a lack of quantitative data on the EV network of the tumor microenvironment (TME). Here, we aimed to establish 3D tumor model systems for live tracking of EV crosstalk. Our goal was to identify the primary communication axes and quantititatively analyse the EV transfer in different tumor types under physiological conditions and chemotherapeutic stress.

Methods. To monitor the EV transfer between cells, we used in‐cell EV‐labelling using CellTracker dyes. We generated three different 3D tumor models co‐culturing one type of tumor cells (T‐47D ductal carcinoma, A375 melanoma, or MG‐63 osteosarcoma) and various stroma cells (MRC‐5 fibroblasts, EA.hy926 endothelial cells and THP‐1 monocytes) in hydrogel matrix. To mimic chemotherapeutic stress, low dose doxorubicin was applied. We analyzed the quantitative features of transfered EVs between different cell types using our patented high‐content screening plates, a Leica SP8 Digital LightSheet microscope and the image processing and machine learning software called BIAS (Biological Image Analysis).

Results. By co‐culturing four different cell types, we have optimized the 3D tumor model sytems for quantitative monitoring of EV crosstalk and mapping the EV network of the TME. The three types of tumor models showed significant differences in their EV crosstalk activity, the primary communication axes and drug‐induced effects as well.

Conclusions. In this study, we identified potential target cells for EV‐blocking drugs, which may be integrated to the medicine as a complementary therapy in tumor diseases. Also, the established 3D model systems and experimental pipeline may help to develop a high‐throughput personalized medicine platform supporting clinical decision‐making.

Funding. TKP2021‐EGA09, OTKA‐K143255, ÚNKP‐23‐4‐SZTE‐639, INKP_2024‐11.

PT01.01. A High‐efficiency isolation system combined with proteomics in studying urinary small extracellular vesicles proteins for improving prostate cancer diagnosis

Dr. Cheng Zhou, Ms. Jie Gong, Mr. Baokun Fan, Ms. Xuan Ding, Dr. Bairen Pang, Prof. Yong Li, Dr. Junhui Jiang, Dr. Zejun Yan, Dr. Yue Cheng, Mr. Yingzhi Chen, Dr. Zhaohui Jiang, Mr. Tiannan Guo

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

Prostate cancer (PCa) is the most common cancer among men globally, particularly in developed countries. The current diagnostic tools such as prostate specific antigen (PSA) test, magnetic resonance imaging and tissue biopsy lack sensitivity and specificity. Extracellular vesicles (EVs) are nano‐sized vesicles and play an important role in cancer communications and metastasis. The advancements in a novel EV separation system such as EXODUS and proteomics are providing an excellent chance in EV protein biomarker discovery The aim of this study was to leverage a robust EV isolation method‐ EXODUS, coupled with data‐independent acquisition mass spectrometry (DIA‐MS), to profile the urinary small EV (sEVs) protein biomarkers for accurate PCa diagnosis.

Methods:

Urinary sEVs were isolated using a novel EXODUS technology, and sEVproteins were identified by DIA‐MS to enhance coverage and quantitation. A comprehensive landscape of urinary sEV‐associated proteins was developed based on samples (n=168), including low‐intermediate risk PCa (n=58), high‐risk PCa (n=58), metastatic PCa (n=22), and controls (n=30). Machine learning models were established and validated in an independent cohort consisting of PCa patients (n=60)and controls (n=20). This cohort also includes both grey zone PCa (n=34) and grey zone controls (n=11). The independent validation set was also performed with ELISA.

Results:

Total 5409 sEV proteins were identified in all samples. The key identified pathways in sEVs associated with PCa incidence, progression, and metastasis. include lysosome, proteasome, and cholesterol metabolism. was employed for classification evaluation, Two machine learning diagnostic models including sEV CD177, PEPD, DNASE1, CTNNB1, SEMA3D, SCGB1A1 proteins were established, demonstrating enhanced diagnostic efficiency for PCa diagnosis, especially in the grey zone PCa.

Summary/Conclusions:

In conclusion, this study presents a translational workflow focusing on identifying urine sEV proteins as molecular markers to advance the clinical diagnosis of PCa. The enriched pathways shed light on the mechanistic aspects of PCa development. Our established diagnostic models hold promise in improving PCa early diagnosis, especially in the grey zone PCa, contributing to the best personalized treatment choice.

PT01.02. A novel bladder cancer liquid biopsy using mutated proteins in urinary extracellular vesicles

M.D., Ph.D. Yuji Hakozaki, M.D., Ph.D. Yuta Yamada, M.D., Ph.D. Haruki Kume, Ph.D. Koji Ueda

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

We have previously identified a group of proteins with quantitatively cancer‐specific high expression in plasma EVs of cancer patients for the purpose of enabling liquid biopsy of renal or colorectal cancer (Int J Cancer (2018);142:607)(Mol Cancer Res (2021);19:834). However, in many cases, these biomarker candidates were also found to be expressed in several normal organs, and their cancer specificity was insufficient.

Therefore in this study, we established an original proteogenomics analysis method and developed a cancer liquid biopsy technology targeting proteins with cancer‐specific somatic mutations, which are expected to have 100% cancer specificity.

Bladder cancer is a cancer with a high risk of recurrence and requires regular follow‐up using cystoscopy and contrast‐enhanced CT after endoscopic resection. In this study, we aimed to develop a new liquid biopsy technique by detecting mutated proteins in urinary extracellular vesicles as a new, minimally invasive method of diagnosing recurrence.

Seven patients with urothelial carcinoma who underwent transurethral resection of bladder tumor at the University of Tokyo Hospital were included in the study. Genomic DNA was extracted from the resected tumors and blood samples from the patients, and somatic mutations were identified by whole exon sequencing. As the result, 55‐444 nonsynonymous mutations and 5‐16 frameshift mutations were identified.

Based on this information, the personalized proteogenomic analysis of bladder cancer tissue was performed, resulting in identification of 4‐20 mutated proteins. More importantly, analysis of the patients’ urinary EVs revealed 1‐3 mutated proteins in common with tissues.

An absolute quantification system using stable isotope‐labeled peptide standards was established for these urinary EV mutated proteins, and the quantitative changes in the patient's treatment prognosis with disease progression were also monitored.

In conclusion, we introduce the potential of EV mutant proteins as a new cancer liquid biopsy modality.

PT01.03. A validated workflow and bioinformatic analysis pipeline for extracellular vesicle‐based RNA biomarker signature discovery in molecular diagnostics

Christian Grätz, Dr. Benedikt Kirchner, PD Dr. Marlene Reithmair, Dr. Florian Brandes, Dr. Agnes S. Meidert, Prof. Dr. Gustav Schelling, Prof. Dr. Michael W. Pfaffl

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Extracellular vesicles (EV) are shed by almost every cell type and can be found in any biofluid. They contain a variety of molecules, including several RNA species, which are protected from degradation. Over the last decade, we developed a validated workflow and analysis pipeline for the discovery and characterisation of EV‐associated transcriptomic biomarkers in molecular diagnostics. Transcriptomic disease indicators offer several advantages over other types of biomarkers used in liquid biopsies. However, EV‐associated RNA from liquid biopsy is limited in amount and quality, making it even more critical to ensure validity, transparency and repeatability throughout the process. In the biomarker analysis pipeline we highlight the key steps and give particular emphasis to the necessary quality control checkpoints, which are linked to numerous international guidelines that should be considered along the workflow, e.g. GMP, MIQE, MISEV, or GLP. Our discovery workflow starts with patient cohort recruitment and continues with liquid biopsy sampling and processing. The purification and characterization of EVs is explained in detail, as well as the isolation and quality control of EV‐associated RNA species. We point out the possible pitfalls during next generation sequencing library preparation and subsequent RNA sequencing. Advanced bioinformatics assist to identify and correct for unwanted bias possibly introduced in an earlier step of the biomarker workflow. Applied multivariate methods, HCA, PCA and PLA‐DS, are useful visualization tools to identify powerful biomarker candidates from the sequencing data set. For EV small‐RNA biomarker studies, isomiRROR and caRNAge are in‐house developed software routines, which were successfully applied for miRNA isoform analysis, batch correction, in‐silico functional validation, and the overall process of small RNA sequencing data evaluation, respectively. Validation of the biomarker signature obtained this way is mandatory using RT‐qPCR / RT‐dPCR and miREV, following the recommendations according to the MIQE guidelines. Numerous studies have demonstrated the validity and diagnostic potential of EVs, and it is only a matter of time before EV‐associated transcriptomic biomarkers will find their way into clinical routine.

PT01.04. Advancements in biomarker development for toxicology and safety assessment studies

Tasvilla Sonallya, Annamária Minus, Ferenc Fekete, Dr. Anikó Gaál, Kinga Ilyés, Dr. Tamás Beke‐Somfai, Dr. Zoltán Varga, Dr. Katalin Monostory

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

INTRODUCTION

Toxicity testing is a crucial aspect of evaluating the potential harm of substances on living organisms. Currently, routine assessments often involve measuring enzymes such as GPT and GOT. However, the quest for more effective methodologies is ongoing, with a focus on developing specific biomarkers that can detect toxicity at earlier stages. Extracellular vesicles (EVs) could be exploited as biomarkers providing more accurate and timely indications of adverse effects, enhancing our ability to assess and mitigate potential risks. Their enhanced potential is owing to their varied contents, stability, and ease of access to different bodily fluids allowing them to be used as potential biomarkers. The microRNAs (miRNAs) in EVs are non‐coding RNA molecules and in extracellular miRNAs can be organ‐specific markers of deleterious lesions affecting a particular organ. EVs may be expressed in different sizes and concentrations, and their miRNA content may vary, which can be an early predictor of adverse effects.

METHODS

EVs were isolated from rat liver cell culture medium and rat blood plasma. They were characterized using MRPS, FACS, and FT‐IR. In in vitro hepatotoxicity studies, various drugs such as paracetamol were used to check the hepatotoxicity at different concentrations, and toxicity was characterized by the miRNA concentration in EVs. We tested the applicability of reference miRNA((let‐7‐a, miR‐103, miR23a, miR‐16, miR‐92, U6)) expression at constant concentrations independent of experimental design, and the expression of miRNAs(miR‐122, miR‐151, miR‐155, miR‐192, miR‐194, miR‐193a, miR‐21, miR1, miR‐375, miR133a, miR‐146a) predictive of potential liver toxicity.

RESULTS

The extracellular vesicle isolation protocol, extracellular vesicle characterization method, and liver‐specific miRNA profiling and concentration determination protocols were standardised to follow the hepatotoxicity. Paracetamol was used as a model compound with a well‐established hepatotoxicity at high concentrations. Paracetam ol toxicity was characterized by the miRNA(miR‐122 & miR146a) concentration in EVs that increased with treatment. We found that the labelling ability of glycoproteins on the surface of EVs is significantly reduced by paracetamol treatment.

SUMMARY

EVs can be employed as toxicity biomarker and the selective expression of miRNAs for hepatic damage and the change in their concentration are the criteria for their applicability as biomarkers.

PT01.05. Advancing precision: Development of extracellular vesicles protein‐based panel for validation of endometrial cancer biomarkers

Dr Anastasiia Artuyants, Martin Middleditch, Deanna Shea, Bianca Nijmeijer, Sophia Bebelman, Dr Cherie Blenkiron

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Endometrial cancer (EC) is the most prevalent gynaecologic malignancy, originating in the lining of the uterus. Existing diagnostic methods, such as ultrasound, hysteroscopy, and endometrial biopsy, while effective, are invasive, affecting patient acceptance and quality of life. This underscores the imperative for a non‐invasive, easily accessible biomarker‐based assay for early EC detection. Extracellular vesicles (EVs) emerge as promising entities in cancer research, offering a less intrusive means of testing due to their unique molecular cargo and systemic presence. Our goal is to develop a multiplex Multiple Reaction Monitoring (MRM)‐based targeted panel for validation of our previously identified EV protein candidates (identified by tissue‐derived EV profiling) that can be translated into routine clinical practice.

Methods

Building on our prior findings, we shortlisted peptides of interest representing diagnostic (EC vs non‐cancer), prognostic (discriminating between EC histotypes endometrioid and serous), and biological (obesity, co‐morbidities) purposes. Rigorous exclusion criteria, such as avoiding oxidizable amino acids and peptides with multiple adjacent cleavage sites, ensure assay specificity. Our approach involves optimising the relative quantitation of the targets using existing datasets of peptide identifications, followed by absolute quantitation using stable isotope‐labelled peptides. Evaluation of the multiplex method includes analytical selectivity, carryover, matrix effects, and linearity. Analytes will be verified on various sample types (SEC isolated EVs from tissue, biofluids, and cell lines) before patient biofluid testing.

Summary

The envisioned multi‐biomarker panel, designed to differentiate between cancer and benign samples and categorise histological subtypes, promises not only improved diagnostic accuracy to complement or replace current EC clinical tools, thereby revolutionising early diagnostic outcomes for people diagnosed with endometrial cancer.

PT01.06. Analysis of secreted small extracellular vesicles from activated human microglial cell line reveals distinct pro‐ and anti‐inflammatory proteomic profiles

Miss Xueming Niu, Dr Zhen Zhang, Mr Quan Zhou, Dr Alain Wuethrich, Dr Richard Lobb, Professor Matt Trau

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Microglia are a specialized population of innate immune cells located in the central nervous system, CNS. In response to physiological and pathological changes in their microenvironment, there are two classical inflammation‐related phenotypes based on in vitro studies: pro‐inflammatory or anti‐inflammatory phenotype microglia. The balance between microglia pro‐ and anti‐inflammatory polarization can be a potential biomarker for the various brain pathologies in the CNS. Here, we perform proteomic analysis of small extracellular vesicles (sEVs) derived from microglia cells to identify sEVs biomarkers indicative of pro‐inflammatory and anti‐inflammatory phenotypic changes.

Method

We have utilized nanoparticle tracking analysis, nanoflow cytometry, western blot and mass spectrometry to characterize sEVs and investigated the alterations in the proteome of sEVs purified from human microglia cell line following lipopolysaccharides, or a cytokine co*cktail of IL‐4, IL‐10 and TGFβ stimulation. Cellular morphological changes and inflammatory gene expression validated the activated microglia cell models. Gene Ontology (GO) analysis was performed to determine the biological and functional properties of enriched proteins.

Result

Our analysis showed a distinct protein profile that influences specific biological pathways involved in inflammation found in sEVs released from LPS and IL‐4/10/TGFβ treated cells compared to control. 188 proteins were identified in LPS‐sEVs and 242 proteins were identified in IL‐4/10/TGFβ‐sEVs via mass spectrometry. According to GO analysis, we found a few proteins in LPS‐sEVs that are related to immune response. Additionally, the enrichment of anti‐inflammatory and tumor‐associated proteins was identified in IL‐4/10/TGFβ‐sEVs. GO enrichment analysis revealed the downregulation of numerous pathways involved with antigen presentation and signaling cascades involved in neuroinflammation.

Conclusion

These results indicate that the activation of microglia through LPS and IL‐4/10/TGFβ can lead to the secretion of sEVs reflective of the pro‐ and anti‐inflammatory phenotype of the cells they were derived from. Although beyond the scope of this investigation, clinical analysis of proteins enriched in LPS‐ and IL‐4/10/TGFβ‐sEVs could provide important information when using advanced approaches to detect trace levels of sEVs. This can provide critical insight into biomarker discovery to monitor the pathogenesis of CNS disorders.

PT01.07. Aquaporin 3 detection in placental extracellular vesicles in normal human pregnancy and preeclampsia

PhD Natalia Szpilbarg, Matías Nicolás Sierra, MD Juan Sebastián Sar, PhD Alicia Ermelinda Damiano

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Preeclampsia (PE) is a human gestational syndrome associated with placental insufficiency. In the most severe cases, trophoblast migration is impaired and the placenta is not adequately formed. The syncytiotrophoblast releases extracellular vesicles (EVs) into maternal circulation from the sixth week of gestation until term. In addition, placental EVs have been reported to increase in number and change their content in PE. Therefore, it has been proposed that they could function as biomarkers. It has been reported that aquaporin 3 (AQP3) is involved in trophoblast migration and its expression is decreased in placentas with PE. The aim of this work was to validate a method to detect AQP3 in EVs from (i) maternal plasma and (ii) supernatant of cultured placental explants to evaluate the potential utility of AQP3 as a PE biomarker.

Methods: This study was approved by the Ethics Committee of the Hospital Naval Pedro Mallo, Ciudad de Buenos Aires, Argentina. EDTA‐anticoagulated maternal blood and placentas from normal and PE pregnancies were collected under informed consent. Placentas were obtained immediately and processed within one hour after cesarean section. Explants from normal and PE placentas were cultured 18 h at 37°C and supernatants were collected. Plasma and explant EVs were obtained by differential centrifugation, filtration and ultracentrifugation. Samples enriched in EVs were characterized by DLS, NTA, transmission electron microscopy and western blot to analyze the presence of CD63 and HSP70. AQP3 protein expression was also determined. Placental alkaline phosphatase (PLAP), a syncytiotrophoblast marker, was analyzed to confirm the presence of EVs of placental origin in plasma EVs samples.

Results: Preliminary results show that samples enriched in EVs were obtained and EVs of placental origin were present in plasma EVs fraction. AQP3 was detectable in both plasma and explant EVs samples.

Conclusion: This work lays the foundations to evaluate whether AQP3 is differentially expressed in placental‐released EVs under normal and pathological conditions. Further studies are needed to confirm if the results obtained in placental explant cultures are reflected in maternal plasma in order to consider the potential evaluation of AQP3 as a PE biomarker.

PT01.08. Automated high‐throughput isolation of extracellular vesicles (EVs) and small RNA sequencing profile in serum of breast cancer patients

Dr Ramin Khanabdali, Dr Scott Zhu, Dr Mathew Moore, Dr Gregory Rice

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Extracellular vesicles (EVs) and their molecular profiling have been regarded as ideal candidates for biomarker developments for the earlier detection of different diseases for diagnostic applications. The current major obstacle for clinical translation is the lack of efficient, robust and high‐throughput EV isolation and downstream analysis method that can be easily integrated into clinical laboratory workflows. In this study, we used a high‐throughput automated bead‐based immunoaffinity system to isolate EVs and Identify differentially expressed miRNAs from serum samples obtained from women diagnosed with breast cancer to identify EV‐associated biomarkers for earlier detection of breast to improve disease prognosis.

Methods: EVs were isolated from 500 µL of serum obtained from women diagnosed with breast cancer (I, II, III, IV n=12 per stage) and age matched normal healthy women (n=48) using EXO‐NET EV isolation kit on high‐throughput automated KingFisher Apex system. Small RNAs were isolated on the same system using Promega Maxwell® HT miRNA Plasma and Serum kit for small RNA sequencing and RT‐PCR analysis.

Results: High‐throughput EVs isolation and their associated miRNAs from 96 serum samples was conducted in less than 2 hours. Small RNA seq analysis identified top 50 significantly differentially expressed miRNAs (log2 =2, p <0.01) between normal healthy women and women diagnosed with breast cancer. Data modelling including GO and KEGG pathway analysis identified molecular pathways associated with breast cancer and EV compartments.

Conclusion: We have established an efficient, robust, and fit‐for‐ purpose high‐throughput and automated system for EVs and their associated RNA and protein isolation on the same system for downstream analysis that holds great promise for facilitating the translation of EV diagnostics into routine clinical practice.

PT01.09. Cargo content in extracellular vesicles from a murine cell model of organotropic metastatic breast cancer

Graduate Student Amélie Nadeau, Graduate Student Thupten Tsering, PhD Kyle Dickinson, PhD Daniela Quail, PhD Peter Siegel, PhD Julia V Burnier

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: While primary disease is often well controlled, metastatic breast cancer (mBC) is fatal and the mechanisms underlying organotropism remain largely unknown. Extracellular vesicles (EVs) carry various biomolecules and are involved in organ‐specific metastasis, but their role as biomarkers is still being explored. Our aim is to study EV‐associated cargo of murine mBC cell lines that possess different metastatic potentials and organotropism to the lung, the bone, the liver, and the brain. This will allow us to identify genomic and proteomic drivers that can potentially be used as informative biomarkers of metastatic disease.

Methods: EVs were isolated from the cell culture media of metastatic 4T1 and non‐metastatic 67NR parental cell lines, as well as lung‐metastatic (533, 537), bone‐metastatic (592, 593), liver‐metastatic (2776, 2792), brain‐metastatic (BP, LM) and normal (NMuMg) cell lines using centrifugal filtration followed by ultracentrifugation. The presence of EVs was confirmed and characterized by nanoparticle tracking analysis, transmission electron microscopy, and western blot. EV‐DNA and cell free (cf)DNA were isolated using the DNA Purification kit and the EZ2 ccfDNA kit, respectively. Both were quantified using Qubit. Digital PCR was used to detect DNA mutations in EV‐DNA and cfDNA. Proteomics analysis of EV proteins from all cell lines was conducted with label‐free mass spectrometry.

Results: Varying levels of EV‐DNA and cfDNA were measured in all parental and metastatic cell‐derived EVs. The genomic alterations of Trp53 P31X and Kit A942S were detected in the EV‐DNA and cfDNA of all cancer cells and absent from the normal cells. When compared to NMuMg, untargeted proteomics revealed that parental and mBC cell‐derived EVs are enriched in several proteins that play important roles in cancer growth and metastasis, including HTRA1, PDCD6, Annexin A11, NT5E, TSPAN14, CPNE8, H2‐L, H2‐D1, and SH3GL1. Moreover, four variants of histones (H2AX, H2A.Z, H1.4, H1.0) were found only in site‐specific cell‐derived EVs and were especially enriched in brain‐metastatic derived EVs.

Summary/Conclusion: We found mutated EV‐DNA, cfDNA and differentially expressed protein cargo in mBC cell‐derived EVs compared to normal cells. Further investigation of EV cargo will help in understanding metastatic properties in organotropism, allowing us to identify potential biomarkers for mBC.

PT01.10. Changes to small and large urinary extracellular vesicles in glioblastoma

Dr Susannah Hallal, Mr Liam Sida, Dr Agota Tűzesi, Dr Elissa Xian, Dr Daniel Madani, Dr Krishna Muralidharan, Dr Brindha Shivalingam, Associate Professor Michael Buckland, Dr Laveniya Satgunaseelan, Dr Kimberley Alexander

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Patients with glioblastoma face major obstacles to effective treatment, partly due to unreliable methods for monitoring tumour progression and recurrence. The availability of a liquid biopsy that can accurately monitor glioblastoma biomarkers from patient urine has great potential enhance patient care. The urine contains diverse extracellular vesicle populations (EV; 30‐1000 nm membrane particles) which undergo alterations in tumour states and mirror tumour pathophysiological changes. As urinary‐EV populations may offer clinically‐relevant diagnostic endpoints for glioblastoma, we have investigated the biochemical differences between small urinary‐EV (< 200 nm) and large urinary‐EV (200‐1000 nm) subtypes, and molecular changes associated with glioblastoma recurrence.

Methods: Urine (15‐60 mL) was collected from 24 catherterised glioblastoma IDH‐wildtype patients at three clinical timepoints; before (Pre‐OP; n=9) and after (Post‐OP; n=7) first surgeries, prior to recurrence surgery (REC; n=6), and from age/gender matched healthy controls (HC; n=13). Urinary‐EVs were isolated by differential ultracentrifugation, separating large and small EVs at 17,000 xg and 100 000 xg, respectively. The size, morphology and integrity of urinary‐EVs was characterised by nanoparticle tracking and cryo‐electron microscopy, while molecular differences were assessed via fourier‐transform infrared (FT‐IR) spectroscopy. Urinary‐EV proteomes were analysed with data independent acquisition mass spectrometry (DIA‐MS) and aligned to a custom glioma protein library comprised of spectral coordinates for 8662‐protein species.

Results: Small and large urinary‐EVs had distinct FT‐IR spectral profiles for regions associated with functional groups of nucleic acids, proteins and lipids. DIA‐MS confidently identified 2294 and 928 proteins in at least 80% of small and large urinary‐EV samples, respectively. Across the three glioblastoma timepoints, there were significant alterations in protein distribution between small and large urinary‐EVs, reflecting changes associated with tumour burden (Pre‐OP vs Post‐OP), treatment resistance (Pre‐OP vs REC) and recurrence (Post‐OP vs REC). Notably, significant large urinary‐EV proteins were highly accurate for glioblastoma (classification accuracy = 96.7%) and displayed consistent trends across the three clinical stages, shifting at Post‐OP and returning to Pre‐OP levels at REC.

Conclusions: Our findings demonstrate significant alterations between small and large urinary‐EVs in glioblastoma. Putative large urinary‐EV biomarkers described here merit further validation with longitudinal cohorts of glioblastoma urine.

PT01.11. Circulating EVs as diagnostic biomarkers of indeterminate thyroid nodules

Dr Nada Ahmed, Dr Kevin Beatson, Dr Jigisha Patel, Dr Mohammad Eddama, Dr Tarek Abdel‐Aziz, Professor Lucie Clapp

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Preoperative differentiation of benign from cancerous indeterminate thyroid nodules (ITN) remains one of the most challenging issues in endocrine oncology. Most patients with an ITN will undergo diagnostic surgical resection with around 80% receiving a benign diagnosis on final histology. Liquid biopsy‐based analysis of circulating extracellular vesicles (EVs) presents a promising diagnostic modality. The aims of this study were to measure the levels of circulating total and subpopulations of EVs and identifying differentially expressed EV miRNAs in patients with benign versus cancerous ITNs. Methods: For EV quantification, EVs where isolated by differential centrifugation from plasma of 28 patients with ITN (14 benign and 14 cancers on final histology) and 16 matched healthy controls (HCs). The total phosphatidylserine+ and subpopulations of cancer related EVs (CXCR7+, CD147+, SDC4+, EpCAM+) were measured in the plasma using a scatter calibrated flow cytometer with a limit of detection of ∼ 210 nm EV diameter. All controls were performed according to MiFlowCyt‐EV guidelines. For EV miRNA profiling, EVs were isolated by membrane affinity spin columns, and total EV‐RNA extracted from 6 HCs, 6 benign and 9 cancer patient plasma samples. miRNA sequencing was performed using DNBseq‐G400 sequencing platform. Results: The concentration of the phosphatidylserine+ plasma EVs was significantly higher in patients with ITN (both cancer and benign) (p<0.0001) compared to HCs. Furthermore, the plasma concentrations of CXCR7+, CD147+, SDC4+, EpCAM+ EVs were all significantly higher in patients with ITN in comparison to HCs (p<0.0001 for all). A total of 650 miRNAs were identified. Two miRNAs (hsa‐miR‐195‐3p, hsa‐miR‐619‐5p) were significantly upregulated (p<0.0001) and 4 were downregulated: hsa‐miR‐3176, hsa‐miR‐205‐5p, a novel miRNA: hsa‐miR208‐3p (p<0.0001 for all 3), and hsa‐let‐7i‐3p (p=0.03) in patients with malignant versus benign ITNs. KEGG pathway analysis of each of the differentially expressed miRNA targets revealed genes involved in cancer pathways. Conclusions: Circulating EVs, especially EV miRNAs, have a high diagnostic value for ITNs and may improve the diagnostic strategy for patients with ITNs.

PT01.13. Detection of MTA1 in plasma sEVs derived from cancer patients

Graduate Research Assistant Kritisha Bhandari, Laboratory Technician Jeng Shi Kong, Physician Scientist Haoyao Sun, Professor Jinchang Wu, Assistant Professor Bethany Hannafon, Professor William Dooley, Professor Wei‐Qun Ding

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Metastasis‐associated protein 1 (MTA1) is overexpressed in different types of human cancer and has been suggested as a cancer therapeutic target and diagnostic biomarker. We have recently reported that MTA1 is contained in small extracellular vesicles (sEVs) released from human cancer cells.

Objective: The objective of this study is to evaluate the potential of the MTA1 levels in plasma sEVs as indicators of human cancer.

Methods: Plasma from the patients with breast (n=10) and pancreatic cancer (n=30) were collected by the Cooperative Human Tissue Network. Nude mice implanted with the breast cancer cell line MDA‐MB‐231 (n=6) were utilized to determine whether sEV‐associated MTA1 is released into the circulation. A Cancer Biomarker Antibody Array was applied to plasma sEVs isolated from cancer patients, and MTA1 level in plasma sEVs was further determined using an ELISA kit. Statistical analysis was performed with the GraphPad Prism 10.

Results: Plasma sEV MTA1 level was significantly elevated in mice implanted with MDA‐MB‐231 cells for 6‐10 weeks compared with mice without implantation, indicating the release of sEV‐associated MTA1 from cancer cells to the circulation. Consistently, MTA1 level was found to be significantly higher in plasma sEVs derived from patients with breast cancer compared with matched healthy controls. The Cancer Biomarker Antibody Array showed that the level of MTA1 is three times higher in plasma sEVs derived from patients with early stage pancreatic cancer compared with that in matched healthy subjects. This was further confirmed by ELISA showing that the level of MTA1 is significantly elevated in plasma sEVs derived from patients with early stage pancreatic cancer.

Summary: We observed that the level of MTA1 in plasma sEVs is significantly elevated in nude mice implanted with MDA‐MB‐231 cells and in patients with breast and pancreatic cancer. These observations indicate that sEV‐associated MTA1 is released from tumor cells to the circulation and that plasma sEV MTA1 may serve as a biomarker indicative of breast and pancreatic cancer.

PT01.14. Developing metabolomic approach in profiling extracellular vesicle biomarkers for prostate cancer diagnosis and progression risk stratification

Mr Mahmoud Hamed, Dr Valerie Wasinger, Mr Qi Wang, Associate Professor Peter Graham, Dr David Malouf, Dr Joseph Bucci, Professor Yong Li

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Background

Current prostate cancer (PCa) diagnosis tools such as serum prostatic‐specific antigens (PSA) test, magnetic resonance imaging and tissue biopsy are not specific and inaccurate. Extracellular vesicles (EVs) are nano‐sized vesicles and secreted by all living cells into the extracellular milieu and contain cellular components encapsulated by lipid membranes. Liquid biopsy is a fast‐growing research area and can provide an alternative solution that is less invasive and more reliable. Advances in metabolomics show a comprehensive analysis of EVs’ cargo could lead to the discovery of more accurate biomarkers. In this study, we hypothesise that EVs’ metabolic profiles are different between PCa cell lines and normal prostate cell line or between small EVs (sEVs) and large EVs (lEVs). Our objective is to isolate sEVs and lEVs from a panel of PCa cell lines and a normal prostate cell line to identify differences in key metabolites using global approaches.

Methods

Using ultracentrifugation (UC), EVs were isolated from different PCa cell lines (PC3, DU145, LNCaP and 22RV1) and a normal prostate epithelial cell line (PNT2). Isolated EVs were characterised into lEVs and sEVs. Confirmation tests including nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and western blotting (WB) were conducted to check the characteristics of isolated EVs. Ultra‐high performance liquid chromatography‐mass spectrometry (UHPLC‐MS) is employed to investigate the metabolomic profiles of EVs isolated from different cell lines.

Results

The characterisation of both sEVs and lEVs is confirmed by NTA, TEM and WB. We found that EVs concentration of at least 108 vesicles is required to achieve reliable metabolomics outcomes. We have preliminarily identified differences in sEVs and lEVs metabolite content between PCa cell lines and a control cell line.

Conclusion

Our established protocol in EVs isolation using UC is confirmed to yield EVs with high purity and quality for metabolomic analysis. We have established the metabolomic analysis in EVs using PC3 cell line and a control cell line. In our following study, additional experiments will be run to test more PCa cell lines and normal control prostate cell line and further validate metabolite candidates identified for PCa diagnosis and risk stratification.

PT01.15. Early cancer detection made easy: liquid biopsy analysis of low‐concentration EGFR mutations in NSCLC using large‐volume plasma and urine

Young‐Hye Seo, Sung‐Kyung Bong, Beomhee Ahn, Hanna Kim, Hwanghee Ryu, Myunghee Jang, Ph.D Seung‐Hak Choi, Ph.D Vijaya Sunkara, Juhee Park, Ph.D Yoon‐Kyoung Cho, Ph.D Kyusang Lee, Ph.D Beomseok Lee

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Lung cancer, a leading cause of cancer mortality, finally improved overall survival thanks to targeted therapies in recent years. This relies on identifying somatic mutations in genes such as EGFR, through tissue biopsies, which is not feasible to early stage patients who will benefit the most. Thus, liquid biopsy, utilizing blood samples, has been gaining interest, and extracellular vesicles (EVs) emerged promising improved sensitivity over cell‐free DNA.

This study explores the utility of large‐volume clinical samples (e.g., plasma exceeding 3 mL and urine reaching 30 mL) for detecting EGFR mutations at low concentration in early‐stage non‐small‐cell lung cancer (NSCLC) patients. We focus on RNA‐based EGFR mutation detection qPCR assays using both plasma and urine‐derived EVs.

The enrichment of extracellular vesicles from sizable plasma and urine volumes preserves scarce nucleic acid in the liquid samples. We employed a quick and high yield EV concentration method that uses ionic strength modulation technique (ExoPRISM) for selective EV precipitation followed by high performance TFF (tangential flow filtration) on a lab‐on‐a‐disc. The low‐molecular‐weight electrolytes used for immediate EV aggregation are washed on the lab‐on‐a‐disc along with soluble proteins.

For validation, we obtained EVs from the cell culture of H1975 and HCC827 cell lines which have EGFR genes with specific mutations (L858R, T790M, Exon19del). The EVs were spiked into plasma and urine samples from normal human donors for EV enrichment and RNA preparation. The RNA samples undergo reverse transcription, pre‐amplification of cDNA, and multiplex quantitative real‐time PCR to determine the limit of detection (LOD), which is consistently below 30 copies (less than 0.01%) for all mutations.

The ongoing clinical assessment of EGFR mutation detection sensitivity across 50 plasma and urine specimens from individual non‐small cell lung cancer patients will establish the clinical potential of this method. Due to the higher concentration of RNAs with mutations than DNA counterparts in the sample, our assay will be more useful for early stage detection, as well as treatment monitoring. The comparative analyses of plasma, urine, and tissue samples aim to provide improved insights into precise management, encompassing treatment response assessment, and minimal residual disease in lung cancer patients.

PT01.16. Effect of X‐ray irradiation on quantity and tetraspanin markers expression of extracellular vesicles (EVs) derived from peripheral blood mononuclear cells (PBMCs) and plasma from patients undergoing total‐body irradiation (TBI)

Zi Huai Chew, Senior Research Scientist Christelle Chua

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

EVs are believed to be involved in cell‐to‐cell communication after exposure to ionising radiation (IR), but while PBMCs are known to be particularly sensitive to IR,it is only lately that researchers have begun to characterise PBMC‐derived EVs Previous studies focused mainly on the impact of EV cargo after high doses of radiation typically applied for radiotherapy, and frequently at later time points of >24h.We sought to explore the impact of TBI on EVs profiles derived from plasma of cancer patients approximately 24h after undergoing their first fraction of TBI treatment (2Gy),and compare this with EVs derived from PBMCs after 2Gy ex vivo irradiation as it is not well understood.

(1) Whole blood or PBMCs collected from healthy donors were exposed to X‐ray irradiation and cultured in plasma for 24 hours.(2) Plasma was collected from cancer patients before and 24 hours after their first 2Gy fraction of TBI.EVs were isolated using size‐exclusion chromatography and analysed by interferometric imaging.

The overall quantity of EVs does not appear to increase significantly, 24 hours after exposure for (1) and (2).This is contrary to expectations that EV populations always increases in response to IR,and suggests that at earlier time points this may be otherwise.We observed that CD9 is generally highly expressed in EVs derived from PBMCs of healthy donors and from plasma of cancer patients. However,while CD81 expression is also highly expressed for EVs derived from plasma of cancer patients before and after exposure,this was not the case for EVs derived from PBMCs of healthy donors,in agreement with other published data reported.CD63 expression increases with exposure for EVs derived from PBMCs of healthy donors while expression of all 3 tetraspanin markers (CD9,CD63,CD81) decreases with exposure for EVs derived from plasma of cancer patients.

Our results suggest that in vivo (TBI for radiotherapy) compared to ex vivo (IR of whole blood) exposure of ionizing radiation have differing extent of effects on the quantity of EVs and expression of various tetraspanin markers.Hence,care must be applied when interpreting results derived from in vivo experiments and comparing it into ex vivo experiments.

PT01.17. Establishing the capacity of liver derived extracellular vesicle cargo to reflect variability in drug exposure and response

Ms Lauren Newman, Dr Zivile Useckaite, Associate Professor Andrew Rowland

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: An individual's exposure and response to a drug is determined by the pharmaco*kinetic (PK) and pharmacodynamic (PD) profiles of the drug. Critically, between subject differences in abundance of drug metabolizing enzymes (DMEs) and drug target proteins underpin variability in PK and PD and impact treatment efficacy and tolerability. Differences may result from normal physiology, genetics, environment or chronic liver disease including metabolic associated fatty liver disease (MAFLD), but have historically been challenging to define on a molecular level in vivo. This study sought to (i) establish the fundamental capacity of liver derived EVs to define between‐subject variability in abundance of DME proteins and MAFLD drug targets and (ii) explore the concordance between absolute abundance in tissue and tissue derived EVs.

Methods: EVs were recovered from human liver tissue samples (LT‐EVs, n=11) using our reported protocol of enzymatic digestion followed by size exclusion chromatography. The presence of EVs in the extracellular space of hepatic cells was observed by TEM. EVs were subject to particle analysis by NTA and targeted liquid chromatography mass spectrometry (LC‐MS) assays to assess EV markers and contaminants and quantify a panel of DMEs and drug targets for MAFLD.

Results: LT‐EVs were enriched in CD81, CD9 and TSG101 and significantly depleted of albumin relative to whole tissue. Of the 26 proteins evaluated in the DME and MAFLD panel, 23 (88%) were detected and quantified in LT‐EV. Moderate‐strong positive correlations (Pearson r > 0.6) between tissue and LT‐EVs were observed for 15 of 23 targets, with greater concordance for proteins with reported liver specificity or enrichment relative to other tissues.

Conclusions: This study developed a workflow to robustly quantify proteins in LT‐EVs associated with interactions of drugs with the liver. Accounting for possible contribution from extra‐hepatic EVs in the tissue, LT‐EVs could report on between‐subject variability in tissue protein abundance. Further work in liver‐derived plasma EVs will address the intriguing potential to track markers of drug exposure and treatment response in vivo by liquid biopsy.

PT01.21. Exploring plasma‐derived small extracellular vesicles as novel biomarkers for early‐stage detection of pancreatic neuroendocrine tumors

Ms Priya Kumari Gorai, Ms Simran Rastogi, Dr Surabhi AS, Dr Seema Singh, Dr Shipra Agarwal, Dr Sujoy Pal, Dr Tapas Chandra Nag, Prof Renu Dhingra, Prof Mehar Chand Sharma, Prof Rakesh Kumar, Dr Saroj Kumar, Dr Neerja Rani

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

The five‐year survival rate of rare Pancreatic Neuroendocrine Tumors (PanNET) is the lowest among neuroendocrine tumors because initial detection is being done at late stages due to the unavailability of appropriate diagnostic markers; therefore, there is an urgent need for an early‐stage biomarker for PanNETs. The small extracellular vesicles (sEV) play a key role in tumor growth and metastasis, owing to their remarkable capacity to induce metastatic behavior and proliferation. For the first time, as proof of concept, this study pioneers the investigation of the relationship between sEV concentration and PanNET grades, as well as the presence of BIRC2/cIAP and the autophagy marker Beclin‐1 as cargo within sEVs derived from plasma. By examining plasma‐derived small extracellular vesicles, this research sheds new light on the potential role of these vesicles as diagnostic biomarkers for PanNETs, offering a promising avenue for early detection and improved patient outcomes.

Materials and methods:

Plasma‐derived sEVs were examined via TEM to characterize their morphology and western blot of CD63 and TSG101 confirmed the presence of sEVs, with calnexin serving as the negative control. Nano Tracking Analysis was employed to quantify their abundance and size. Western blot analysis was used to probe the presence of BIRC2 and Beclin‐1 in sEVs. Additionally, IHC assessed the expression of BIRC2 and Beclin‐1 in both PanNET and control tissues.

Results:

This study presents compelling evidence of elevated plasma secretion of sEVs in PanNETs (Grade I & II) individuals compared to healthy controls (HCs) (p<0.0001) with a sensitivity of 100%. Moreover, higher protein expression levels of cancer marker BIRC2/cIAP1 (p= 0.002) and autophagy marker Beclin‐1 (p= 0.02) were observed in PanNETs compared to HCs. Notably, the immunohistochemistry (IHC) analysis of PanNET tissue revealed a parallel expression pattern for both proteins.

Conclusion:

The findings unveil a potential correlation between elevated plasma secretion of sEVs and PanNET pathogenesis. Heightened expression of BIRC2 and Beclin‐1 proteins further highlights their potential as important PanNET biomarkers. This study provides valuable insights into PanNET biology, paving the way for new diagnostic and therapeutic approaches.

PT01.22. Extracellular vesicle‐derived RNA profiling predicts melanoma and non‐small cell lung cancer (NSCLC) response to immune checkpoint inhibitors

Ms Lidia Medhin, Doctor Lydia Warburton, Professor Benhur Amanuel, Doctor Leslie Beasley, Professor Elin Gray

Poster Pitches (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:45 PM ‐ 1:00 PM

Introduction: Melanoma's aggressiveness underscores the need for a reliable and accessible biomarker. Our preliminary analysis identified a circulating extracellular vesicle‐derived gene signature (cEVsig) from plasma and linked to response to immune checkpoint inhibitors. This study aims to validate the predictive role of the EV signature in an independent cohort of melanoma and NSCLC patients undergoing immunotherapy.

Methods: Samples were collected before treatment and EVs were isolated from plasma. EV‐RNA was extracted for qRT‐PCR analysis of a 21‐gene set signature (previously identified). To evaluate differences in gene expression between responders and non‐responders to treatment, Mann‐Whitney U‐test was conducted. Logistic regression models were assessed including all 21 genes or a subset thereof, and their Receiver Operating Characteristic‐ Area Under the Curve (ROC‐AUC) values were determined. Cox proportional hazard models and log‐rank tests were used to assess the predictive value of the gene signature on patient survival.

Results: We analyzed a total of 50 eligible metastatic melanoma patient samples, comprising 37 patients commencing treatment with ipilimumab/nivolumab, 11 pembrolizumab, and 2 nivolumab. Notably, there was a significant difference in gene expression between responders and non‐responders using Mann‐Whitney U‐test analysis. Our predictive model exhibited strong performance with a model quality score of 0.82, AUC of 0.902, sensitivity of 71%, specificity of 93%, PPV of 96%, and NPV of 58%. Survival analysis indicated that the presence of the gene signature was linked to improved PFS (HR:0.13 (95%CI: 0.04‐0.39), p=0.0019) and OS (HR:0.16 (95%CI: 0.05‐0.55), p=0.0093). Analysis of a cohort of 54 NSCLC treated with pembrolizumab monotherapy is ongoing to ascertain the biomarker's predictive potential across diverse cancers.

Conclusion: Our validation study underscores the predictive potential of the identified EV‐derived RNA signature in categorizing melanoma patients as responders or non‐responders to immune checkpoint inhibitors. Determining the likelihood of response before commencing therapy can enable personalizing treatment selection, minimizing unnecessary toxicity and expediting patients' alternative therapies and participation in clinical trials.

Disclosure note: The Abstract outlines EV‐derived genes with pending patent, restricting disclosure of their specific names.

PT01.23. Extracellular vesicles are diagnostic and predictive of blood pressure before and during exercise in people with hypertension

Samantha Upson, Dr. Sabrina LaSalvia, Eric Trillaud, Dr. Emily Heiston, Nathan Stewart, Dr. Steven Malin, Dr. Uta Erdbrügger

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Hypertension (HTN) is a leading modifiable risk factor for cardiovascular disease. Although plasma extracellular vesicles (EVs) have been investigated for diagnostic purposes, limitations in methodology have raised uncertainty for EV's role in HTN. Therefore, we utilized advanced EV flow cytometry to characterize a range of EVs in patients across HTN stages as well as analyze their relation to exercise blood pressure responses.

Using a cross‐sectional design, adults with obesity (n=62, 83% F, 53.2±1.2yr, BMI:36.0±0.7kg/m2, VO2max: 22.0±0.5mg/kg/min) underwent clinical testing. HTN was defined based on systolic blood pressure (SBP): Normotensive (n=12, 92% F, <120mmHg), Elevated (n=15, 80% F, 120‐129mmHg), and HTN (n=35, 80% F, >129mmHg). Blood work was done after an overnight fast to assess tetraspanin EVs+, platelet (CD41+), endothelial (CD105, CD31+/CD41‐), platelet endothelial cell adhesion molecule (CD31), and leukocyte derived EVs (CD45+) using nanoflow cytometry (Northern Light, Cytek) following MyFlowCyt guidelines. A subgroup of 34 individuals underwent a VO2max (maximal oxygen consumption aerobic fitness) test at self‐selected speeds and treadmill incline rising every 2 minutes until exhaustion to assess SBP responses (VO2max‐0min, ΔSBP). Individuals were characterized as having normo‐ (n=12, 92% F) and hyper‐tensive (n=22, 73% F; men: >60 and women: >50mmHg) ΔSBP responses to exercise.

There were no group differences in age, BMI, or VO2max (all p>0.05). However, fasting total EV+ counts varied between BP groups (p=0.0037). EVs were elevated in people with HTN compared to those with elevated (p=0.015) and normotensive (p=0.022) SBP. CD105+ EVs varied among BP groups (p=0.0144) and were higher in those with HTN compared to with Elevated SBP(p=0.042). As expected, ΔSBP to exercise was greater in those with hypertensive compared to normotensive (75.0±19.9 vs. 43.8±5.7mmHg, p<0.001) responses. Interestingly, platelet and endothelial EVs+ only correlated with ΔSBP for those with hypertensive (r=0.53, r=0.56, r=0.68 and r=0.50 respectively, all p<0.02), but not normotensive (r=0.00, r=0.01, r=0.01 and r=0.20, all p>0.05) responses.

EVs were related to HTN status, exercise, and blood pressure responses. While this work supports EVs as possible diagnostic markers of vascular health, additional work in plasma and urine is warranted to determine how EVs impact vascular physiology for optimization of chronic disease risk reduction.

PT01.24. Extracellular vesicles as potential biomarkers for non‐alcoholic fatty liver disease (NAFLD)

Malene Joergensen, Anders Askeland, Rikke Bæk, Charlotte Sten, Rikke Wehner Rasmussen, Morten Hjuler Nielsen, Nahuel Garcia, Maiken Mellergaard, Aase Handberg

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1) Introduction:

Non‐alcoholic fatty liver disease (NAFLD) is a common obesity‐related metabolic disorder lacking non‐invasive diagnostic biomarkers. Extracellular vesicles (EVs) have emerged as potential sources of tissue‐ and disease‐specific biomarkers, for which EV Array (Jørgensen etal. 2013, JEV) provides a high‐throughput method for screening of candidate biomarkers. Here, we present preliminary findings for identifying EV‐based biomarkers for NAFLD utilizing the EV Array platform.

2) Methods:

Plasma was collected from obese individuals with NAFLD and the metabolic syndrome (obese NAFLD‐MetS; n=33), obese without these conditions (obese controls; n=28), and lean controls (n=35). Obese individuals with NAFLD‐MetS were followed and samples prepared during weight loss intervention (baseline, visit 1 (1 month), visit 2 (5‐6 month).

Microarray technology (EV Array) was used to screen the plasma samples for their content of small EVs against a panel of 90 surface‐ or surface‐associated markers. The investigated markers can be categorized as general EV markers, tissue‐specific markers (liver), fatty acid transporters, chemokine receptors (CXCR), Notch receptors, inflammatory (TNF), and immune markers (Garcia etal., 2023, Int. J. Mol).

3) Result:

Data from the EV Array analysis was analyzed both in univariate and multivariate manners and in comparison with Proton Density Fat Fraction (PDFF) obtained by MRI scanning as well as several other clinical and metabolic characteristics (Askeland et al, submitted). Comparing the three groups at baseline showed that the EV phenotypes can indeed distinguish the groups. In addition, several markers showed tendency to decrease during weight loss in the obese NAFLD‐MetS group. Specific results are provided to the extent it is allowed according to a non‐disclosure agreement as a biomarker patent is pending.

4) Summary/Conclusion:

Our findings suggest that EV Array analysis could facilitate novel NAFLD biomarker discovery. Several markers demonstrated potential as biomarkers for NAFLD progression. However, analysis of the full dataset and subsequent validation are required to confirm the clinical utility of EVs as non‐invasive biomarkers to screen for NAFLD.

PT01.25. Forecasting post‐COVID syndrome: leveraging molecular signatures of extracellular vesicles for pedictive analysis

Dr Edina Gyukity‐Sebestyen, Gabriella Dobra, Matyas Bukva, Dr Maria Harmati, Timea Boroczky, Dr Szabolcs Nyiraty, Dr Barbara Bordács, Dr Margareta Korsos, Dr Zoltan Szabo, Dr Gabor Kecskemeti, Prof. Dr Tamas Varkonyi, Prof. Dr Zoltan Konya, Prof. Dr Marta Szell, Dr Peter Horvath, Dr Krisztina Buzás

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: The post‐COVID‐19 condition (also known as long COVID) refers to long‐term symptoms that some people experience more than 12 weeks after recovering from COVID‐19. These symptoms may persist from the onset of their initial illness, or may develop after their recovery, and may disappear over time and then recur. We hypothesized that the molecular pattern of small extracellular vesicles (sEV) found in the plasma of patients during acute SARS‐CoV‐2 infection could predict the course of COVID‐19 disease, the risk of developing post‐COVID syndrome, and its symptoms.

Methods: We conducted a detailed investigation involving 60 volunteers, from whom we isolated ‐ by size exclusion chromatography ‐ small extracellular vesicles (sEVs) from serum samples collected during their acute phase of COVID‐19 infection. Based on the symptoms occurred after COVID‐19, we perform hierarchical clustering classifying the patients into 3 groups. Subsequent analyses are carried out on the 3 groups: i) a group exhibiting a wide range of symptoms in a cumulative manner, ii) a group manifesting a limited set of symptoms, and iii) a group showing no residual symptoms post‐infection. Following a characterization of sEV samples, we employed advanced Raman spectroscopic measurement, liquid chromatography–mass spectrometry (LC‐MS) analysis and enzyme–linked immunosorbent assay (ELISA) to examine their properties.

Results: The Raman spectra of the 3 patients group showed different characteristics allowing us to draw predictions over the potential complications. According to a proteomic analysis of sEVs, more than 25 proteins were significantly enriched in the two patient groups exhibiting post‐COVID symptoms compared to the symptom‐free control group. These proteins include some members of the complement system, such as C2, C4, C5, C1 inhibitors.

Conlusion: As the protein composition of sEVs isolated from serum collected during acute SARS‐CoV‐2 infection shows a significant difference between patients exhibiting post‐COVID symptoms and those who are asymptomatic, plasma EV analysis might be suitable for forecasting the post‐COVID syndrome and may help the patient care.

PT01.26. Glioblastoma biomarkers in urinary extracellular vesicles reveal the potential for a ‘liquid gold’ biopsy

Dr Susannah Hallal, Dr Agota Tuzesi, Mr Liam Sida, Dr Elissa Xian, Dr Daniel Madani, Dr Krishna Muralidharan, Associate Professor Brindha Shivalingam, Associate Professor Michael Buckland, Dr Laveniya Satgunaseelan, Dr Kimberley Alexander

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Readily accessible biomarkers that reflect tumour activity and treatment response are urgently needed to improve the clinical management of the aggressive primary brain tumour glioblastoma, especially for recurrent cases. Given that the urinary system is a major clearance route for circulating extracellular vesicles (EVs; 30‐1000 nm membranous particles), we investigated whether sampling urinary‐EVs could offer a reliable non‐invasive liquid biopsy strategy for diagnosing and monitoring glioblastoma.

Methods: Fifty urine specimens (15‐60 mL) were collected from 24 catheterised glioblastoma IDH‐wildtype patients at three clinical timepoints; before (Pre‐OP, n=17) and after (Post‐OP, n=9) gross‐total‐resection of a de novo glioblastoma tumour, and prior to recurrence surgery (REC, n=7). We also collected urine samples from age/gender‐matched healthy controls (HC, n=14). The urinary‐EVs were isolated by differential ultracentrifugation, and characterised by nanoparticle tracking analysis and cryo‐transmission electron microscopy. The urinary‐EV proteomes were analysed by high‐resolution data‐independent acquisition mass spectrometry (DIA‐MS), and the data was extracted by alignment to our custom glioma protein library comprised of 8662‐protein species.

Results: Overall, 6857 proteins were confidently identified in urinary‐EVs (q‐value≤0.01). A stepwise logistic regression identified five urinary‐EV biomarker proteins with significantly elevated levels in Pre‐OP compared to HCs, with an excellent cumulative diagnostic performance of 95.8% (AUC=0.958). Strikingly, urinary‐EV protein levels effectively distinguished glioblastoma patients at the three clinical stages (FC≥|2|, adj.p‐val≤0.05, AUC>0.9). Many significant urinary‐EV proteins aligned with previously defined EV biomarkers from GBM cell culture, neurosurgical fluids and plasma, and showed consistent trends across the three clinical timepoints, altering at Post‐OP and reverting to Pre‐OP levels at REC. Notably, we identified three urinary‐EV proteins, GGH, GRN and ITM2B, with excellent sensitivity and specificity for glioblastoma recurrence (AUC>0.92), and known links to glioblastoma progression and/or treatment‐resistance.

Summary/Conclusion: Comprehensive DIA‐MS characterisation confirms the urine as a viable source of EV‐associated biomarkers for glioblastoma diagnostics and monitoring. The urinary‐EV biomarkers discovered here warrant further investigation using large longitudinal cohorts of glioblastoma urine specimens.

PT01.27. Glycosignatures of small extracellular vesicles secreted by breast cancer cells

Lifang Yang, Benjamin Johnson, Caleb Smack, Professor Eric Feliberti

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1) Introduction: Breast cancer (BC) is a highly heterogenous disease with many subtypes that differ in clinical behavior. However, current tissue‐based diagnostic routines are simplification of the inherent biology of BC and provide limited information for personalized diagnosis. Extracellular vesicles (EVs) have been recognized as a mode of intercellular communication. Like their parent cell, EVs are heavily glycosylated. Glycoconjugates of EVs has been shown to play a role in EV protein sorting, cell targeting, and recognition. As aberrant glycosylation is a hallmark of cancer cells, little is known about the molecular basis of glycosylated EV cargos as well as their presentiveness of known cancer characteristics. Here, we characterized the EV glycoproteins released by different BC cell lines to define subtype‐specific signatures.

2) Methods: Breast cell lines representing normal breast cells and 4 BC subtypes (luminal A, luminal B, HER2 enriched, and triple negative) were cultured in appropriate cell media. Small extracellular vesicles (sEVs) were isolated via a differential ultracentrifugation approach. The morphology and size of isolated sEVs were determined by transmission Electron Microscopy and NanoSight. The sEV purity was assessed by Western blotting with a panel of markers. Lectin blots were performed to examine the glycosylation patterns of specific carbohydrate moieties in the protein lysates from sEVs relative to their parent cells. In addition, metabolic labeling and click chemistry were employed to further characterize cellular and sEV surface sialoglycoproteins.

3) Results: Collected particles with nanoscale size (50–150 nm) harbored membrane‐encapsulated vesicular structure and presented typical small EV markers. Lectin blot analysis showed distinct glycosylation patterns in various BC subtypes. Comparison of the glycosignatures of sEVs with their parent cells revealed both enrichment and depletion of specific glycosproteins in these vesicles. Furthermore, abundant sialylated glycoproteins were detected on the surface of cells and sEVs. Sialylation patterns and levels on the sEV surface, which reflect the cell of origin, were correlated with the aggressiveness of BC subtypes.

4) Conclusions: sEVs from BC cells display specific glycosignatures and are enriched in subtype‐associated sialoglycoproteins on their surface. The identification of these glycoproteins could provide novel biomarkers to improve BC stratification and diagnosis.

PT01.28. High‐throughput and automated isolation of plasma derived extracellular vesicles to identify microRNAs with diagnostic potential for ovarian cancer

Dr Ramin Khanabdali, Dr Carlos Palma, Miss Siena Barton, Professor Greg Rice

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Despite the growing number of disease‐associated extracellular vesicle (EV) biomarkers being identified, the translation of EV diagnostics into routine clinical laboratory tests remains limited. To effectively leverage EVs as diagnostics requires identification of disease‐associated biomarkers in specific EV subpopulations; and rapid, reproducible, and scalable sample processing. Here, we have developed a high‐throughput bead‐based immunoaffinity system that captures a highly enriched subpopulation of EVs to Identify biomolecular profile of any biofluids including plasma, serum, urine and saliva samples for biomarker discovery and clinical translation for diagnostic applications.

Methods: EVs were isolated from 500 µL of plasma obtained from women diagnosed with ovarian cancer (I, II, III n=10 each stage) and age matched normal healthy women (n=30) using EXO‐NET EV isolation kit on high‐throughput automated KingFisher system. Following EXO‐NET EV isolation, small RNAs were isolated on the same system using Promega Maxwell® HT miRNA Plasma and Serum kit for microRNAs (miRNAs) sequencing and RT‐PCR analysis.

Results: High‐throughput EXO‐NET EVs isolation and their associated miRNAs from 60 clinical plasma samples was performed on the KingFisher system in less than 2 hours. Small RNA sequencing analysis identified top 27 significantly differentially expressed miRNAs (log2 =2, p <0.01) between normal healthy women and women diagnosed with ovarian cancer across different stages. Data modelling identified top GO molecular pathways associated with ovarian cancer and EV compartments.

Conclusion: We have established an efficient, robust, and fit‐for‐purpose high‐throughput and automated system for EVs and their associated RNA and protein isolation for downstream analysis which holds great promise for facilitating the integration of EV diagnostics into routine clinical practice.

PT01.30. Identification of extracellular vesicles and particles derived proteins as novel biomarkers for prostate cancer diagnosis, risk stratification and monitoring metastasis

Mr Qi Wang, Dr Bairen Pang, Dr Cheng Zhou, Dr Meng Han, Jie Ni, David Malouf, Joseph Bucci, Peter Graham, Tiannan Guo, Junhui Jiang, Yong Li

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1. Introduction

Prostate cancer (PCa) is the second leading cause of death in men in Australia. Current diagnostic tool blood prostate‐specific antigen test is insufficient in PCa diagnosis and risk stratification. There is an unmet need to develop novel approaches for more cost‐effective and accurate biomarkers for PCa early diagnosis and treatment stratification to enable personalised medicine. PCa‐derived extracellular vesicles have garnered attention in recent years due to their important role in communications in tumour microenvironment as well as in invasion, progression, metastasis, therapeutic resistance, and immune escape. Our objective in this study was to identify different protein profiles in four different extracellular vesicles and particles (EVPs) subpopulations, including large extracellular vesicles, small extracellular vesicles, exomeres and supermeres, from a panel of PCa cell lines and PCa patients’ plasma using proteomic analysis for PCa diagnosis and progression risk stratification.

2. Methods

PC3, DU145, LNCaP and 22Rv1 PCa cell lines as well as a normal prostate epithelial cell line RWPE‐1 were cultured, and the supernatants were collected for EVPs isolation. PCa patients were selected based on a series of criteria and divided into four groups (control, low‐risk, intermediate‐risk, high‐risk, metastasis, n=3 in each group) following the NCCN guideline 2023. All EVPs were isolated by ultracentrifugation and then characterised by transmission electron microscopy, nanoparticle tracking analysis and western blotting, atomic force microscopy, zeta potential analyzer and nano‐flowcytometry. LC‐MS/MS proteomics and bioinformation analysis was applied for the pathway analysis of PCa derived EVPs proteins.

3. Results

A series of gradient ultracentrifugation was applied for isolating PCa derived EVPs. The EVPs isolated were characterised by morphology, size, classical protein biomarkers and some new physical parameters for confirmation. A group of potential EVP proteins were identified and need to be further validated in PCa cell lines and an independent set of PCa clinical samples.

4. Summary/Conclusion

We have successfully established a reliable and reproducible method for isolating distinct EVP populations from PCa cells and human plasma samples and comprehensively characterised their properties. Investigation of the details of protein profiles from EVPs is ongoing and holds promise for PCa early diagnosis, risk stratification and metastasis monitoring.

PT01.32. Investigation of the immunopeptidome carried by MHC class I molecules on extracellular vesicles (EV) released from lung cancer cells

Miss Debra Lennox, Dr Caitlin Boyne, Dr Sally Shirran, Dr Simon Powis

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

As cancer cells produce extracellular vesicles (EV) more abundantly than normal cells it is possible that EVs from solid tumours could be detected in the patient bloodstream, which has already been shown in breast cancer and melanoma. Characterisation of the peptides from HLA‐I molecules present on the surface of these EVs could provide information on treatment efficacy and gain more insight into the molecular mechanism and more accurate prognostic indicators in a minimally invasive procedure compared to biopsy. Within the scope of EV immunopeptidome derived for lung cancer (cell line or patient blood) we will seek the presence of tumour associated and tumour specific antigens, which can be the target for immune attack of cancer cells.

Methods

Isolation of HLA‐I molecules from EVs via size exclusion chromatography and immunoprecipitation before eluting bound peptides and performing LC/MS/MS as described by Synowsky etal., 2017. Then bioinformatic analysis through various software programmes including but not limited to the TAA database and Pather.

Results

So far, we have determined from 4 different cells lines that we can detect Tumour Associated Antigens (TAAs) from the peptides found on EVs and that the is an overlap in what is presented. 7 distinct peptides and many TAAs such as NCOR, EEF2, SON were found in more than one cell line and had similar prominent pathways detected. We have also began the analysis on 17 patient blood samples which will be compared to donor blood and is currently suggesting similar TAAs.

Summary/Conclusion

EV released by lung cancer cells do indeed have HLA‐I molecules on the surface for antigen presentation that is detectable in both cell models and in patient blood samples. The HLA‐I bound peptides present relevant TAAs can also give some indication of the molecular processes likely occurring in the cells of origin in both cell lines and patient samples which with further investigation could allude to possible future biomarkers for diagnosis/prognosis the possibility of a non‐invasive method for determining prognosis in the future.

PT01.33. Isocitrate dehydrogenase 1 is increased in urinary extracellular vesicles from type 2 diabetic model rats

Ph.D. Student Haruka Sei, M.S. Naoya Hirade, Ph.D. Fumie Nakashima, Ph.D. Takahiro Shibata

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Diabetic nephropathy (DN) is one of the complications of diabetes and is the main cause of dialysis. In this study, we attempted to discover the candidate(s) of the DN markers from urinary extracellular vesicles (uEVs).

Method: The uEVs isolated from type 2 diabetic (T2D) model rats were subjected to proteomic analysis. We also investigated the effects of calorie restriction (CR) on the levels of a potential marker candidate in uEVs from T2D model rats since it was reported that calorie restriction effectively attenuated the promotion of diabetes in the T2D model rats. The candidate expression in the renal cortex was assessed by Western blotting and real‐time PCR. To identify the origin of uEVs containing the candidate, we focused on the differences in the renal cell surface sugar chains between renal segments. We performed lectin affinity enrichment of uEVs and immunohistochemical staining.

Results: Proteomic analysis revealed that isocitrate dehydrogenase 1 (IDH1) in uEVs from T2D model rats was significantly upregulated compared to control rats. The upregulation was significantly suppressed by CR. Western blotting and real‐time PCR revealed that IDH1 was significantly upregulated in the renal cortex of T2D model rats. Using immunohistochemical staining, we found that IDH1 was highly expressed in proximal tubular cells of the renal cortex and collecting duct cells of the inner medulla. To confirm that IDH1‐containing uEVs came from proximal tubular cells, we developed the lectin enrichment method using biotinylated Lentil culinaris lectin (LCA). Biotinylated LCA enabled to enrich IDH1‐containing uEVs and proximal tubular cells‐derived uEVs. Furthermore, immunohistochemical staining showed that IDH1‐positive cells and LCA‐positive cells were colocalized in the renal cortex.

Summary/Conclusion: IDH1 was significantly upregulated in uEVs from T2D model rats. The upregulation was suppressed by CR. We also identified proximal tubular cells as the main host cells of IDH1‐containing uEVs. These findings indicate that IDH1 might be one of the candidates of the DN markers in uEVs.

PT01.34. Isolation and characterisation of extracellular vesicles from tumour and non‐tumour lung tissues for next generation sequencing

Edward Stephens, Dr Tian Mun Chee, Mr Vihanga Dharmasena, Professor Kwun Fong, Professor Ian Yang

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Lung cancer is the leading cause of cancer death worldwide, largely due to late‐stage diagnoses. Biomarkers of disease detectable using minimally invasive methods, such as blood tests, could improve lung cancer detection and screening capabilities. Extracellular vesicles (EVs) have emerged as leading candidates for biomarker studies as EVs contain clinically relevant bioactive cargo that bears notable resemblance to their parent cells, and therefore make them attractive targets for cancer biomarker studies. In this study, we aimed to isolate and characterise EVs derived from tumour and non‐tumour lung tissue to demonstrate their utility as a novel bioresource for an EV‐specific signature of lung cancer tumour biomarkers.

Methods: Thirty milligrams of frozen lung tissue was incubated in a collagenase/dispase‐based digestion buffer for one hour at 37°C and then filtered through a 40µm cell strainer. Digested tissue was then centrifuged at low speeds to remove cells, large vesicles and other debris, followed by ultracentrifugation for 100 minutes at 100,000xg at 4°C. The resulting EV pellet was then purified using size exclusion chromatography, and concentrated further using ultrafiltration. Prior to ultrafiltration, a small volume of sample was reserved for EV characterisation using the IZON Exoid instrument to measure size and concentration, western blotting to qualitatively assess the EVs present, and electron microscopy to visualise the EVs. RNA and DNA were extracted from EVs using QIAGEN AllPrep Mini kits. The resulting RNA and DNA fractions were quantified using a Qubit 4 Fluorometer and Agilent Tapestation.

Results: EV concentration and size analyses revealed an average particle diameter of 121nm and a concentration of 4.59x10¹⁰ particles/mL. Western blotting indicated the presence of EVs with positive staining of CD9 and FLOT1 antibodies. Preliminary concentration analyses of EV‐extracted DNA and RNA yielded DNA concentrations of 37.4ng/µL and 39.4ng/µL, and RNA concentrations of 2.9ng/µL and 8.5ng/µL for tumour and non‐tumour tissues, respectively.

Conclusion: Here, we demonstrate a lung tissue digestion technique to isolate EVs from tumour and non‐tumour lung tissues. Ample concentrations of DNA and RNA were obtained from lung tissue EVs, enabling downstream sequencing experiments to interrogate EV‐specific signatures of lung cancer tumour biomarkers.

PT01.36. Leveraging extracellular vesicle glycan signatures for prostate cancer detection

MS Trevor Enright, PhD Kai Tao, PhD Sinan Sabuncu, PhD Emek Demir, MD Mark Garzotto, BS Randall Armstrong, PhD Michelle Gomes

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Levels of Prostate‐specific antigen (PSA) in the blood used to screen patients for prostate cancer, but lacks specificity. Of the patients referred for diagnostic biopsy a third are cancer‐negative. Minimally invasive fluid‐based biomarkers distinguishing between low‐risk, high‐risk, or benign prostatic conditions are needed to reduce the incidence of unnecessary biopsies. Tumor‐derived extracellular vesicles (EVs) are a fingerprint of biomarkers on cancer cells. Since cancer cells exhibit glycosylation aberrancies, we hypothesize that the glycan landscape of prostate‐derived EVs from expressed prostatic fluid (DRE‐urine) may provide unique markers for early detection and disease progression.

Methods: EVs were enriched from DRE‐urine samples of low‐risk (Gleason <7; n=25), high‐risk (Gleason >7; n=25) prostate cancer patients and benign controls (n=25) using density gradient ultracentrifugation and characterized by Nanoparticle Tracking Analysis (NTA), and Transmission Electron Microscopy (TEM). Immunoblotting confirmed EV markers. To test our hypothesis, we profiled and compared the glycosylation patterns of prostate‐derived DRE urine‐enriched EVs from prostate cancer patients and controls by multi‐parametric flow cytometry using panels of fluorescently conjugated lectins and an antibody to the prostate cancer marker, PSMA.

Results: All 75 patient samples had >1x 10¹⁰ EV particles. Patient EVs were in the typical size ranges of 50‐150nm and showed characteristic cup‐shaped morphology. Computational analysis of lectin/antibody intensities on individual prostate‐derived EVs showed glycan patterns associated with prostate cancer. Using glycan biomarkers in a Random Forest Classifier, we could classify patients into the relevant cohort with high sensitivity and specificity. Furthermore, Shapley Analysis quantifies the contribution of each glycan biomarker and shows distinct glycan signatures for prostate cancer patients and controls.

Conclusion: Taken together, our data indicates that EV glycan signatures can distinguish high‐risk, and low‐risk prostate cancer patients from benign/screen‐negative disease status suggesting EV glycosylation is a promising biomarker for prostate cancer detection.

Limited Information Disclosure: The lectin panels are retained by OHSU as a patent candidate, preventing detailed disclosure of the lectin and glycan signature identities.

Ethical Statement: Human subject samples were collected from consenting individuals for the CEDAR Biorepository under OHSU and VA clinic Institutional Review boards.

(IRB#18048, VAIRB#4214)

Funding: CEDAR‐ Full 2020‐1251, Full 2023‐1688

PT01.37. Lipidomic and proteomic approaches revealed glycerophospholipids as a signatures of hypoxic small extracellular vesicles from head and neck squamous cell carcinoma

Dr Alicja Głuszko, dr. hab. Mirosław Szczepański, dr. Andrzej Ciechanowicz, Prof. Theresa Whiteside, dr. Nils Ludwig

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Hypoxia, a hallmark of solid tumors, including head and neck squamous cell carcinomas (HNSCCs), molecularly and functionally modulates activity of cancer cells. In our prior experiments, we demonstrated that HNSCC cell lines exposed to hypoxia release an increased number of small extracellular vesicles (sEVs). This study aims to analyze metabolic shift by combining lipidomic and proteomic profiles of sEVs released from HNSCC cell cultures under normoxic and hypoxic conditions, comparing them with plasma samples from HNSCC patients and normal donors.

Materials and Methods: We collected sEVs from plasma of HNSCC and normal donors, as well as from the supernatants of HNSCC cells (PCI‐30) and normal control cells (HaCaT keratinocytes) exposed to 21 % (normoxia) and 1 % (hypoxia) oxygen supply. sEVs were isolated from plasma and supernatants using size exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis, electron microscopy, immunoblotting, and, for cell line‐derived sEVs, high‐resolution mass spectrometry lipidomic and proteomic analysis.

Results: The analyzed sEVs had an average diameter of 125–135 nm and carried CD63 and CD9 but not Grp94. The analysis of hypoxia‐derived sEVs in tumor demonstrated 815 and 735 significantly dysregulated lipids compared to normal counterparts exposed to hypoxia and to tumor‐derived under normoxic conditions, respectively. Among the 263 lipids in common, the most abundant were glycerophospholipids (GPLs). Hypoxia determined the distribution of 281 unique lipids in tumor‐derived sEVs compared to normal keratinocytes. The adaptation of HNSCC cells to hypoxia was also associated with an enrichment in the increased number of unique GPLs.

This data was overlaid on the proteomic level. The expression of proteins involved in the catabolism of GPLs to lysophosphatidic acid increased in both hypoxic cell line‐derived sEVs and HNSCC plasma‐derived sEVs but not in those from normal donors. Choline transporter was characteristic feature of hypoxic HNSCC‐derived sEVs.

Conclusion: Adaptation to environmental stress such as hypoxia, unveils the plasticity of the lipid profile in HNSCC‐derived sEVs, marked by the upregulation of GPLs. This lipid signature correlates on the proteomic level, suggesting that sEV‐associated lipids may serve as a signature for tissue hypoxia in HNSCC and act as signaling molecules in tumor progression.

PT01.38. Lipidomic identification of novel small extracellular vesicle biomarkers for prostate cancer early diagnosis and risk progression stratification

PhD Meng Han, PhD Jie Gong, Professor Qi Wang, PhD Bairen Pang, PhD Cheng Zhou, PhD Zhihan Liu, Professor Junhui Jiang, Professor Yong Li

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1. Introduction:

Current diagnosis and risk progression stratification using blood prostate specific antigen (PSA) test for prostate cancer (PCa) is inaccurate and unreliable. Tissue biopsy is harmful and cannot cover tumor heterogeneity. Therefore, developing innovative approaches for accurate PCa diagnosis and risk stratification is critically important in choosing the best treatment and personalized medicine. Extracellular vesicles (sEVs) play an important role in regulating cell‐to‐cell communication and tumor initiation, progression, and metastasis positioning them as an important source of biomarkers for liquid biopsy. Lipidomic analysis of EVs for cancer biomarker discovery is a new developing research area and holds promise for PCa diagnosis and personalized therapy. Our Aim in this study was to identify novel lipid biomarkers from plasma sEVs of different stages of PCa patients for personalized treatment choice.in.

2. Methods: sEVs from plasma and urine samples of 6 controls and PCa patients (n=6. low‐risk: 3 and high‐risk: 3) and control subjects (n=6) were isolated. Employing nanoflow and liquid chromatography‐mass spectrometry (LC‐MS/MS) were applied for lipid biomarker discovery and targeted parallel reaction monitoring (PRM) was use for lipid biomarker validation using urine samples including control, low‐risk, and high‐risk groups (n=15 each group).

3. Results: Total 727 distinct lipids were identified form sEVs of urine and plasma samples by LC‐MS/MS. The lipid markers identified was found more variable in urinary sEVs than in plasma sEVs. A significant upregulation of most lipid types was observed in the urine and plasma sEVs of PCa patients compared to control subjects. In addition, lipid significant reduction of sEV lipids expression in thigh‐risk PCa group was found compared to low‐risk PCa group.

4. Conclusion: Our findings indicate that a panel of sEV lipid biomarkers identified such as carnitine C14:1, carnitine C16:1‐OH, hold potential for PCa early diagnosis and risk stratification. Validation of these biomarkers are ongoing in an independent set of urine samples for clinical value confirmation.

Keywords: Prostate cancer, extracellular vesicles, lipidomics, liquid biopsy, diagnosis, risk stratification

PT01.39. Multiplex profiling of endometriosis‐derived extracellular vesicles reveals novel potential biomarkers for endometriosis

MSc Karolina Soroczyńska, Tobias Tertel, Bernd Giebel, Małgorzata Czystowska‐Kuźmicz

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Endometriosis, a prevalent and challenging gynecological disorder, poses therapeutic challenges with a lack of effective treatments and early diagnostic biomarkers. The multifaceted nature of this condition calls for innovative approaches, highlighting the pivotal role of exploring a diverse panel of biomarkers for accurate diagnostics and tailored therapeutic interventions. In this context, extracellular vesicles (EVs), present in various body fluids, emerge as promising candidates for liquid biopsy biomarkers, given their accessibility and potential diagnostic value. Implementing a comprehensive strategy that integrates state‐of‐the‐art EV analysis techniques, including high‐resolution molecular profiling of EVs using proteomics and single‐vesicle imaging flow cytometry (IFCM) analysis, is crucial for identifying novel EV‐based biomarkers or therapeutic targets for this complex and heterogeneous condition.

Methods

EVs were isolated from plasma and peritoneal fluid (PF) of endometriosis and control patients using SEC and were verified by WB, fluorescent mode NTA (F‐NTA), imaging flow cytometry, and TMT‐based quantitative proteomics analysis. Molecular profiling of EVs directly in plasma and PF samples was conducted using high‐throughput IFCM with a specialized antibody panel. This panel included detection of antigens elevated during chronic inflammatory states (CD152), associated with early endometriotic lesions (CD82; CD44), immune suppression (CD16; CD206), and endometrial receptivity leading to infertility (CD227).

Results

A heterogeneous collection of EVs was identified in plasma and PF samples from both endometriosis patients and controls. These vesicles exhibited typical characteristics of small EVs and contained bona fide EV markers. Single EV analyses on the IFCM platform, along with EV proteomics analysis, revealed that EV populations derived from endometriosis patients contain a wide range of molecules, with some associated with the pathogenesis of endometriosis.

Summary/Conclusion

In summary, our study highlights the potential of EVs as promising liquid biopsy biomarkers for endometriosis. The presence of diverse EV populations, along with identified endometriosis‐specific signatures, suggests promising applications in diagnostics, prognostics, and therapeutics. Further evaluation of these EV signatures is crucial for advancing non‐invasive approaches in managing this complex gynecological disorder, providing valuable insights into potential diagnostic and therapeutic avenues for improved clinical outcomes.

PT01.40. Novel set of extracellular vesicle proteins as biomarkers for early detection of high grade serous ovarian cancer

Kalpana Deepa Priya Dorayappan, Dr. Michelle Lightfoot, Dr. Lianbo Yu, Dr. Colin Hisey, Dr. Takahiko Sakaue, Dr Muralidharan Anbalagan, Dr Casey Cosgrove, Dr Larry Maxwell, Dr Premal Thaker, Dr Beth Y. Karlan, Dr David O'Malley, Dr Raphael E. Pollock, Dr David E. Cohn, Dr Rajan Gogna, Dr Selvendiran Karuppaiyah

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

High‐grade serous ovarian cancer (HGSOC) accounts for over 75% of all epithelial ovarian cancer and has a high mortality rate due to a lack of early detection methods. Many biomarkers have been proposed; however, none have been shown to improve detection at an early stage in this patient population. The purpose of this study is to assess whether there are unique extracellular vesicle (EV) protein signatures of early‐stage HGSOC that could serve as early detection biomarkers.

Methods:

250 serum samples were obtained from a multi‐institutional retrospective cohort of patients with all stages of HGSOC and healthy controls. LC‐MS/MS and PEA of serum samples from patients with early‐stage HGSOC identified differentially expressed EVs proteins in HGSOC versus controls. Tob candidate proteins are validated by ELISA.

Results:

We have identified the top 10 EVs candidate proteins based on their fold change and statistical significance. When comparing early‐stage HGSOC and controls using ELISA, CFH, PCP, and CCNE1 exhibited the highest Area Under the Curve (AUC) values of 0.94, 0.91, and 0.83, respectively. In contrast, MUC16 (also known as CA‐125) exhibited an AUC of 0.78 in EVs but only 0.42 in whole serum. Furthermore, MUC16 distinguished between controls and both early and advanced‐stage HGSOC in EVs, while in whole serum, it was only differentially expressed between controls and advanced‐stage disease. Our results revealed generally weak correlations between pairs of biomarkers. In this study, we integrated the identified ten biomarkers, deriving the optimal combination biomarker by maximizing the corresponding Sum of Harmonic Means (SHUM). The overall true positive rate (TPR) of 0.943, false positive rate (FPR) of 0.000, and Mathew's correlation coefficient (MCC) were presented for both the combined biomarker approach and individual biomarkers. The results demonstrated that the predictive performance of the combined biomarkers surpassed that of any individual biomarker.

Conclusion:

Expression of EV‐based protein biomarkers is significantly different in early‐stage HGSOC compared to both control and late‐stage HGSOC. This work can be readily translated to clinical use and potentially improve the early detection of HGSOC substantially.

PT01.41. Proteomic analysis of cerebrospinal fluid in medulloblastoma and associated extracellular vesicle protein ‐ TKT as a potential biomarker

Research Professor Seung Ah Choi, Professor Seung‐Ki Kim, Professor Ji Hoon Phi

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Cerebrospinal fluid (CSF) is a crucial factor in brain tumor studies, particularly medulloblastoma (MBL). Previous CSF proteomics research has predominantly concentrated on extracellular vesicle (EV) proteins rather than the comprehensive CSF proteome. Recent progress in mass spectrometry systems and ‘Omics’ data analysis techniques has opened the door to unbiased and in‐depth exploration of the proteome. Our goal was to discover specific diagnostic biomarkers associated with EV proteins through proteomic profiling of the comprehensive CSF. Through liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) analysis of CSF samples from MBL (n=31) and hydrocephalus (HC, control, n=19) patients, we identified 1,112 quantified proteins per CSF sample. Enrichment analysis and differential expression profiling revealed 273 differentially expressed proteins (DEPs). Feature selection highlighted four upregulated soluble proteins (SPTBN1, HSP90AA1, TKT, and NME1‐NME2) in MBL CSF. Validation with ELISA confirmed elevated levels of TKT in MBL compared to HC, particularly in Group 4 MBL and leptomeningeal seeding (LMS). Moreover, TKT‐positive EVs were significantly enriched in MBL CSF compared to HC CSF and correlated with LMS burden. These results provide significant insights into the proteomic profile of the total CSF in MBL patients, underscoring the potential of TKT as a meaningful biomarker for MBL, particularly in the diagnosis of LMS.

PT01.41. Plasma extracellular vesicle miR‐512‐3p modulates the GTPase activity and the angiogenic function of endothelial colony‐forming cells by targeting ARHGEF3 in pediatric Moyamoya disease

Research Professor Seung Ah Choi, Professor Eun Jung Koh, Professor Seung‐Ki Kim

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Background

Moyamoya disease (MMD) is a chronic occlusive cerebrovascular disease known to be a major cause of pediatric stroke. Emerging evidence suggests that circulating extracellular vesicle (EV) containing miRNAs in cerebrovascular disease plays a significant role in intercellular communication by delivering RNA cargo involved in biological processes. This study aimed to investigate the specific miRNAs loaded in EVs from MMD plasma, followed by identification of their roles and mechanisms.

Methods

EVs were isolated from plasma of normal group and MMD patients. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, ExoView, RT‐qPCR and western blot. Profiling of miRNAs in EVs were determined using NanoString nCounter miRNAs analysis. Guanosine triphosphatase (GTPase) activity, tubule formation and cell viability were observed by transfection of miR‐512‐3p inhibitor in MMD endothelial colony forming cells (ECFCs).

Results

EVs were successfully isolated from normal and MMD plasma. Compared with normal EVs, the number of MMD EVs were small, but there was no difference in size. miRNA profiling demonstrated that miR‐512‐3p was significantly upregulated in MMD EVs. The target prediction analysis of EV‐miR‐512‐3p suggested that rho guanine nucleotide exchange factor 3 (ARHGEF3) was down‐regulated and could be targeted in MMD ECFCs. Inhibition of miR‐512‐3p in MMD ECFCs resulted in increased expression of ARHGEF3 and its downstream effector RHOA, enhanced GTPase activity, and restoration of tubule formation.

Conclusion

This study suggests the potential of miR‐512‐3p in EVs from plasma as a diagnostic biomarker of MMD. Downregulation of ARHGEF3, a target gene of miR‐512‐3p, may be involved in the aberrant angiogenesis of MMD through downstream RHOA signaling.

PT01.42. Proteomic profiling of extracellular vesicles from lymphatic drainage fluid after optimized isolation reveals enriched tumor‐associated markers compared to plasma

Dr XINYU QU, Dr Leanne Leung, Dr Bojie Chen, Professor Zigui Chen, Professor Katie Meehan, Professor Jason Chan

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Oral cancers tend to metastasize to locoregional, tumor‐draining lymph nodes other than distant sites, and lymph node metastasis is the well‐known prognostic factor. Therefore, it is assumed that lymph‐rich drainage fluid is the proximal biofluid to the surgically resected tumor, and tumor‐related markers are more highly enriched in lymphatic fluid than in blood.

Methods: Matched plasma and lymphatic drainage fluid (LDF) samples were collected from oral cancer patients (n=9). Extracellular vesicles (EVs) were isolated by iodixanol density gradient ultracentrifugation followed by size‐exclusion chromatography, and then purified EVs were treated with trypsin. All EV samples were characterized and validated based on guidelines. The protein cargo of EVs was determined using a TMT‐based high‐resolution quantitative proteomic approach.

Results: Through an optimized isolation strategy, most lipoproteins and albumin were eliminated from EV samples. In LDF samples, a total of 3,771 EV proteins were identified and 3,596 of them were quantified, while in plasma samples, only 1,256 EV proteins were identified and 822 of them were quantified. More importantly, the plasma EV proteome is completely contained in the LDF EV proteome, and LDF EVs contain more head and neck cancer‐associated protein markers compared with plasma.

Summary/Conclusion: LDF is a better source of EV protein biomarkers and is superior to plasma. By combining iodixanol density gradient ultracentrifugation and size‐exclusion chromatography, we successfully isolated and purified EVs from LDF for proteomics analysis. Lymph liquid biopsy will enable earlier and more precise care following surgery and aid in post‐surgical treatment decisions.

PT01.45. Raman spectroscopy‐based profiling of plasma‐derived extracellular vesicles: a novel approach for differentiating cancerous diseases

Timea Boroczky, Matyas Bukva, Gabriella Dobra, Maria Harmati, Edina Sebestyen‐Gyukity, Yasmin Ranjous, Laszlo Szivos, Katalin Hideghety, Krisztina Budai, Judit Olah, Peter Horvath, Gyorgy Lazar, Zoltan Konya, Pal Barzo, Almos Klekner, Krisztina Buzas

Poster Pitches (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:45 PM ‐ 1:00 PM

Introduction

Investigation of the molecular composition of small extracellular vesicles (sEVs) for tumor diagnostic purposes is gaining popularity, particularly for diseases with difficult diagnosis, such as central nervous system malignancies. Using spectroscopy analysis for diagnosing cancerous diseases is promising, but yet an underexplored method. Therefore our aim is to elucidate the potential role of plasma‐derived sEVs in diagnosing seven distinct patient groups using a sufficient number of clinical samples and Raman spectroscopy.

Methods

The study was conducted in accordance with the Declaration of Helsinki, informed consent forms were collected and the study was approved by national ethics committee. Up to 532 plasma samples from seven patient groups (glioblastoma multiforme, meningioma, melanoma and non‐melanoma brain metastasis, colorectal tumors, melanoma and lumbal disc herniation patients as a control group) were collected. SEV isolation was performed through differential centrifugation. The isolates were characterized by Western Blot, transmission electron microscopy and nanoparticle tracking analysis. For Raman spectra classification Principal Component Analysis–Support Vector Machine algorithm was used. Classification accuracy, sensitivity, specificity and the Area Under the Curve (AUC) value were used to evaluate the performance of classification.

Results

According to our results, there are no significant differences in the particle numbers belonging to the 7 patient groups. Raman measurements indicate that the patient groups are distinguishable with 80–95% sensitivity and 61–100% specificity. AUC scores of 0.63–1 suggest excellent classification performance. The highest sensitivity and specificity can be reached with the comparison of the malignant brain tumors and the control group.

Summary/Conclusion

Our findings indicate that Raman spectroscopic analysis of sEV‐enriched plasma isolates is a promising strategy for the development of noninvasive and cost‐effective methods supporting the clinical diagnosis of various cancers.

PT01.46. Revealing urinary exosomal eiomarkers in progressive NAFLD: proteomic analysis in a rat model

Chao‐Yuan Chang, Visiting Staff Chun‐Jen Huang, Visiting Staff Syuan‐Hao Syu, Visiting Staff Tze‐Sian Chan

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

Disease progress of non‐alcoholic fatty liver disease (NAFLD) to liver fibrosis is often asymptomatic, challenging the effectiveness of blood‐based biomarker tests. The use of urine samples for biomarker measurement presents a promising alternative. Exosomes, nanosized double‐membrane particles containing nucleic acids, microRNAs, and proteins, hold potential as clinical biomarkers. However, the cargo of urine exosomes in NAFLD remains largely unexplored. This study aims to elucidate established biomarkers in urine exosomes across three stages of NAFLD, employing a high‐fat diet/fructose‐induced NAFLD and liver fibrosis rat model.

Methods:

Male Sprague‐Dawley rats were randomly assigned to receive a normal diet (ND group) or high‐fat diet/fructose feeding for 6, 12, or 18 weeks (HFr/HFD_E, HFr/HFD_M, or HFr/HFD_L groups, respectively). Following euthanasia, liver tissues and urine samples were collected. Liver histopathological assessment included hematoxylin & eosin, Oil Red, and Masson's trichrome staining for NAFLD activity score, fat accumulation, and fibrosis. Urine exosomes were isolated through ultra‐centrifugation. Proteomic analysis of urinary exosomes utilized liquid chromatography–tandem mass spectrometry, followed by MetaboAnalyst 5.0 and Ingenuity Pathway Analysis (IPA).

Results:

Histopathological analysis revealed a significant increase in NAFLD activity score in the HFr/HFD_E, HFr/HFD_M, and HFr/HFD_L groups compared to the ND group as the disease progressed with longer feeding duration (all p<0.05). Oil Red staining showed significantly higher levels of fat accumulation in the HFr/HFD_M and HFr/HFD_L groups compared to both the HFr/HFD_E and ND groups (all p<0.05). Masson's trichrome staining depicted significantly higher fibrosis levels in the HFr/HFD_L group than the other 3 groups (all p<0.05). These data confirmed that a high‐fat diet/fructose diet induces NAFLD in rats, progressing to liver fibrosis in 16 weeks. Proteomic profiling identified 675 proteins with expression variations among the groups. MetaboAnalyst 5.0 demonstrated distinct clusters, and IPA revealed altered upstream regulators in urinary exosomes. Notably, hepatocyte nuclear factor 4 alpha, angiotensin, tumor necrosis factor‐α, interleukin‐1β, and interleukin‐6 displayed increased expression with disease progression.

Conclusion:

This study explores potential NAFLD biomarkers in urine exosomes across disease stages using proteomic analysis, offering insights for non‐invasive detection and monitoring of NAFLD progression.

PT01.48. Small extracellular vesicle (sEV) proteins as a potential biomarker for endometriosis

Dr Hannah Nazri, Dr Raphael Heilig, Associate Professor Roman Fischer, Professor Benedikt Kessler, Dr Kavita S Subramaniam, Professor Christian Becker, Dr Thomas Tapmeier

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

Endometriosis, defined as ectopic endometrial‐like tissue, causes pain and/or subfertility in 10% of reproductive‐age women. The cause is unknown, resulting in inadequate diagnostic methods (no clinically relevant biomarker) and treatment. The sEV‐protein cargo, described in cancer, diabetes and pre‐eclampsia, could similarly serve as an endometriosis biomarker.

Methods:

Peritoneal fluid (PF) samples were obtained from 18‐49‐year‐old women (n=63) who were investigated for abdominal/pelvic pain and/or subfertility via diagnostic laparoscopy within the ENDOX study at the Endometriosis CaRe Centre, Nuffield Department of Women's and Reproductive Health, University of Oxford (REC 09/H0604/58). PF was collected and classified according to cycle phase (proliferative/secretory/menstrual) and endometriosis severity (ASRM stages I+II/III+IV) according to WERF EPHect standards. Exclusion criteria were hormonal treatment, malignancy, pregnancy, breastfeeding, and an inability to understand the consent form. PF was centrifuged to remove cells, debris, and microvesicles. sEVs were isolated using size exclusion chromatography (SEC) and analysed by nanoparticle tracking analysis (NTA), immunoblotting, and mass spectrometry.

Results:

We confirmed the presence of sEVs in PF of women at different endometriosis stages and from disease‐free women in different menstrual cycle phases by NTA, immunoblotting and mass spectrometry. Enriched sEVs were positive for ALIX, CD9, and syntenin. The endometriosis PF‐derived sEV mode size was 130±8.7 nm (n=44), compared to 134±2.12 nm in controls (n=19). Irrespective of the menstrual cycle phase, sEV concentrations were highest in stage III‐IV endometriosis (n=19), followed by stage I‐II endometriosis (n=25) and controls (P=0.0210) (n=19). sEV concentrations in stage I‐II endometriosis were highest in proliferative compared to secretory cycle phases. Proteomic analysis found CD44, in sEVs a protein unique to endometriosis, contributing most to the separation of endometriosis and control samples (high variable importance projection, VIP score).

Conclusions:

PF‐derived sEV concentrations vary regardless of cycle phase and disease stage, and this difference appears to be reflected in the protein cargo. Correlating these findings to other, easily accessible biological fluids such as blood or urine will aid our understanding of endometriosis and in endometriosis biomarker identification.

PT01.49. Storage stability study of human urinary extracellular vesicles

MD Cahyani Gita Ambarsari, Professor MW Taal, MRCPCH MD(res) JJ Kim, Assistant Professor Dong‐Hyun Kim, Assistant Professor AM Piccinini

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Urinary extracellular vesicles (uEVs) are potentially clinically valuable biomarkers, particularly in patients with kidney and urinary tract problems. uEVs encapsulate their cargoes, including nucleic acids, proteins and metabolites, protecting them from enzymatic degradation. We implemented methods reported by previous studies for collecting, storing, and preserving uEVs to examine particle concentration stability in random morning urine samples stored for 6 months.

Methods

Fresh urine samples were collected from seven healthy volunteers, three females and four males, 100 mL each. Two tablets of cOmpleteä Mini Protease Inhibitor co*cktail tablet (Product No: 11836153001 Roche) were added to each urine container. Pre‐processing to remove cells and cell debris was done within 4 hours from sample collection by centrifugation at 800xg at 4°C for 10 minutes with a swing‐bucket rotor. Samples were divided into aliquots of 10 mLs each for uEV isolation at 0 and 6 months post collection and stored at ‐80°C. We isolated uEVs using three different commercial kits, which use precipitation‐ (Total Exosome Isolation (from urine) Reagent (TEIR; Invitrogen)), pH and precipitation (Urine Exosome Purification Kit (UEPK; Norgen)), and size‐exclusion chromatography (SEC)‐based methods (IZON), respectively. Cell‐free urine samples were defrosted overnight at 4°C on ice for uEV isolation and quantification. uEV isolation was conducted following manufacturers’ instructions. uEV characterization, including quantification and size distribution assessment, was done using nanoparticle tracking analysis (NTA) with Zetaview PMX‐120.

Results

Performing NTA analysis, we found that particle concentration after 6 months of storage was not different from baseline. uEV particle size distributions at 0‐month and 6‐month post collection were also similar. Among the three kits utilized, Total Exosome Isolation (from urine) Reagent (TEIR; Invitrogen) resulted in the highest uEV concentration, followed by SEC (IZON) and Urine Exosome Purification Kit (UEPK; Norgen).

Summary/Conclusion

Protease inhibitors for preservation, pre‐processing done within 4 hours after sample collection, and storage at ‐80°C may be useful for future long‐term uEV‐derived biomarker studies.

PT01.51. SWATH‐MS identified differentially expressed proteins in extracellular vesicles isolated from pleural effusions of Malignant Pleural Mesothelioma

Dr. Kelly Tian Mun Chee, Prof. Kwun M Fong, Prof. Ian A Yang, Assoc. Prof. Rayleen V Bowman

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Malignant pleural mesothelioma (MPM) is a rare type of cancer affecting serosa that lines body cavities, most commonly pleura. The extracellular vesicle (EV) compartment of pleural fluid could be selectively enriched with protein cargo specific for pathologic entities responsible for pleural effusions. The protein content of the EV contained within pleural fluid has not been extensively studied. Therefore, the protein cargo of pleural fluid EV was studied with the aim of identifying specific protein candidates of MPM capable of distinguishing it from other causes of pleural effusion.

Methods

This study was approved by The Prince Charles Hospital ethics committee (HREC/18/QPCH/312). Pleural fluid was centrifuged at 800 x g for 10 minutes at 4 ⁰C, followed by ultracentrifugation at 100,000 x g (w2t = 5.46e10) for 1 hour 40 minutes at 4⁰C to isolate EV. Sequential Window Acquisition of all Theoretical fragment‐ion spectra ‐ Mass Spectrometry (SWATH‐MS) was performed on 19 pleural effusion cases of MPM (n=6), lung adenocarcinoma metastatic to the pleura (LUAD; n=5), breast cancer metastatic to the pleura (BRCA; n=4) and non‐malignant conditions (NM; n=4). R package LIMMA algorithm based on empirical Bayesian methods was used for differential expression analysis.

Results

Analysis of all 19 PFEV samples collectively showed a total of 830 proteins, 10477 peptides, and 76042 spectra detected at 5% local FDR. The number of proteins detected in each disease state were 630 (MPM), 439 (LUAD), 406 (BRCA) and 625 (NM), respectively. Four proteins (LG3BP, FCGBP, MXRA5 and STOM) demonstrated greatest potential as protein marker candidate for MPM. The level of LG3BP was higher in MPM than in any other cause of pleural effusion: p‐values of 0.0087 (MPM‐LUAD) and 0.0095 (MPM‐BRCA), and 0.0095 (MPM‐NM). FCGBP was higher in MPM than in LUAD and BRCA (p‐values of 0.03 and 0.038, respectively), while MXRA5 and STOM levels were reduced in MPM PFEV compared with BRCA (p=0.019 and p= 0.038, respectively) and NM (p=0.038 and p=0.038, respectively).

Conclusions

The protein candidates warrant further testing to determine whether they could be useful components of a panel of protein biomarkers with diagnostic utility for patients with pleural effusions.

PT01.52. Systemic changes in Immune System‐Related Plasma Extracellular Vesicles During Healthy Aging

Dr. Xin Zhang, Dr. Sisi Ma, Syeda Iffat Naz, Janet Huebner, Dr. Erik Soderblom, Noor Alnemer, Dr. Constantin Aliferis, Dr. Virginia Kraus

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: We previously reported that multiple immune cell‐associated circulating extracellular vesicle (EV) subpopulations declined with aging in healthy humans and mice. In this study, we aimed to identify systemic changes in immune system‐related plasma EVs during healthy aging.

Methods: Plasma was isolated from blood of younger (18‐31 years, n=6) and older (47‐83 years, n=22) healthy donors by centrifugation at 3000 rpm for 15 minutes at 4°C to remove cells and debris, and stored at −80°C. Frozen plasma was completely thawed and centrifuged at 2000 g for 10 minutes at 4°C to remove remaining debris; EVs and EV‐depleted supernatants were separated by polymer‐based precipitation. EVs were validated to have a bilayer structure and size diversity, and carry mitochondria, traditional EV markers (CD81, CD9, CD63, CD29), and hematopoietic cell‐related markers (CD4, CD8, CD56, CD15, CD14, CD68, CD19, CD235a, CD41a, CD31, CD34, HLA‐ABC, HLA‐G, HLA‐DRDPDQ) with various frequencies. We measured concentrations of endo‐EV (in EVs) and exo‐EV (in EV‐depleted supernatants) cytokines by ELISA. The endo‐EV proteome was quantified by high‐resolution mass spectrometry. High‐resolution flow cytometry was used to quantify the frequency of EV subpopulations carrying the tested surface markers, and internalization of PKH67 pre‐labeled EVs by WI‐38 fibroblasts and their change in proliferation.

Results: Compared with matched exo‐EV supernatants, endo‐EV contents had higher TNF‐α and IL‐27, lower IL‐6, IL‐11, IFN‐γ, and IL‐17A/F, and similar concentrations of IL‐1β, IL‐21, and IL‐22. There were significant correlations of endo‐EV and exo‐EV TNF‐α, IL‐27, IL‐6, IL‐1β, and IFN‐γ. Endo‐EV IFN‐γ and exo‐EV IL‐17A/F and IL‐21 significantly declined with age. Age was significantly associated with EV peptides, positively (n=37) and negatively (n=257); the corresponding age‐related EV proteins were predominately enriched in liver and innate immune system. WI‐38 fibroblasts (>95%) internalized similar amounts of both young and old EVs. Compared to cells that did not take up PKH67‐EVs, the cells with PKH67‐EVs (that internalized EVs), particularly young EVs, underwent greater cell proliferation.

Conclusions: Our results identify EV phenotypes reflecting immunosenescence during healthy aging and the ability of plasma EVs of young donors to stimulate proliferation of recipient cells in vitro. These EV biomarkers are candidates for future aging studies.

PT01.53. The altered levels of urinary extracellular vesicles pre‐ and post‐surgery relative to proteomics change in breast cancer patients

Miss Nilobon Jeanmard, Dr. Rassanee Bissanum, Mr. Kittinun Leetanap*rn, Mr. Pongsakorn Choochuen, Assoc.Prof. Hutcha Sriplun, Miss Sawanya Charoenlappanit, Dr. Sittiruk Roytrakul, Assoc.Prof. Raphatphorn Navakanitworakul

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Urinary extracellular vesicles (uEVs) have shown great promise as non‐invasive biomarkers for various cancerous diseases due to their ability to reflect pathological states along the course of the disease and treatment. Thus, this study aimed to evaluate the uEVs concentration and uEVs proteomes in BC patients before and after surgery.

Methods: Urine samples were collected from BC patients (n = 30) at two time points: before surgery and after surgery. The uEVs were isolated using differential ultracentrifugation and were then characterized by western blot and TEM. The concentration of uEVs was measured by nanoparticle tracking analysis (NTA). LC‐MS/MS was utilized for proteomic analysis of uEVs.

Results: The uEVs concentrations of individual BC patients were compared before and after surgery. In comparison to pre‐surgery, we observed two trends in the uEVs level after surgery: an increase and a decrease from pre‐surgery. The eight BC patients had an elevated uEV level, whereas the twenty‐two BC patients had a decrease of uEVs concentration after surgery. Proteomic analysis of uEVs clarified the differentially expressed proteins (DEPs) between pre‐ and post‐surgery samples, which were grouped into an increasing trend (sig‐up 26, sig‐down 35) and a decreasing trend (sig‐down 121, sig‐up 121). The heatmap between the pre‐ and post‐surgery samples revealed remarkable patterns of protein expression. It's interesting to note that while cancer‐associated proteins were down‐regulated in post‐surgery samples, which showed a declining trend of uEVs concentration, the significantly up‐regulated proteins in post‐surgery samples were mapped to proteins in cancer‐related pathways annotated by the KEGG database that might be related to the increase of uEVs after surgery.

Conclusion: The alteration of uEVs concentration pre‐ and post‐surgery revealed the dissimilarity of uEVs proteomes and might be relative to pathological changes in breast cancer patients.

PT01.54. The circulating extracellular vesicles in ovarian cancer study

Dr Andrew Lai, Dr Dominic Guanzon, Dr Shayna Sharma, Mrs Katherin Scholz‐Romero, Dr Yaowu He, Mr Weitong Huang, Dr Tanja Pejovic, Dr Carmen Winters, Professor Terry Morgan, Professor Jermaine Coward, Associate Professor Amy McCart Reed, Professor Sunil Lakhani, Professor Andreas Obermair, Professor Amanda Barnard, Professor Anna deFazio, Professor Lewis Perrin, Professor John Hooper, Professor Gregory Rice, Professor Carlos Salomon

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Extracellular vesicles (EVs) contain bioactive molecules, making them strong candidates as disease biomarkers. Ovarian cancer (OVCA) survival rates have remained stagnant over the past two decades due to the absence of clinically useful biomarkers for early detection. This study aims to evaluate their sensitivity and specificity in accurately classifying women with OVCA using Machine Learning (ML).

Methods: A total of 926 patients were analysed between 2016 and 2022. Plasma samples were collected, and patients were randomly assigned into three groups: 1) Training set with cross‐validated models (n=220), 2) Validation set (n=235), and 3) Assay refinement (n=471). EVs were isolated using size exclusion chromatography and a capture immunoaffinity magnetic bead‐based system compatible with routine pathology testing and characterised in compliance with MISEV2018. The EV content (miRNAs, proteins, lncRNA, miscRNA, MtrRNA, MttRNA, rRNA, scaRNA, snRNA, and tRNA) was used as ML classifiers. EV biomarker stability was evaluated in a heterogeneous population of OVCA cell lines in the absence or presence of RNase/proteinaseK. All procedures were conducted in compliance with ISO17025.

Results: Ten classifiers were trained in group 1 to detect cancer, benign, and healthy patients. Across all groups, a sensitivity of 98% specificity (2% false‐positive rate) was reported, with statistical comparisons made for each of the 10 classifiers independently and in combination. The most sensitive classifiers at 98% specificity for detecting cancer were miRNAs (training: 78%, validation: 82%), proteins (training: 85%, validation: 90%), and the pan‐feature classifier (training: 90%, validation: 95%). Notably, identifying healthy controls exhibited high sensitivity (95% CI 84%‐100%) compared to cancer and benign conditions. An Early Identification Multivariate Index Assay was developed using data from stages I and II OVCA patients and healthy controls, achieving consistent performance in the training set (95%CI, 88%‐100%), validation set (95%CI, 90%‐100%), and assay refinement (95%CI, 80%‐95%). Hierarchical clustering identified two distinct clusters based on OVCA cell line stability: A) CAOV‐3, TOV‐112D; B) HEY, OVCA‐420, OVCAR‐3, SKOV‐3, OVCA‐429, OVTOKO, OV90.

Conclusions: This study is the most comprehensive investigation of EVs in ovarian cancer. Given the potential value of early detection in OVCA, further evaluation of this test is warranted in prospective population‐level studies.

PT01.58. Unique lipidomic profile sets Extracellular vesicles apart from other cellular fractions in ovarian cancer

Ms Shikha Rani, Dr Andrew Lai, Dr Dominic Guanzon, Mr Kaltin Ferguson, A/Prof Lewis C. Perrin, Prof John D. Hooper, Prof Carlos Salomon

Poster Pitches (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:45 PM ‐ 1:00 PM

Introduction:

Lipids serve a dual role, acting as fundamental structural components of extracellular vesicles (EVs) and vital messengers in intercellular communication. They potentially influence the reprogramming of recipient cells during cancer progression. This study aimed to optimize a lipidomic approach for identifying and quantifying a broad range of plasma‐derived EV‐associated lipid classes, including phospholipids, sphingolipids, glycerolipids, and cholesterols. It aimed to distinguish EVs from other cellular reservoirs and evaluate the EV‐lipid signature across the progression of ovarian cancer.

Methods:

EV and non‐EV fractions were isolated from plasma samples of healthy donors using size exclusion chromatography (SEC). Characterization involved size analysis, the abundance of classical protein markers, and morphology assessment using nanoparticle tracking analysis (NTA), immunoblotting, and transmission electron microscopy. Samples were spiked with SPLASH® II LIPIDOMIX® Mass Spec Standard, and lipid extraction used solid‐phase extraction (SPE) cartridges. Targeted lipidomic analysis was performed on a Shimadzu Nexera UHPLC/5500QTRP tandem mass spectrometer system. Clinical relevance was assessed in a high‐risk ovarian cancer population (n=97).

Results:

Comparative lipidomic mass spectrometry (LC‐MS) analysis revealed distinct enrichment of phospholipids, sphingolipids, and glycerolipids within plasma‐derived EVs compared to non‐EV fractions. Abundant phospholipids such as phosphatidylcholine, lyso‐phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylserine were identified in EV fractions. Additionally, EVs exhibited notable enrichment of sphingomyelins, ceramides, and diacylglycerol, distinguishing them from other vesicular pools. Specific changes in EV lipid composition associated with disease stage and ovarian cancer histotype were identified.

Summary:

Our findings highlight the unique abundance of molecular lipid species within plasma‐derived EVs. Characterizing the EV lipidome holds potential for the development of improved diagnostic and prognostic biomarkers for ovarian and various gynaecological cancers.

PT01.59. Unlocking ovarian cancer detection: Long‐read sequencing reveals promising biomarkers using extracellular vesicle DNA methylation and mutation patterns

Dr Dominic Guanzon, Dr Subash Rai, Mr Rakesh Sankar, Ms Pragati Lodha, Ms Vidya Gummagatta, Dr Andrew Lai, Professor Lewis Perrin, Professor John Hooper, Professor Carlos Salomon

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

Extracellular vesicles (EVs) have emerged as significant carriers of molecular biomarkers and mediators of intercellular communication. While research has concentrated on understanding the protein and RNA content of these EVs, the role of EV DNA remains inadequately explored. The potential functional aspects of EV DNA have been proposed in pathological conditions such as cancer, highlighting its relevance as a promising biomarker. Moreover, increasing utilization of cell‐free DNA as a diagnostic tool in liquid biopsy applications underscores the potential importance of EV DNA as a novel and noteworthy biomarker for ovarian cancer (OVCA) detection.

Methods:

For initial optimisation, the EV and cell‐free components were isolated from normal (n=3) and ovarian cancer (n=3) plasma using size‐exclusion chromatography, and characterized using Nanoparticle Tracking Analysis and Bicinchoninic acid assay. The DNA was extracted, quantified and sizing of DNA fragments performed using Qubit dsDNA and DNA ScreenTape assays. Long‐read Oxford Nanopore sequencing was used to comprehensively characterize and compare EV DNA with cell‐free DNA. A larger independent cohort (n=30) with different OVCA histotypes will be used to refine DNA methylation and mutation signatures in circulating EVs.

Results:

The average particle concentration within the EV fraction was 1.96e+09 particles/mL for normal and 5.4e+09 particles/mL for cancer patients. Among normal patients, the average DNA concentration was 1.68 ng/µL and 2.29 ng/µL for EV DNA and cell‐free DNA, respectively. Conversely for cancer patients, the average DNA concentration was 1.35 ng/µL and 1.63 ng/µL for EV DNA and cell‐free DNA, respectively. Nanopore long‐read sequencing revealed a median read length of 200 base pairs for cell‐free DNA, and 350 base pairs for EV DNA. Notably, methylation signatures were observed in EV DNA of cancer patients, contrasted with the absence of this signature in normal patients, for specific genomic loci.

Conclusion:

This research marks the first effort to utilize long‐read sequencing for EV DNA analysis to investigate methylation and mutation patterns in ovarian cancer. The results indicate that EV DNA holds significant promise as a biomarker for diagnosing ovarian cancer. This study lays a foundation for further exploration and validation of EV‐based DNA biomarkers in cancer diagnostics and treatment monitoring.

PT02.01. Altered protein nitrosylation patterns in extracellular vesicles isolated from activated microglia

Dr Natasha Vassileff, Dr Jereme Spiers, Miss Sarah Bamford, Dr Rohan Lowe, Dr Keshava Data, Professor Paul Pigram, Professor Andrew Hill

Poster Pitches (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:45 PM ‐ 1:00 PM

Introduction:

Neuroinflammation is a common feature of neurodegenerative conditions, often appearing very early in disease pathogenesis. Microglial activation is known to be a prominent initiator of neuroinflammation and can be induced through lipopolysaccharide treatment (LPS). This activation leads to the expression of the inducible form of nitric oxide synthase (iNOS), resulting in the production of nitric oxide (NO). NO targets cysteine thiols resulting in post‐translational S‐nitrosylation, which can alter the target protein's function. Furthermore, packaging of these NO‐modified proteins into Extracellular Vesicles (EVs) propagates the neuroinflammatory phenotype by allowing the exertion of NO signalling in distant locations. Despite this, the NO‐modified proteome of activated microglial EVs has not been investigated. Therefore, this study aimed to identify the effect NO signalling exerts through protein post translational modifications in neuroinflammation.

Methods:

Extracellular Vesicles (EVs) were isolated from LPS‐treated microglia using differential ultracentrifugation with a sucrose density gradient. This was followed by characterisation to meet the Minimal Information for Studies of EVs 2018, including nanoparticle tracking analysis, electron microscopy and western blot analysis. The EVs subsequently underwent novel advanced surface imaging using time of flight‐secondary ion mass spectrometry (ToF‐SIMS) in addition to, iodolabelling and comparative proteomic analysis to identify post‐translation nitrosylation changes.

Results:

ToF‐SIMS imaging successfully identified NO modified cysteine thiol side chains in the EV proteins isolated from LPS treated microglia. Additionally, the iodolabelling proteomic analysis revealed the EVs from LPS‐treated microglia carried nitrosylated proteins indicative of neuroinflammation, microglial activation, and protein clearance pathway regulation. These included known NO‐modified proteins and those associated with LPS‐induced microglial activation that may play an important role in neuroinflammatory communication.

Conclusions:

Together, these results show activated microglia are capable of exerting broad signalling changes through the selective packaging of EVs during neuroinflammation.

PT02.02. Antidepressant effects of aerobic exercise: are circulating EVs responsible?

Reine Khoury, Dr. Dariusz Zurawek, Gabriella Frosi, Assistant Professor Corina Nagy

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Exercise, a potent non‐drug therapy, maintains mental health and has been used in the treatment of depression, anxiety, and schizophrenia, among others. Exercise is acknowledged for promoting brain plasticity, memory, and delaying neurodegeneration. Aerobic exercise's positive effects are thought to be driven by “exerkines” (exercise factors) including metabolites, proteins, nucleic acids, and hormones. The precise mechanisms behind exercise's benefits for MDD are not fully understood. The literature indicates that exercise might directly or indirectly affect the brain by releasing small extracellular vesicles (sEVs) into the bloodstream. However, studies on exercise‐induced sEVs often lack control in timepoints, sex, exercise duration, and sEV collection timing.

Methods: Mice underwent a light/dark switch for two weeks before starting a treadmill exercise regimen. A week of low‐intensity training preceded a 2‐week treadmill program at 10m/min x 60min/d x 5d/week, followed by 15m/min x 60min/d x 5d/week. Sedentary mice spent an hour daily on a stationary treadmill for two weeks. Daily body weight was recorded. Mice were sacrificed pre‐exercise, immediately post‐exercise, and 3h, 9h, and 18h post‐exercise. Trunk blood was collected, centrifuged, and plasma obtained. Skeletal muscle, liver, and brain tissues were snap‐frozen on dry ice. Plasma was clarified, and EVs were isolated using 70nm qEV. EVs were isolated directly from muscle using collagenase D, centrifugation, and 70nm qEV columns. Western blotting, TEM, and NanoSight assessed EV quality. Small RNA was assessed using sequencing adaptors, and protein was quantified using LC‐MS/MS.

Results: We anticipate EVs from exercise will differ from sedentary conditions, primarily in cargo rather than size or concentration. Given the sex specific effects of exercise on health and behaviour, we expect sex‐specific effects of exercise on EV cargo, including an initial increase in inflammatory markers in females and a later increase in males.

Summary: Examining EVs and their contents within controlled exercise paradigms in vivo is an essential initial exploration into how context‐dependent EV release may contribute to exercise's beneficial effects. The crucial next phase involves exploring whether myocyte‐released EVs can cross the blood‐brain barrier, potentially exerting therapeutic effects.

PT02.03. BDNF/TrkB system dysregulation at the cell environment: extracellular vesicles as carriers of TrkB‐ICD in Alzheimer's disease

Mr. Tiago Costa‐Coelho, João Fonseca‐Gomes, Gonçalo Garcia, Mafalda Ferreira‐Manso, Catarina B. Ferreira, Carolina de Almeida‐Borlido, Juzoh Umemori, Mikko Hiltunen, Eero Castrén, Ana M. Sebastião, Alexandre de Mendonça, Dora Brites, Maria José Diógenes

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: The neuroprotective BDNF/TrkB‐FL system is compromised in Alzheimer's disease (AD). Amyloid‐beta triggers calpain‐mediated TrkB‐FL receptor cleavage, giving rise to the formation of TrkB‐ICD, a novel intracellular toxic fragment. Biological samples are used to pinpoint potential disease biomarkers and within these, extracellular vesicles (EVs) are cell‐specific carriers of promising pathological hallmarks. Thus, this work aims to investigate the presence of extracellular TrkB‐ICD in the post‐mortem brain and CSF samples from AD patients, as well as in EVs from plasma samples of AD patients and neuroblastoma cells.

Methods: Human AD patient post‐mortem brain samples were collected for RNA and protein extraction and classified according to the Braak staging – stages 0‐II, III‐IV. For CSF and plasma samples, patients fulfilled the criteria for Mild Cognitive Impairment (MCI, controls) or MCI due to AD (MCI/AD). Such controls reported cognitive complaints despite showing no Aβ deposition nor neuronal injury. CSF AD biomarkers, neuropsychological analysis, and brain imaging were used for patient characterization. Plasma‐derived EVs (pdEVs) from the same MCI/AD cohort were isolated using the ExoQuick reagent and characterized. In parallel, EVs from 48‐hour conditioned medium of control, GFP‐ and TrkB‐ICD‐V5‐transduced (ICD‐V5) differentiated SH‐SY5Y cells were isolated through differential ultracentrifugation. Large (lEVs) and small (sEVs) EVs were characterized and, together with the concentrated EV‐depleted secretome, probed for ICD‐V5 and the endogenous TrkB‐ICD fragments.

Results: Human post‐mortem samples revealed a decrease in TrkB‐FL protein levels concomitant with an increase in the TrkB‐ICD fragment levels (p=7.3 x10‐3 and 3.9 x10‐3, n=7‐11). CSF analysis showed increased TrkB‐ICD immunoreactivity in MCI/AD patients (p=7.55x10‐3, n=23‐47), and a negative correlation between the levels of Aβ1‐42 and TrkB‐ICD (ρ=‐0.47, n=47). Regarding EV presence, plasma of MCI/AD patients contained higher levels of TrkB‐ICD (p=0.010, n=17‐18). TrkB‐ICD and ICD‐V5 were detected in both SH‐SY5Y EV subpopulations equally (p>0.05, n=3). Importantly only the endogenous TrkB‐ICD fragment was detected in the EV‐depleted secretome.

Conclusions: Altogether, these data demonstrate TrkB‐ICD extracellular secretion, alluding for its potential toxicity dissemination. TrkB‐ICD as a proxy of BDNF/TrkB‐FL dysregulation may prove itself as a relevant hallmark of AD in the future.

PT02.04. Brain region‐specific changes in extracellular vesicles release and composition in tau R406W human organoid tauopathy model

Dr. Tina Bilousova, Nina Knitowski, Dr. Qing Cao, Shengkai Zhao, Swetha Atluri, Mikhail Melnik, Achyutha Kodavatikanti, Dr. Ranmal Samarasinghe, Dr. Jessica Rexach, Dr. Karen Gylys

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Progress in the development of organoid culture models opens up a new avenue for investigating extracellular vesicle (EV) biogenesis, cargos, and their role in intercellular communications in health and diseases allowing a more natural 3D environment without a risk of EV sample contamination with intracellular vesicles from tissue dissociation. Utilizing an organoid tauopathy model, our overall goal is to evaluate the role of EVs in immune cell reprogramming, leading to the establishment of a permissive environment for tau pathology spread and neuronal circuit disruption.

Methods: We compared four different methods of EV isolation from brain organoid culture media, including ultracentrifugation, polymer‐based precipitation, size exclusive chromatography, and immunoprecipitation. Levels of exosomal markers and immune checkpoint molecules were evaluated in EVs isolated from cortical (CTX), ganglionic eminence (GE), and fused CTX+GE organoid cultures derived from tau R406W (frontotemporal dementia mutation) and isogenic control human iPSCs.

Results: Pan‐exosomal immunoprecipitation, utilizing three tetraspanins enriched in exosomes (CD63, CD9, and CD81), was chosen for analysis as the most successful method of organoid EV isolation. We observed region‐ and genotype‐specific changes in CD63 and CD47 expression in EVs isolated from the organoid cultures. There was a significant upregulation of CD63 in EV fractions from mutant CTX and CTX+GE organoid cultures compared to isogenic control, coinciding with a decrease in CD63 levels in mutant GE‐derived EVs. These results indicate a differential effect of the tau R406W mutation on EV biogenesis and/or cell viability of excitatory (CTX) and inhibitory (GE) neurons and/or a region‐specific effect on glial cells. An upregulation of CD47, but not another immune checkpoint molecule, PDL‐1, in EVs isolated from mutant CTX+GE fused organoid cultures (Day 120), suggests a potential role of exosomal CD47 in immune cell regulation through interactions with its cognate receptor, signal regulatory protein alpha (SIRPα). Further experiments are required to evaluate this hypothesis.

Summary: Most neurodegenerative disorders, including tauopathies, are characterized by endo‐lysosomal dysfunctions and dysregulation of intercellular communications. The accompanied changes in EV biogenesis and composition are part of the disease mechanism, and a better understanding of their role can provide potential therapeutic targets and biomarkers for disease progression.

PT02.05. Cerebrospinal fluid extracellular vesicle miRNAs identify synaptic transmission alterations in Alzheimer's disease

PhD Ursula Sandau, Trevor McFarland, PhD Sierra Smith, MD Douglas Galasko, MD Joseph Quinn, MD, PhD Randy Woltjer, PhD Julie Saugstad

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Alzheimer's disease (AD) is the most common form of dementia and is the fifth leading cause of death for individuals aged 65 and older. With only six FDA approved therapies, none of which cure the disease, there is an urgent need to identify new therapeutic targets. Cerebrospinal fluid (CSF) contains brain derived EVs with cargo that reflects disease alterations. Thus, we performed a target prediction analysis with human CSF EV miRNA data from AD and control (CTL) donors, to identify relevant gene targets that we show are altered in human AD brain and may contribute to pathogenesis.

Methods: CSF from living donors (AD n=28; CTL n=28) was fractionated by size exclusion chromatography and characterized by transmission electron microscopy; immunoblot for EV and non‐EV markers (e.g., flotillin, CD81, apolipoproteins); and tunable resistive pulse sensing. 190 miRNAs were assessed by qPCR using primer‐probe arrays and those significantly changed in AD (FDR corrected) were used for target prediction and Ingenuity Pathway Analysis. Human postmortem frontal cortex (FC) and hippocampus from AD and CTL was used for miR‐16‐5p staining and immunoblot analysis of SNAP‐25 and MUNC18‐1.

Results: In AD CSF EVs, four miRNAs (miR‐16‐5p, ‐331‐3p, ‐409‐3p, ‐454‐3p) were significantly increased compared to CTLs. CSF EV miR‐16‐5 expression was also found to be sex and genotype dependent, with significant increases specific to APOE4 positive AD females. We also found the miR‐16‐5p was expressed in human brain and trends towards increased expression in AD (n=4) white matter, compared with CTLs (n=3). Relevant to synaptic dysfunction in AD, miR‐16‐5p predicted targets included mRNAs integral to synaptic transmission (SNAP‐25, MUNC18‐1). In AD females both SNAP‐25 and MUNC18‐1 were significantly decreased in the hippocampus (n=17) and FC (n=12) compared with control females (hippocampus and FC, n=11). While in AD males there was only a significant decrease in FC (n=10) SNAP‐25 compared with CTL males (n=12) and no other differences (hippocampus: CTL n=11, AD n=17).

Summary/Conclusion: We demonstrate that miRNAs altered in CSF EVs are informative to changes that occur in brain and have the potential to identify targets that may serve as novel therapeutics to treat AD.

PT02.06. Eukaryotic and microbiota‐derived extracellular vesicles in Parkinson's disease

Tiana Koukoulis, Purnianto Adityas, David Finkelstein, Leah Beauchamp, Kevin Barnham, Dr Laura Vella

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Parkinson's disease (PD) is an increasingly common neurodegenerative disease. The etiology of idiopathic PD is complex and multifactorial involving environmental contributions, such as viral or bacterial infections and microbial dysbiosis, in genetically predisposed individuals.

A limited understanding of the molecular events that drive neuroinflammation and neurodegeneration causes a major hindrance in the development of efficacious treatments for PD. Understanding these systems and triggers of disease may provide novel biological drug targets for the development of neuroprotective treatments as well the discovery of biochemical biomarkers of preclinical disease.

Here, we will report on our preclinical Parkinson's disease research program that uses cell (bacterial, human and mouse) and animal models to elucidate the contribution of eukaryotic and microbiota‐derived extracellular vesicles to disease progression.

PT02.07. Exploitation of vitreous‐derived extracellular vesicles to study the central nervous system dynamics

Lien Cools, Dr. Cristiano Lucci, Sam Noppen, Dr. Charysse Vandendriessche, Drs. Kaat Verleye, Drs. Laura Raes, Elien Van Wonterghem, Prof. Inge Mertens, Prof. Dominique Schols, Prof. Roosmarijn E Vandenbroucke, Prof. Lies De Groef

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

The evolving role of extracellular vesicles (EVs) in intercellular communication within the central nervous systems (CNS) has sparked interest in using EVs as biomarkers, drug delivery tools and therapeutic targets for neurodegenerative diseases. Since established protocols to isolate EVs from CNS tissue are lacking, current research mainly uses cerebrospinal fluid or plasma, albeit both having drawbacks. Here, we propose using vitreous‐derived EVs to investigate CNS EVs. Being an integral part of the CNS, the retina has a similar cellular composition as the brain and (patho)physiological processes are conserved. Only separated by the inner limiting membrane, the vitreous humor is suggested to be a liquid biopsy of the retina, and is known to be rich in EVs. Although vitreous‐derived EVs are being studied in ophthalmological diseases, their application to study neurodegenerative processes has remained unexplored thus far.

Methods

Mouse vitreous‐derived EVs were isolated via size exclusion chromatography, and their concentration and size were characterized with nanoparticle tracking analysis and electron microscopy. Western blot for tetraspanin markers, TSG101 and calnexin was performed on retinal lysates and EV fractions. Lastly, glial and neuronal cell markers were assessed via a surface plasmon resonance bioassay. In a second step, retinal neuroinflammation and ‐degeneration was induced via lipopolysaccharide injections or optic nerve crush, and changes in EV dynamics were evaluated. An ongoing proteomic analysis will assess the EV cargo in all experimental conditions.

Results

EV concentrations isolated from 30µL vitreous comprised on average 10E10 particles/mL, with diverse morphologies and sizes ranging between 50‐350nm. EV fractions displayed several tetraspanin markers and TSG101, but not calnexin. Moreover, neuronal and glial EVs were identified in the vitreous humor. Lastly, neuroinflammatory or neurodegenerative stimuli impacted the EV release/uptake ratio, yet no discernible changes in EV size were found.

Summary

This study demonstrates the potential of the vitreous humor as a liquid biopsy to obtain and study retinal EVs. The presence of neuronal and glial EVs suggests that this biofluid provides unique opportunities to study CNS‐derived EVs, including their involvement in neuroinflammatory and neurodegenerative processes. Ongoing proteomics analysis will help to further unravel their cargo and functions.

PT02.09. How do tumour derived EVs interact with the maturing nervous system and lead to altered pain processing in cancer survivors?

Dr Hannah Jackson, Dr Anna Grabowska, Dr Victoria James, Dr Federico Dajas‐Bailador, Dr Beth Coyle, Dr Gareth Hathway

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

Childhood cancer‐related pain and its treatment lack sufficient clinical attention due to limited understanding of the underlying mechanisms. While adult literature has extensively studied chemotherapy‐induced peripheral neuropathic pain (CIPN), mechanisms in early life remain unexplored. In the CIPN literature there is a growing body of evidence for tumour derived factors altering pain processing. Our study aims to understand how tumour derived EVs interact with the developing nervous system and impact pain processing in childhood brain tumour survivors.

Methods:

We determined dose‐response curves and IC50 values for several medulloblastoma (MB) cell lines using standard‐of‐care chemotherapy drugs: vincristine, etoposide, cisplatin, and lomustine. EVs were isolated via size exclusion chromatography and characterised through western blotting, flow cytometry and transmission electron microscopy (TEM). We quantified the effects of chemotherapy on EV release using ZetaView analysis. We then tested whether EVs from relevant MB cell lines could influence axon development in primary mouse embryonic day 16.5 dorsal root ganglion (DRG) neurons in vitro after pre‐treatment with chemotherapy‐treated EVs.

Results:

EVs from chemotherapy‐treated and untreated cells exhibited similar size and shape. However, a combination of vincristine, etoposide, and cisplatin at low concentrations significantly increased EV secretion by MB cell lines. We are currently assessing the impact of these EVs on axonal length and growth in early‐phase neuronal cultures of primary DRG neurons.

Conclusion:

Standard chemotherapy drugs substantially enhanced EV release from MB cells, and co‐culture of embryonic DRG neurons with chemotherapy exposed EVs resulted in neuronal death. Our next step is to investigate whether administering these selected EVs in healthy animals alters pain responses and pain maturation, and to study the biodistribution of these EVs within the nervous system and body, after infusion into the cerebrospinal fluid as well as how EV cargo is altered by chemotherapeutics.

PT02.10. Inflammation‐associated microglial EVs exhibit morphological differences and enrichment for ribosomes

Mr William Phillips, Ms Irumi Amarasinghe, Dr Ebony Monson, Dr Nicholas Reynolds, Prof Karla Helbig, Dr Lesley Cheng, Prof Andrew F Hill

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Neuroinflammation is a fundamental aspect of multiple neurological conditions, including neurodegenerative conditions such as Alzheimer's disease and chronic traumatic encephalopathy (CTE). Microglia are the brain's primary immune cells involved with inflammation. Extracellular vesicles (EVs) are bilayered lipid nanoparticles that reflect a cell's state, making them a rich source of biomarkers. Omics methods, including proteomics, lipidomics, and small RNAseq, were used to evaluate changes in EV populations from LPS‐treated microglia. Small‐angle X‐ray scattering (SAXS) and cryo transmission electron microscopy (Cryo‐TEM) were used to comprehensively assess EVs from activated microglia and any associated morphology changes.

Methods

EVs were isolated from 200 mL of serum‐free conditioned media from either control or 50 ng/mL LPS‐treated SIM‐A9 murine microglial cells by tangential flow filtration and size‐exclusion chromatography. EV physical characteristics were examined using Zetaview nanoparticle tracking analysis and SAXS at the Australian Synchrotron. 30 Cryo‐TEM images were taken per replicate, and vesicles were quantified. LC/MS proteomics and lipidomics were conducted at the Bio21 Melbourne Mass Spectrometry and Proteomics Facility. Small RNA isolation was performed using established protocols, and RNAseq conducted an Ion GeneStudio S5.

Results

NTA measurements from treated and untreated cells did not reveal any significant changes in the size profile of the EVs. A lower particle count but larger size in LPS versus control was detected in both SAXS and Cryo‐TEM. Additionally, Cryo‐TEM saw more multi‐membraned vesicles compared to control. Demonstrating the utility of SAXS and Cryo‐TEM for evaluating EV morphology.

Proteomic analysis revealed enrichment for ribosomal components in EVs isolated from LPS‐treated cells and immune response components such as IL‐1β and the inflammasome component NLRP3. Comparing lipid composition between LPS treated and control also provided further insight into compositional EV changes during inflammation. Finally, small RNA analysis found multiple enriched small RNA, including mir‐146a, upregulated in LPS EVs compared to control.

Conclusions

This study reveals the dynamic and heterogeneous nature of EVs, both compositionally and morphologically, while highlighting their potential as valuable biomarkers for evaluating inflammation in the brain. These findings contribute significantly to understanding the role of microglial EVs during neuroinflammation and their potential role in a greater systemic context.

PT02.11. Isolation of spontaneously‐released brain extracellular vesicles: implications for stress‐driven brain pathologies

Dr Ioannis Sotiropoulos, Dr Patricia Gomes, Dr Cristian Bodo, Dr Carlos Noguera‐Ortiz, Dr Martina Samiotaki, Dr Minghao Chen, Dr Carina Soares‐Cunha, Dr Joana M. Silva, Dr Bárbara Coimbra, Dr George Stamatakis, Dr Liliana Santos, Dr George Panayotou, Professor Clarissa L. Waites, Proffessor Christos Gatsogiannis, Professor Nuno Sousa, Professor Dimitrios Kapogiannis, Dr Bruno Costa‐Silva

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: In the era of Precision Medicine, extracellular vesicles (EVs) exhibit great potential for the theragnostic of brain disorders such as Alzheimer's disease (AD), a complex disease with no effective treatment and poorly understood risk factors, where pathological heterogeneity and diverse clinical presentations complicate the development of precise patient‐tailored therapies. Thus, the collection and characterization of physiologically relevant EVs, as well as the study of its precipitating/risk factors, are of the utmost importance. However, standard brain EV isolation approaches rely on tissue dissociation, which can contaminate EV preparations with intracellular vesicles.

Methods: Based on a multiscale analysis, including cryo‐EM, label‐free proteomics, and ExoView, we hereby present a novel isolation method of small EVs (sEVs), named “release method”, based on their spontaneous release from the human and mouse brain tissue. Moreover, we have also tested the release method under conditions of chronic psychological stress, a known risk factor of AD.

Results: Our advanced EVs analysis demonstrated that the release method represents an efficient method that captures a small EV‐enriched population spontaneously released by brain tissue. In addition, we tested the significance of the release method under conditions where biogenesis/secretion of sEVs was pharmacologically manipulated and under exposure to chronic stress, a clinically‐relevant precipitant of AD. Here we found that the release method monitors the drug‐evoked inhibition or enhancement of sEVs secretion in a very sensitive manner, while chronic stress induced the secretion of Tau‐carrying brain‐derived EVs accompanied by memory loss and mood deficits suggesting a potential role of sEVs in the brain response to stress and progression of related stress‐driven brain pathologies, such as AD.

Summary/Conclusion: This spontaneous release method may contribute to the characterization and biomarker profile of physiologically relevant brain‐derived exosomes in brain function and pathology. Also, the clarification of the “footprints” of chronic stress on brain EVs may contribute to the identification of stress biomarkers helping towards early detection of stress‐related disorders. Given that the modern lifestyle increasingly exposes individuals to high stress loads, the understanding of the mechanistic link(s) between chronic stress and AD pathogenesis may facilitate both AD treatment and prevention.

PT02.12. Metabolic analysis of extracellular vesicles isolated from human brain tissue in Alzheimer's disease context

PhD student Patricia Hernández‐López, Dr. Elisabeth Rackles, Dr. Oihane E. Albóniga, Dr. Juan Manuel Falcon‐Pérez

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Alzheimer's disease (AD) is the most common form of dementia, which affects more than 55 million people worldwide (according to WHO in 2023). It is characterized by loss of cognitive functions through the accumulation of insoluble deposits (hyperphosphorylated‐tau and amyloid‐β‐peptide‐42) in brain. In the context of metabolism, pathological changes are also observed consistently during the progression of the disease. Recently, a link between the presence of specific metabolic pathway intermediates in the cell and the development of AD has been described (González‐Domínguez etal. 2021). Extracellular vesicles (EVs) secreted by Central Nervous System (CNS)‐derived cells are considered to be involved in AD pathogenesis as they carry a cargo including AD intermediators. The exosomal cargo, which varies on the biological‐pathological state of the secretory cell, is considered as a potential source of reliable biomarkers for different diseases. So, we hypothesize that comparing the metabolic cargo of CNS‐derived EVs between AD patients and healthy controls could be a tool to understand the metabolic changes in AD.

Methods: EVs were isolated from post‐mortem human temporal cortex tissues using in‐house size exclusion chromatography. Two groups of samples were included: AD patients, displaying Braak stages III‐V (n=14); and healthy controls with no evidence of dementia (n=10). Lipid and metabolite profiling of EVs were analysed by ultra‐high performance liquid chromatography coupled to a time of flight mass spectrometer (UHPLC‐ToF‐MS) using positive and negative ionization modes to find those compounds that were carried in these EVs and were involved in group separation.

Results: In total, we found 8 metabolic features from polar positive and 43 from lipidomics negative analysis that are responsible of the separation observed between the two EV groups (AD patients and healthy controls).

Summary/Conclusion: Untargeted metabolomics and lipidomics analysis of EVs showed some features that are different between AD patients and healthy control.

Keywords: extracellular vesicles, Alzheimer's disease, metabolomics, lipidomics

PT02.13. Mitochondrial proteins are exported from cells via sEVs in Parkinson's disease

Mr Adityas Purnianto, Ms Mitali Kulkarni, Professor Scott Ayton, Professor Catriona McLean, Professor Ashley Bush, Professor David Finkelstein, Professor Kevin Barnham, Dr Laura Vella

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Under oxidative stress, cells release EVs with an altered cargo composition, potentially as a protective response against oxidative stress. This phenomenon has been reported in various diseases, especially those associated with inflammation and aging. However, this has not been thoroughly investigated in Parkinson's disease (PD), despite the prominence of oxidative stress and mitochondrial dysfunction as features of the disease. Therefore, this study has investigated the impact of oxidative stress on EV composition within the context of PD.

Methods

Small EVs (sEVs) were isolated using size exclusion chromatography from two in vitro models of oxidative stress relevant to PD (rotenone, a mitochondrial toxin, and erastin, an inducer of iron‐dependent cell death) as well as post‐mortem caudate tissue of PD subjects. sEVs were validated by immunoblotting, nanoparticle tracking analysis, and transmission electron microscopy. The EVs’ content of proteins with lipid peroxidation adducts and mitochondrial proteins were examined using LC‐MS‐MS bottom up proteomic and immunoblotting. Density gradient ultracentrifugation (DGUC) was used to isolate a subpopulation of sEV with a mitochondrial origin.

Results

Rotenone and erastin‐induced oxidative stress significantly increased mitochondrial proteins and proteins with lipid peroxidation adducts in sEVs while minimal changes were observed in the cells. The change in mitochondrial protein content in sEVs, particularly prominent in the rotenone model, suggests a phenomenon highly sensitive to oxidative stress originating from mitochondria. These findings were supported by identification of increased mitochondrial oxidative phosphorylation complexes in the sEVs isolated from post‐mortem caudate tissue of PD subjects. A subpopulation of sEVs, separated using DGUC, contained the mitochondrial proteins and proteins with lipid peroxidation adducts. This suggests a common origin for these types of protein cargo within sEVs.

Conclusion

This study demonstrates that sEVs can serve as sensitive indicators of oxidative stress and mitochondrial dysfunction in PD, owing to their ability to export cellular components damaged by oxidative stress, such as mitochondrial components. Hence, EVs may offer a promising avenue as a potential source of biomarker for early stages of PD when cellular changes are relatively minimal and for investigating early neuropathological changes linked to oxidative stress and mitochondrial dysfunction in PD.

PT02.14. Plasma‐derived small extracellular vesicles in alzheimer's disease progression: insights into synaptic dysfunction and neuroinflammation

Mr Rishabh Singh, Ms Sanskriti Rai, Dr Prahalad Singh Bharti, Dr Prasun Chatterjee, Dr Saroj Kumar

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Alzheimer's disease (AD) is a neurodegenerative disease characterized by Aβ plaques and neurofibrillary tangles, with chronic inflammation and synaptic dysfunction playing a significant contributor to disease progression and cognitive decline. Small extracellular vesicles (sEVs) are implicated in AD progression by facilitating the spread of pathological proteins and inflammatory cytokines. This study investigates the role of plasma‐derived sEVs (PsEVs) in synaptic dysfunction and neuroinflammation and their association with amyloid‐β and tau pathologies in AD progression.

Method: A total of 45 [15 each in AD, mild cognitive impairment (MCI), and age‐matched healthy control (AMC)] subjects were recruited, and written informed consent was obtained (Ethics Ref No: IECPG‐670/25.08.2022). PsEVs were isolated using a chemical precipitation method, and their morphology was characterized by transmission electron microscopy. The size and concentration of PsEVs were determined using nanoparticle tracking analysis (NTA). Antibody‐based validation of PsEVs was done using CD63, CD81, TSG101, and L1CAM antibodies. Synaptic dysfunction and neuroinflammation were evaluated with Synaptophysin, Glial Fibrillary Acidic Protein (GFAP), IL‐1β, and TNF‐α antibodies. AD‐specific markers, Aβ‐42, and pTau were examined using Western blot and ELISA.

Results: Our findings reveal higher concentrations of PsEVs in AD and MCI compared to AMC (p<0.0001). Aβ42 and pTau expression are significantly elevated in MCI (AUC = 0.9711, sn=100%, sp=80%, p<0.0001; AUC = 0.9689, sn=100%, sp=93.3%, p<0.0001, respectively); AD (AUC = 1, sn=100%, sp=100%, p= p<0.0001; AUC = 1, sn=100%, sp=100%, p<0.0001, respectively). Synaptophysin exhibits decreased expression from AMC to MCI to AD (p=0.047), whereas, IL‐1β, TNF‐α, and GFAP showed increased expression in MCI and AD compared to AMC (p=0.002, 0.0006, 0.0184, respectively). The increased levels of PsEVs correlate with synaptic dysfunction and neuroinflammation.

Conclusion: Elevated PsEVs and upregulated Aβ42 and pTau expression show high diagnostic accuracy in AD. The decreasing synaptophysin expression and increased neuroinflammatory markers in AD and MCI patients suggest synaptic degeneration and neuroinflammation as potential indicators. These findings support the potential of sEV‐associated biomarkers for AD diagnosis and highlight synaptic dysfunction and neuroinflammation in disease progression.

Funding: CSIR [09/006(0533)/2021‐EMR‐I], ICMR (2020‐1194), DHR (YSS/2020/000158).

Keywords: Alzheimer's disease, mild cognitive impairment, small extracellular vesicles, synaptic dysfunction, neuroinflammation.

PT02.15. Primary rat cortical tri‐culture to study cellular response to cancer EVs

Mrs. Rachel Rachel, Dr. Randy Carney, Hyehyun Kim, Dr. Erkin Seker

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Understanding interaction of EVs with CNS cells will be important for identifying mechanisms of disease, as well as for developing EV‐based therapies for them. However, models to study this often use isolated single cell types that lack the intercellular signaling that may mediate EV function and fate, such as the complex processes in neuroinflammation. Critically, metastatic niche formation in the brain is hypothesized to be mediated in part by cancer‐derived EVs, though details are poorly understood. Here, we employ primary rat cortical tri‐culture (neurons, astrocytes, microglia) and contrasting co‐culture (neurons, astrocytes) to compare interactions of EVs from breast cancer cells (MDA‐MB‐231) to their brain‐tropic variant (231‐Br), and a non‐cancer control (HEK293T), to identify EV fate and neuroinflammatory response.

Methods

EVs were isolated via differential ultracentrifugation and characterized via nanoparticle tracking analysis, flow cytometry, resistive pulse sensing, and interferometric imaging. Cells from rat‐pup neocortices were dissociated and grown in chambered coverslips. For uptake, EVs were fluorescently labeled, separated by size exclusion chromatography, and incubated at 7.5*109 EVs/mL in cultures for 4‐24 hrs. Cells were immunostained and imaged by confocal microscopy to assess uptake, while cell culture media was collected and probed for cytokines to assess inflammatory response.

Results

231‐Br and HEK293T EVs were taken up at similar levels while MDA‐MB‐231 had lower overall uptake in co‐ and tri‐culture. EVs mainly associated with neurons and astrocytes. Despite low uptake, MDA‐MB‐231 EVs resulted in the highest increase in inflammatory cytokines (e.g., MCP‐1 and MIP‐1α) in tri‐culture. Though 231‐Br EVs had similar cytokine trends, increases were not significant compared to controls. In contrast, HEK293T EVs decreased VEGF, MCP‐1, and IP‐10 expression. Cytokine expression (except of fractalkine, produced by neurons) did not change in co‐culture.

Discussion

These results suggest that functional effect and broad uptake rate, rather than cell targeting, could be an important contributor to EV‐mediated metastasis, with EVs from brain‐metastatic cells causing less inflammation yet having higher overall uptake. More broadly, the interaction of EVs with each cell type suggests that EV function may be multifaceted, highlighting the importance of complex in vitro systems to better understand EV function in the CNS.

PT02.16. Protein nitrosothiol patterns altered in extracellular vesicles from Alzheimer's disease brain cortex

Dr Natasha Vassileff, Dr Rohan Lowe, Dr Keshava Datta, Professor Catriona McLean, Professor Andrew Hill, Dr Jereme Spiers

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: The cargo of extracellular vesicles (EVs) is known to be altered in neurodegenerative conditions like Alzheimer's disease (AD) and may facilitate disease pathology. Neuroinflammation is an underlying feature of neurodegenerative conditions including AD and is characterised by the production of numerous cytokines and free radicals such as nitric oxide (NO). NO exerts its effect through post‐translational S‐nitrosylation of target cysteine thiols (nitrosothiols), altering the protein's function and subcellular localisation, which may include packaging into EVs. Despite this, the NO‐modified proteome of EVs in AD has not been investigated. Therefore, this study aimed to determine the NO signalling changes occurring through post‐translational modifications on EV protein cargo in AD.

Methods: Brain derived EVs (BDEVs) isolated from the frontal cortex of human post‐mortem AD brain tissue samples using enzymatic digestion and sucrose density gradient separation were thoroughly characterised, to meet the Minimal Information for Studies of Extracellular Vesicles guidelines, before undergoing iodolabelling and comparative proteomic analysis to identify NO‐induced post‐translational changes.

Results: The iodolabelling proteomic analysis revealed a protein profile heavily representative of microglial and antioxidant proteins indicative of neuroinflammation in the AD BDEVs. These included inflammatory, membrane surface, and mitochondrial proteins, suggesting protein nitrosothiols may play an important role in neuroinflammatory communication via EVs in AD.

Conclusions: Together, this study is the first to show NO‐induced S‐nitrosothiol post‐translational changes in BDEVs, demonstrating the effect NO signalling exerts on the protein cargo of EVs during AD. Further work should investigate the functional consequence of protein nitrosothiolation of EV cargo and how this may affect cellular processes in AD.

PT02.17. Proteomic analysis of brain‐dervied extracellular vesicles in Huntington's disease

Miss Mitali Manish Kulkarni, Mr. Adityas Purnianto, Miss Tiana Koukoulis, Miss Huaqi Su, Miss Geraldine Kong, Professor Anthony Hannan, Dr. Laura.J Vella

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Huntington's disease (HD) is a genetic neurological disorder that is characterised by decline in movement, cognition, and behavior. It is imperative to understand the progression of HD due to the absence of treatment and therapeutic targets. Despite the genetic cause of HD being known, there is still considerable progress to be made to understand the cellular and molecular mechanism associated with the disease pathogenesis. In the past decade, research has demonstrated that extracellular vesicles (EVs) play a role in the pathogenesis of neurodegenerative diseases and that the protein content of EVs can provide insights into underlying disease mechanisms. To elucidate the potential role of EVs in HD, we investigated the protein cargo of EVs isolated from human tissue and a mouse model of HD.

Methods: EVs were isolated from the extracellular matrix of human HD and neurological control parietal brain tissue (n=8) or the cortex, striatum, and hippocampus of the R6/1 transgenic mouse model of HD and wild‐type controls (n=10) by differential centrifugation, size exclusion chromatography and ultrafiltration. EVs were characterised by immunoblotting, transmission electron microscopy and liquid chromatography mass spectrometry‐based proteomics.

Results: Our studies highlighted the enrichment of EV markers in immunoblotting and proteomics demonstrating successful isolation of EVs. We identified an overrepresentation of proteins associated with mRNA splicing, via the spliceosome in human HD EVs. These novel findings demonstrated that spliceosome and spliceosomal components are exported by the cells via EVs in HD. In the R6/1 mice model, we identified overrepresentation of proteins associated with the MAPK signaling cascade revealing that EVs export proteins associated with the MAPK signaling pathway.

Summary/Conclusions: This is the first study to comprehensively characterise the cargo of EVs in HD human and mouse brain. Our findings illustrate the potential of EVs to offer insights into both early and late stage disease mechanisms in HD.

PT02.19. Uncovering the composition of extracellular vesicles (EVs) regulated by Translin‐Associated Factor X (TRAX) that modulates microglial identity

Dr. Yu‐Ting Weng, Ph.D. Yijuang Chern

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

Translin‐Associated Factor X (TRAX) controls the expression of a specific set of miRNAs. Ablation of TRAX in striatum leads to the disruption of miRNA‐mRNA networks, accelerating the progression of Huntington's disease (HD), an inherited neurodegenerative disease linked to the elongated glutamine‐containing mutant huntingtin protein. Unexpectedly, reducing TRAX expression, primarily expressed in neurons, decreases several signature genes in microglia that lack TRAX expression, suggesting the role of TRAX in neuron‐microglia communication. Our investigation focuses on the potential of TRAX to modify the composition of extracellular vesicles (EVs), thereby impacting the identity of microglia receiving these EVs.

Methods:

To examine the effect of TRAX on EV composition, we established TRAX knockout WT and HD striatal cell lines using CRISPR‐Cas9. EVs were isolated from control and TRAX knockout cell culture medium containing 10% of EV‐depleted FBS by differential centrifugation followed by total exosome isolation kit (Invitrogen) and characterized by nanoparticle tracking (NTA), western blotting and miRNA sequencing. To assess the effect of EVs secreted by TRAX knockout cells on microglial identity, WT primary microglia were treated with EVs secreted by control and TRAX knockout cells and the expression of microglial signature genes was evaluated by qRT‐PCR.

Results:

The isolated EVs exhibited enrichment of EV markers, CD63 and TSG101, in comparison to the total cell lysate. Surprisingly, TRAX was detected in EVs secreted by both WT and HD striatal cells, suggesting the potential function of TRAX within EVs. Treatment of WT primary microglia with EVs secreted by TRAX knockout HD striatal cells resulted in the loss of microglial identity. We identified 53 differentially expressed (DE) miRNAs, with a log2 fold change > 1 or < ‐1 and p < 0.05, between EVs secreted by control and TRAX knockout HD striatal cell lines. Further analysis of the predicted miRNA‐targeted genes suggested the involvement of several these genes in regulating microglial signature genes. The characterization of the TRAX‐regulated EV miRNA‐mRNA axis in modulation of microglia identity will be discussed.

Summary/Conclusions:

We demonstrated that TRAX affects the identity of microglia that uptake neuron‐secreted EVs by potentially regulating the miRNA content within the EVs.

PT02.22. Extracellular vesicles in mood disorders: a systematic review

Dr Cristian‐Daniel Llach¹, Ms Gia Han Le¹, Dr Gerard Anmella², Dr Joshua Rosenblat¹, Dr Anna Gimenez‐Palomo², Dr Isabella Pacchiarotti², Dr Eduard Vieta², Dr Roger McIntyre¹, Dr Rodrigo Mansur¹

¹University Of Toronto, Toronto, Canada, ²Bipolar and Depressive Disorders Unit, Hospital Clinic de Barcelona, Barcelona, Spain

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: EVs hold promise as CNS biomarkers, traversing the blood‐brain barrier with diverse cargo and offering non‐invasive “liquid biopsies” for brain pathology assessment. This systematic review consolidates EV research in mood disorders, highlighting their biomarker and therapeutic potential.

Methods: Studies of extracellular vesicles in both pre‐clinical and clinical research on mood disorders were identified from PubMed, Scopus, Web of Science and Psycinfo from 2010 to December 2023. Risk of bias was assessed, and results are presented as a systematic review following PRISMA guidelines.

Results: Limited research has explored extracellular vesicles in mood disorders, with the existing evidence lacking reproducibility. Besides, heterogeneity in materials and methods and overall lack of adherence to MISEV guidelines makes it difficult to synthetize all results. Most studies focused on exosome mi‐RNA cargo, reporting interesting molecules such as miR‐139‐5p, miR‐26a, miR‐335‐5p or miR‐1292‐3p, that could help differentiate between healthy subjects and mood disorders, as well as among diverse subtypes of mood disorders. Some of them may be useful to identify subthreshold depression or predict antidepressant response, especially those regulating TLR4. Noteworthy proteins and metabolites in EVs, including SERPINF1 and various metabolites, exhibited potential as biomarkers for MDD and BD. Several studies linked their findings to synaptic plasticity and neurogenesis, to the theory of neuroinflammation, insulin resistance, mitochondrial or circadian rhythms dysfunction, as well as to the functioning of key molecules like GSK‐3, BDNF, VEGF or second messenger systems such as MAPK, Ras and PI3K‐AKT. A work under the umbrella of the gut‐brain axis paradigm also focused on bacteria‐derived exosomes to differentiate MDD from non‐depressive patients. On the other hand, engineered EVs, like RVG‐circDYM EVs, hold promise for targeted gene therapy in MDD, exhibiting alleviation of depressive symptoms in mouse models.

Summary: EVs, and neural EVs in particular, exhibit significant potential in aiding the diagnosis and treatment of intricate mood disorders. Enhanced EV‐based methodologies are gaining traction, paving the way for personalized treatment strategies. However, technical limitations remain and regulatory approval for EV‐based therapies is pending. Integration of various data sources could enhance precision in biomarker discovery, heralding a potential era of EV‐based brain diagnostics and therapeutics.

PT03.01. Analysis of the phenotypical changes of plasma EVs over time in healthy donors

Rikke Bæk, Maiken Mellergaard, Rikke Wehner Rasmussen, Rikke Bülow Eschen, Evo Lindersson Søndergaard, Aase Handberg, Malene Møller Jørgensen

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1) Introduction

Extracellular vesicles (EVs) in plasma have great diagnostic potential as biomarkers for several diseases. To use EVs in clinical settings, it is of great importance to establish whether EV phenotypes and numbers change over time in a healthy cohort. The aim of this study was to determine both the long‐term (week‐to‐week) as well as the short‐term (day‐to‐day) variation of EV concentration and composition in plasma from healthy donors and to compare the findings with blood cell counts.

2) Methods

Venous peripheral blood (citrate and EDTA) was obtained from 4 fasting healthy donors once a week over a period of 6 weeks. Furthermore, blood samples were drawn daily for one week. Blood cell counts were measured using a Sysmex XN‐1000. Small EV concentration and composition were analyzed by the EV Array (Jørgensen etal., 2013, JEV) using antibodies against 23 selected surface‐markers for capture in combination with detection by antibodies against CD9, CD63, CD81 or a co*cktail of these. Capture antibodies included antibodies against EVs in general (CD9, CD63, CD81, Alix, Flotilin‐1 etc.), and immunological and inflammatory markers (CD4, CD8, CD80, HLA ABC, HLA DR/DP/DQ, TNF RI and RII). In comparison, high resolution flow cytometry (hfcm, Sanden etal., 2018) was used for analyzing EV populations based on the expression of CD9, CD63, and CD81 as well as colocalization of these markers. Nanoparticle Tracing analysis was performed to determine size and numbers of EVs.

3) Results

In total, 40 plasma samples were collected and analyzed. Large inter‐individual variation was found in EVs analyzed by EV Array and hFCM. For certain blood cell types, the long‐term intra‐individual variation varied significantly over time, which was not seen in the context of small EVs. Minor short‐term and intra‐individual variation (day‐to‐day) was observed in cellular composition but was not reflected in the obtained phenotypes of small EVs.

4) Summary/Conclusion:

A few of the selected EV surface markers from circulating blood cells showed minor changes over time, although this did not reflect the significant changes identified at the cellular level.

PT03.02. Comparison of primed mesenchymal stromal cells secretome following different methods of purification with a large panel of characterization tools

PHD Student Guillaume Valade, PHD Student Marine DE TADDEO, Mrs Muriel NIVET, Mrs Marion GROSBOT, Mrs Claire LANGLE, Mrs Sylvie GOULINET, PHD Philippe MAUDUIT, Mr Vincent JUNG JUNG, PHD Chiara GUERRERA, MD, PHD Sébastien BANZET, PHD Juliette PELTZER, PHD Marina TROUILLAS

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Acellular therapies have recently emerged as a new therapeutic option in the field of tissue repair. Extracellular Vesicles (EV) and, more generally, the secretome derived from Mesenchymal Stromal Cells (MSC) possess immunomodulatory and pro‐healing properties similar to those of MSC themselves. Moreover, MSC are able to adapt their secretion profile in response to stimuli of their environment and we have previously shown that IL‐1β priming of MSC is effective to improve the properties of their secretome. This study aims to characterize the impact of IL‐1β priming doses and different secretome purifications containing EV more and less enriched in soluble factors (EV+SF), with a wide range of analytical methods, and to better define our products.

Methods

MSC are primed with 1 or 5ng/mL of IL‐1β for 24h. After 72 hours of secretion in serum‐free medium, the conditioned medium is collected. Tangential flow filtration is used to isolate and concentrate EV or EV+FS, using filters with respective cut‐off sizes of 500 or 10kDa. Particles concentration and size are measured using nanoparticle tracking analysis and interferometric light microscopy technology. Cryo‐electron microscopy is used to confirm EV structure. Total protein is quantified by BCA technic. Phenotype is analyzed by mass spectrometry and flow cytometry for nanoparticle analysis.

Results

Our results indicate that neither IL‐1β priming doses nor concentration methods induce major variations in particle concentrations and sizes. However, significant differences are observed between NTA, Videodrop and NanoFCM methods. As expected, our products express CD9, CD63, CD81 but also MSC specific markers. Similar mass spectrometry profiles are obtained between conditions with the different doses of priming. However, major differences are observed between EV and EV+FS. EV+FS are more enriched in growth factors, interleukins, chemokines, proteins linked to matrix remodeling and also metabolic enzyme. The EV fraction contains more proteins implicated in vesicles trafficking.

Summary/Conclusion

This study, using a broad spectrum of characterization methods, allowed to improve our understanding of the impact of production/purification processes on the characteristics of our acellular products. This knowledge will allow us to develop scalable and efficient manufacture of medicinal products for tissue repair.

PT03.03. Evaluation of the physical properties and pharmaco*kinetics of EVs purified by the microfiltration membrane with ion exchange function

Ms. Ayano Higaki, Mr. Keita Inoue, Ms. Mizuki Kobayashi, Ms. Makiko Hiraoka, Mr. Yosh*taka Kawakami, Ph.D. Naohiro Seo

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Among extracellular vesicles (EVs), exosomes (EXO) derived from multivesicular bodies of late endosome are highly expected to be used in clinical treatment and diagnosis. However, there is no existing technology to easily purify EXO from biological samples. Since it has been reported that EXO and other EVs can be separated by differences in surface negative charge [1], we developed a new microfiltration membrane (developed purification membrane) with ion‐exchange groups and micropores through which EXO can pass, and evaluated its accuracy in EXO purification.

Methods: EVs were prepared by passing HEK293 culture supernatant through the developed purification membrane followed by eluting with buffer solutions containing high salt. The obtained EVs were characterized by measurement of physical properties such as zeta potential, proteome analysis, western blot analysis, and DNA content. In addition, we conducted administration experiments of the obtained EVs in mice to investigate their pharmaco*kinetics.

Results: By using the developed membrane, EVs that contained the EXO markers such as Tsg101, and were free of actin and DNA, could be prepared in a short time with a high unnecessary protein removal rate. In a pharmaco*kinetic study, it was confirmed that the EVs purified by the developed membrane circulated systemically and localized in various organs, whereas most of the EVs prepared by ultracentrifugation were localized mainly in the liver via macrophages.

Summary/Conclusion: This method has enabled us to purify EXO‐rich EVs that have the ability to circulate systemically. Our technology can be applied not only to basic research areas but also to clinical applications by incorporating it into GMP‐manufacturing processes, and has the potential to become indispensable for future EV research.

[1] N. Seo, etal., J. Extracell Vesicles, (2022) 11(3): e12205.

PT03.04. Is it feasible to distinguish extracellular vesicles by their biophysical properties?

Mr Fredrik Stridfeldt, MSc Hanna Kylhammar, Dr Vipin Agrawal, MSc VIkash Pandey, Dr André Görgens, Professor Samir El Andaloussi, Professor Dhrubaditya Mitra, Professor Apurba Dev

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Studies have shown that the stiffness properties of extracellular vesicles might be important in disease and drug monitoring as the mechanical properties may change as a response to treatment or because of disease progression. However, multiple reasons including their small size, liquid‐like membrane, and inhom*ogeneous composition complicate the application of traditional and well‐established methods. Atomic Force Microscopy (AFM) is a high‐resolution technique that enables precise single EV analysis. It can extract both physical properties such as height and radius but also biomechanical properties such as linear stiffness K and bending modulus κ. However, there exists no satisfiable model explaining the vesicle's response to indentation and earlier studies report a large spread of stiffness values.

Methods

4 EV samples (WT, WT‐SEC, CD63‐KO, and Pan‐KO) were adhered to poly‐l‐lysine‐coated substrates and imaged in AFM. Individual EVs were scanned at a resolution of 4 nm/pixel. 150 indentations were carried out up to a set force of 800 pN. From there, linear stiffness was extracted from all approach curves and the bending modulus was extracted from the retract curves showing membrane tether formations to the AFM tip. A liquid shell theory was developed to describe indentation experiments.

Results

45 WT, 36 WT‐SEC, 24 CD63‐KO, and 18 Pan‐KO EVs were investigated. T‐testing reveals an increase in mean linear stiffness in the CD63‐KO vesicles (11.7 mN/m) compared to the WT vesicles (7.2 mN/m). Analysis of one individual EV reveals a spread of stiffness values. By comparing the possible linear stiffness distributions, we discover a larger uncertainty within the samples than across different samples. This complexity and heterogeneity on a single EV level would have been missed without repeated measurements. No difference could be found in the bending modulus (27‐34 kBT).

Summary/Conclusion

This study highlights the importance of repeated measurements on a single vesicle when analyzing biomechanical properties. Our data suggest that both K and κ can vary significantly on both individual and ensemble levels. We present a theoretical model that explains most of the indentation behavior. We also suggest an alternative statistical method to better handle the low throughput AFM data.

PT03.06. Characterization of human MSC‐derived extracellular vesicle preparations using size‐exclusion HPLC and ion‐exchange HPLC coupled with multi‐angle light scattering detection

Dr. Hirotaka Nishimura, Dr. Tomofumi Yamamoto, Dr. Noritaka Hashii, Dr. Akiko Ishii‐Watabe

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Clinical applications of human mesenchymal stem cell (MSC)‐derived EVs are being actively investigated. EV preparations often contain a variety of extracellular vesicles, lipoproteins, and other particulate matter. Furthermore, there is heterogeneity in the physical properties and contents of EVs of the same size range. These variabilities may impact their pharmacological activity and safety, necessitating robust quality control, which is not established yet. This study aimed to evaluate the heterogeneity of particle size and charge in MSC‐derived EV samples using high‐performance liquid chromatography (HPLC) and multi‐angle light scattering (MALS).

Methods: Several batches of EV preparations purified and condensed from MSC culture supernatants using 100k MWCO tangential flow filtration (TFF) were used. Size exclusion HPLC (SEC)‐UV‐MALS was employed to analyze soluble protein impurities and particle size variation across batches and enrichment rates. Fractions of SEC‐MALS were analyzed by CD9/CD63 ELISA to identify particle types in each fraction. Anion exchange HPLC (AEX)‐MALS was utilized for the assessment of surface charge heterogeneity of EV preparations. Fractions of AEX‐MALS were analyzed by SEC‐MALS, CD9/CD63, and phosphatidylserine (PS)/CD63 ELISA.

Results: SEC‐UV‐MALS revealed that the EV samples contained soluble proteins and three particle populations with different sizes, and the particle composition and the amount of protein impurities varied with the production batch and concentration rate. CD9/CD63 ELISA revealed that exosomes were mainly contained in the main peak in the SEC‐MALS chromatogram, with very few in the other peaks. The analysis by AEX‐MALS revealed the presence of several types of particles with different charges in the EV samples, and that their composition varied with the enrichment rate in TFF. SEC‐MALS analysis of each AEX‐fraction revealed that the fractions contained the same size particles with different charges. These fractions were assessed by ELISA and found to contain exosomes and to differ in PS presentation, which suggested that PS contributes to the charge diversity of exosomes.

Conclusion: SEC‐MALS and AEX‐MALS effectively characterized size and charge heterogeneity in EV samples. Variances due to production batches and enrichment rates were readily discernible. These methodologies would help quality control of EV‐based therapies.

Funding: This work was supported by AMED under Grant No.JP23mk0101218.

PT03.07. EVs during zebrafish larvae development

Dr.med. Linda‐marie Mulzer¹, Tim Felger¹, PD Dr. med. habil. Dr. rer. nat. Luis Muñoz², Gesa Engl¹, Prof. Dr. med. Heiko Reutter¹, Leila Pourtalebijahromi³, Prof. Dr. Gregor Fuhrmann³, Philipp Arnold⁴, Dr. med. Alina Hilger¹

¹Department of Pediatrics and Adolescent Medicine, Friedrich‐Alexander University of Erlangen‐Nürnberg, Erlangen, Germany, ²Department of Rheumatology and Immunology, Friedrich‐Alexander University Erlangen‐Nürnberg, Erlangen, Germany, ³Friedrich‐Alexander University Erlangen‐Nürnberg, Department of Biology, Chair of Pharmaceutical Biology, Erlangen, Germany, ⁴Friedrich‐Alexander University Erlangen‐Nürnberg, Institute for Functional and Clinical Anatomy, Erlangen, Germany

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

EVs play a crucial role in cell migration and differentiation. Despite zebrafish being a well‐established vertebrate model, limited knowledge exists about EVs in zebrafish embryogenesis. This pioneering study investigates large (lEVs) and small (sEVs) EVs throughout the first 96 hours of zebrafish larvae (zfl) development, analysing size and number changes over time.

Methods:

Wild‐type AB strain zfl were collected at 24, 48, 72 and 96 hours post fertilization (hpf) and mechanically hom*ogenized. LEVs and sEVs were isolated by differential centrifugation and characterized using transmission electron microscopy (TEM), flow cytometry, and nanoparticle tracking analysis (NTA). AxV‐FITC and FM4‐64 were used during flow cytometry to detect phosphatidylserine and a lipid bilayer. Statistics were conducted using Prism 6.0. Results were considered significant at p<0.05.

Results:

TEM showed a clear population of lEVs and sEVs. NTA revealed a significant separation of the lEV and sEV fractions based on their size at every observation point and showed a significantly increasing number of lEVs and sEVs over time with a peak at 72hpf. No correlation of the increase in numbers of EVs with the length development of the zfl was detected. Further NTA indicated that sEVs become significantly larger over time. Flow cytometry showed high positivity of lEVs for AxV‐FITC (99,8%) and FM4‐64 (99,0%).

Conclusion:

The significant size difference observed via NTA indicates the quality of the isolation method. Although the number of EVs increase over time, no correlation with length growth of the zfl was detected. With 72hpf the highest number of EVs was observes, at this time most organs have formed, mainly maturation and growth occur; the decreasing number of EVs afterwards could be due to a reduced need for EV‐mediated cross talk. EVs did not only increase in number but also in size. Since EVs serve as cargo deliveries, larger EVs may reflect a higher transport capacity. Flow cytometry showed a high purity of the lEV samples with a strong expression of phosphatidylserine and the presence of a lipid bilayer. We suggest that an increase in EV number and EV size is necessary to orchestrate the maturing zfl.

PT03.08. Activated human mast cells produce extracellular vesicles that change the metabolic function of target cells

Senior Investigator Marianna Kulka, Dr. Marcelo Marcet‐Palacios, Sabrina Rodrigues Meira

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Small extracellular vesicles (EV), or exosomes, are 30‐150 nm phospholipid‐encased nanoparticles that carry information between cells. Mast cells are myeloid‐derived immune cells that mediate innate/adaptive immunity, tissue remodeling, allergic inflammation and clearance of parasitic infection. Methods: Human mast cells (HMC‐1 and LAD2) were activated and their production of exosomes was analyzed by proteomic profiling, transmission electron microscopy (TEM), nuclear magnetic resonance (NMR) metabolomic profiling and flow cytometry. Results: Activated mast cells produced at least three distinct subpopulations of EVs with unique structures observed by EM. Atom scale modeling indicated that each EV size could potentially carry specific protein configurations. Proteomic profiling showed unique protein content, and a comparison of EV proteomes from activated and resting EVs showed that 16 proteins were significantly more abundant and 32 less abundant in the activated EVs. A STRING analysis of the more abundant proteins revealed a correlation with mitochondria biology (COX6A1 – Cytochrome c oxidase subunit 6A1), catalytic activity (MAP4K4 – Mitogen‐activated protein kinase 4), calcium mediated signaling (PPP3CB ‐ Serine/threonine‐protein phosphatase), as well as regulators of intracellular membrane trafficking (RAB31 – Ras‐related protein) and exon junction complex components (CCDC9 – Coiled‐coil domain containing protein 9). On the other hand, the STRING analysis of the less abundant proteins revealed strong correlation with the protein kinase domain, microtubule turnover regulation (KIF2C – Kinesin‐like protein), and chromatin assembly mediator (CHAF1B – Chromatin assembly factor 1 subunit B). The proteomic profiling also revealed the presence of the markers CD9, CD81, CD63, ALIX, and TSG101 in both activated and resting samples, which indicates the presence of characteristic EVs and that the cell activation process does not significantly affect EV marker expression. EVs from activated cells induced an increase in the metabolic rate of target cells, suggesting that the EV contents modified the mitochondrial function of target cells; the proliferation and viability of the target cells remained unchanged. Conclusions: This data suggests that activated cells produce different populations of EVs that differ in size and cargo that activates the metabolomic profile of target cells.

PT03.09. Advancing ultra‐low, ultra‐deep extracellular vesicle proteomics

Prof David Greening, Mr Alin Rai, Ms Haoyun Fang, Ms Bethany Claridge, Mr David Greening

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

In recent decades the study of extracellular vesicles (EV) has gained attention due to their function and role in intercellular communication and cargo transfer. Advancements in liquid chromatography and mass spectrometry (LC‐MS/MS) has demonstrated the ability to comprehensively define EV proteomes. However, these studies are often limited by sample availability, requiring upscaled EV production from cell culture or biofluids, limiting its applicability to lower yield EV sources.

Methods:

Here, we establish a workflow to enable precise and comprehensive proteomic characterisation of small EVs (sEVs) from ultralow starting quantities. This pipeline is defined by its optimised sample preparation methods, short chromatography lengths, and high‐resolution quantitative proteomics using data‐independent acquisition (DIA). This refined DIA approach combined robust single‐pot, solid‐phase‐enhanced sample preparation with temporally optimised enzymatic digestion and short chromatography gradients using ultra‐low input loads of 0.5 to 50 ng of EV peptide across each LC gradient.

Results:

For 50 ng loading, more than 3730 proteins for all gradient lengths were observed, with 4599 identifications in our 44 min workflow. The short gradient lengths favoured low peptide loads, with a 15 min gradient consistently quantifying >1100 protein groups from 500 pg of EV peptide, and >3800 protein groups from 50 ng, including the robust quantification of 22 core EV marker proteins. Furthermore, we optimised bead‐based sample preparation for ultra‐low quantities of EV (0.5 to 1 µg) to obtain sufficient peptide source for MS quantification. Our easy to apply approach enables the generation of meaningful proteome insights from <1 µg starting EV protein, encompassing the identification of >1900 protein groups and capturing sufficient proteomic diversity of EV from different cell sources to determine known EV biology.

Summary/Conclusion:

This optimised workflow addresses the pressing need to capture precise and comprehensive proteomes of EVs from ultralow sample quantities, without compromising depth and accuracy. Our workflow pipeline is straightforward and can be implemented to suit various laboratory conditions. This adaptability facilitates the characterisation of EVs, particularly where sample availability is constrained.

PT03.10. Amniotic fluid EV proteome is a clear representation of gestational age‐dependent fetal development

Dr Ishara Atukorala, Dr Ching‐Seng Ang, Ms Sally Beard, Ms Bianca Fato, Dr Natasha de Alwis, Dr Hamish Brown, Professor Natalie Hannan, Professor Lisa Hui

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Amniotic fluid (AF) surrounds the developing fetus, providing nutrients and protecting the fetus from biological and mechanical dangers. In addition to a myriad of proteins, immunoglobulins, electrolytes and growth factors, the AF is a rich source of extracellular vesicles (EVs). As the keratinization of the fetal skin begins around 20 weeks of gestation, fetal urination and lung secretions become the main sources of increasing AF volume. This makes the amniotic fluid a rich source of fetal biological material including EVs.

Methods

Second trimester and term amniotic fluid samples (6 each) were obtained from routine amniocentesis and Caesarean section, consecutively, with written informed consent from patients. EVs were isolated using differential centrifugation coupled with filtration and ultracentrifugation. EVs were characterized using nano‐particle tracking analysis, cryo‐electron microscopy and Western blotting for Alix, CD9 and CD63. EVs were subjected to label‐free proteomics and data analysis was performed using Spectronaut® and MaxQuant‐Perseus.

Results

The number of EVs per 1 mL of amniotic fluid and the protein amount per EV did not significantly change according to gestation. Cryo‐electron microscopy revealed the presence of unilamellar, multilamellar, multicompartmental and granular‐centred EVs in each sample. The analysis of 220 EVs using ImageJ revealed that there is no significant gestation‐dependent difference in the ratio of these diverse types of EVs. The proteomic signature showed a stark difference according to the gestation of the amniotic fluid EVs. Moreover, majority of the proteins uniquely expressed in the second trimester was involved in the development of the central nervous system, with cardiac development scoring the second place. The proteins upregulated in the term amniotic fluid EVs corresponded to the impending newborn functions such as breathing and eating.

Conclusions

The EV proteome can be considered as an accurate representation of the fetal developmental processes according to gestation. Therefore, amniotic fluid derived EVs can be used as a liquid biopsy for fetal physiology and pathology.

PT03.11. Assessing the compartmentalisation of small non‐coding RNAs in the circulation

Dr I‐Jou Teng, Dr Kaloyan Takov, Dr Clemens Gutmann, Prof. Manuel Mayr

Poster Pitches (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:45 PM ‐ 1:00 PM

Extracellular vesicles (EVs) have attracted attention as carriers of proteins and nucleic acids that are important for intercellular communication and as biomarkers in the circulation. Small non‐coding RNAs (sncRNAs), such as microRNAs (miRNAs) and YRNAs, have been described as part of the functional cargo of the EVs. However, lipoproteins and RNA‐binding proteins have also been demonstrated as putative carriers of sncRNAs. The lack of reliable methods to resolve these carriers from human blood hampers the assignment of sncRNA signatures to different carriers.

In this study, automatised high‐performance size‐exclusion chromatography (HP‐SEC) workflow was established to improve the separation of nanoparticles from plasma and minimise the co‐isolation of lipoproteins with small EVs. Immunoblotting for small EV markers (CD9 tetraspanin) and apolipoproteins (ApoA1 and ApoB for high‐ and low‐density lipoproteins, respectively) was used to characterise the isolates. Quantitative real‐time PCR of HP‐SEC fractions showed that miRNAs were mainly detected in the late‐eluting fractions enriched in ApoA1 and small, soluble proteins. Contrarily, YRNAs were predominantly found in the albumin‐rich and ApoB‐containing fractions. To further investigate whether lipoproteins are the main carriers, HDL and ApoB immunodepletion methods were employed. Both failed to substantially deplete sncRNAs from plasma indicating that lipoproteins are minor carriers of circulating sncRNAs. To identify other potential carriers, conditioned medium from human hepatoma cells was subjected to HP‐SEC. In agreement with plasma experiments, sncRNAs were primarily found in fractions that did not contain EV markers (CD63, CD81, CD9, syntenin‐1). Instead, most of the sncRNAs were identified in the late‐eluting fractions containing smaller, soluble protein complexes, including numerous RNA‐binding proteins.

In summary, we observed only a minor role for lipoproteins and EVs as carriers of circulating sncRNAs. Instead, soluble RNA‐binding proteins, co‐eluted with sncRNAs from plasma suggestive of a role in the secretion and protection of sncRNAs. The current study established an automatic and reproducible HP‐SEC procedure for a better understanding of the compartmentalisation of circulating sncRNAs and EV cargo. In the future, methods established here can determine changes in sncRNA distribution across different carriers and help delineate the function of circulating sncRNAs in the intercellular communication.

PT03.12. Astrocyte‐enriched extracellular vesicle protein concentrations after proteinase K treatment

Dr. Leandra Figueroa‐Hall, Dr. Kaiping Burrows, Dr. Ahlam Alarbi, Dr. Chibing Tan, Dr. Bethany Hannafon, Dr. Rajagopal Ramesh, Dr. Victoria Risbrougn, Dr. T. Kent Teague, Dr. Martin Paulus

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Extracellular vesicles (EVs) are nanoparticles whose cargo is highly enriched with microribonucleic acids, and other biomolecules like lipids, carbohydrates, and proteins. EVs diverse cargo allows for cell‐to‐cell communication, and their ability to cross the blood brain barrier bi‐directionally enables enrichment of EVs from brain cells to examine central nervous system mechanisms. Here, we investigated the feasibility of measuring proteins expressed in AEEVs with high‐sensitivity multiplex assays.

Methods: A human serum sample (one subject) was treated with or without proteinase K (PK; 0.5 mg/mL‐final concentration) and EVs were isolated with various isolation methods (precipitation, precipitation + purification, resin, and size exclusion). Astrocyte‐enriched (AE)EVs were enriched with an astrocyte‐specific, glutamate transporter (GLAST). AEEVs on beads were lysed and protein used for detection of tetraspanins (CD9, CD81, CD63), apolipoprotein E (APOE), and neural cell adhesion molecule (NCAM) protein concentrations. All samples were run in duplicate, and leftover human serum was used as a positive control for all proteins.

Results: AEEV protein concentrations were reduced with PK treatment, with Norgen at below detectable limit (BDL). All proteins were detectable in AEEVs except for NCAM, which was BDL. CD9 concentrations were detectable in all methods, CD81 concentrations were above the lower limit of detection (LLOD) for ExoQuick, ExoQuick Ultra, and SmartSEC, and CD63 concentrations were above standard 7 for all methods. All methods were comparable for APOE concentrations after PK treatment. Interassay coefficients of variation for samples not treated and treated with PK: CD9 (2.04, 10.8), CD81 (7.02, 24.02), CD63 (2.85, 20.55), APOE (5.19, 5.44) and NCAM (2.16, NA).

Summary: Tetraspanins were detectable in AEEVs for all methods indicating the presence of EVs. APOE, a protein mainly produced in astrocytes, was highly expressed in AEEVs confirming the origin of our EV‐enriched population. NCAM, a neuronal marker, was not expressed in AEEVs, confirming the absence of neuronal type. Treatment with PK is necessary to remove lipoproteins, but care must be taken when measuring proteins of interest including tetraspanins, as PK treatment decreased these in AEEVs. Our preliminary results indicate that all methods are appropriate for APOE detection and ExoQuick and ExoQuick Ultra for tetraspanins.

PT03.13. Characterization of extracellular vesicles with capillary electrophoresis

Aleksandra Steć, Ph.D. Joanna Jońca, Ph.D. Agata Płoska, Prof. Leszek Kalinowski, Assoc. Prof. Bartosz Wielgomas, Ph.D. Krzysztof Waleron, Prof. Bogdan Lewczuk, Mr. Grzegorz Czyrski, Ph.D. Andrea Heinz, Ph.D. Szymon Dziomba

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Most of the techniques currently used for the characterization of extracellular vesicles (EVs) are low selective and ineffective in the assessment of vesicles’ heterogeneity. Impurities profiling is also strongly limited and usually requires the application of additional technique like Western Blotting or mass spectrometry. To address these issues, a novel approach to EVs characterization was developed with capillary electrophoresis (CE) technique. The advantage of CE lies in its simultaneous separation of various analytes such as small molecules, polysaccharides, proteins and particles, which provides complete information on the presence of contaminants in the analyzed isolates [1,2]. Moreover, good agreement between the EVs quantitation results obtained by CE, nanoparticles tracking analysis (NTA) and bicinchoninic acid assay (BCA) has already been proved [1,3].

Methods

EVs were isolated from bacteria culturing media (Pectobacterium sp., E.coli) [1,2], plant material (Citrus limon) [3], and mammalian cell cultures (CHO cells) [4] using different method – ultracentrifugation (bacterial vesicles), and combination of size exclusion chromatography with ultrafiltration (for plant material and mammalian samples). The obtained isolates were characterized using NTA, BCA, microscopy (Cryo‐EM or TEM), and CE equipped in UV and LIF detection.

Results

CE was able to separate subpopulations of EVs in the isolates obtained from various sources. The difference in electrophoretic mobility of certain EVs was related to their cargo composition. It has also been shown that staining of selected vesicle components with fluorescent dyes might be utilized for the identity confirmation of EVs with CE.

Conclusion

CE was proven to be able to solve numerous problems encountered in today's EVs‐focused research. It might be used for the monitoring of the isolation process [3,4] or for the quality control of the isolates.

[1] Steć A, etal. Int J Mol Sci. 23: 4347 (2022).

[2] Jońca J, etal. Int J Mol Sci. 22: 12574 (2021).

[3] Steć A, etal. Food Chem. 424, 136333 (2023).

[4] Steć A, etal. Anal Bioanal Chem. 415, 3167–3176 (2023).

PT03.14. Circadian mass spectrometry‐based proteome profiling of salivary extracellular vesicles

Dr Carlos Andres Palma Henriquez, Ms Siena Barton, Dr Sara Nikseresht, Mr Sadman Bhuiyan, Dr Mozhgan Shojaee, Dr Kartini Asari, Dr Pingping Han, Dr Ramin Khanabdali, Dr Gregory Rice

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Saliva is an easily accessible and non‐invasive biofluid with huge potential for biomarker discovery and diagnostic applications. However, using whole saliva requires considering a few factors, including sample collection time, methods, food contamination, individual variability, and relatively lower concentrations of specific biomolecules. Salivary extracellular vesicles (EVs) have gained significant attention recently to overcome these barriers. However, collection time may play an important role in the quantity, quality, and compositions of saliva derived EVs.

Methods: Saliva samples were collected from 3 volunteers using spitting method at 07:00 (A), 13:00 (B), 19:00 (C) and 01:00 (D). Saliva (1mL) was diluted with 1 mL of PBS and centrifuged at 10,000 x g for 5 min to remove cell/membrane and food debris. EVs were isolated using 30 µL of EXO‐NET® according to the manufacturer's instructions. EXO‐NET captured salivary EVs were then lysed in 1% SDS before processing for mass spectrometry and western blot analysis.

Results: Western blot analysis confirmed canonical EV markers including CD9, CD81 and CD63 across all time points. Mass spec analysis identified around 903 proteins in (A), 813 in (B), 803 in (C) and 871 in (D). Out of that, 718 proteins were present in all 4 times, with 16 proteins identified only in (A), 3 in (B), 2 in (C) and nothing uniquely expressed in (D). Data modelling identified 62 EV‐associated proteins out of the top 100 proteins including Alix, CD63, ENO1, Annexins, HSP90, Rab7a, Actin, and GAPDH. In terms of differential expression of proteins among the 4 times, RAB10 and ALDH4A1 showed to be significantly high during the morning and reduced by the end of day, while GOLM1, FUBP1 and TFF3 showed significantly low in the morning compared to the evening time.

Summary/Conclusion: Immunoaffinity bead‐based method represent a simple and rapid method for isolating salivary EVs for proteomic profiling and downstream analysis. This study identified diurnal variation in the number and composition of salivary EV proteins identified. Time of sample collection thus should be considered an important covariate when analyzing salivary EV cargo.

PT03.15. FunRich enables enrichment analysis of extracellular vesicles OMICs datasets

Mr Sriram Gummadi

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: High‐throughput methods to profile the genome, transcriptome, proteome and metabolome of various systems has become a routine in multiple research laboratories around the world. Hence, user‐friendly software tools to analyse, integrate and interpret multi‐OMICs datasets are essential.

Methodology: FunRich tool was developed in C# language using Microsoft.NET library and is compatible with Microsoft Windows. Hypergeometric distribution test was performed to check the statistical significance of enriched and depleted terms. In addition, Bonferroni and Benjamini–Hochberg (BH) aka FDR (false discovery rate) method is also implemented to correct for multiple testing.

Results: We report FunRich tool that enables biologists to perform functional enrichment analysis on the generated datasets.

Users can perform enrichment analysis with a variety of background databases and have complete control in updating or modifying the content in most of the databases. Specifically, users can download and update the background database from UniProt at any time thereby allowing a robust background database that can support annotations from >18 taxonomies.

The new additional features implemented in FunRich include miRNA enrichment analysis, plugin to analyse extracellular vesicle datasets from Vesiclepedia database, customisable heatmaps, comparison of oncogenes using COSMIC database, ID conversion and customisable colour for all the publication quality graphs.

Importantly, since 2015, FunRich website has been visited by >82,173 users around the world with >248,000 page views. FunRich has also been widely used for analysis extracellular vesicles dataset and users have compared their data with Vesiclepedia.

Conclusion: Overall, FunRich (http://www.funrich.org) tool is user‐friendly and enables users to perform various analysis on their datasets with minimal or no aid from bioinformaticians.

PT03.16. Modulating nonspecific uptake of engineered extracellular vesicles

Beth DiBiase^{Chemical and Biological Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}, Roxana Mitrut^{Chemical and Biological Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}, Taylor Gunnels^{Biomedical Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}, Dr. Neha Kamat^{Biomedical Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}, Dr. Joshua Leonard^{Chemical and Biological Engineering, Northwestern University, Evanston, IL; Center for Synthetic Biology, Northwestern University, Evanston, IL}

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Engineered extracellular vesicle (EV) therapies are emerging treatment modalities for many diseases. An open challenge for clinical use is the susceptibility of EVs to nonspecific uptake by the mononuclear phagocytic system, leading to fast clearance times. Extending circulation time and decreasing nonspecific uptake would provide therapeutics with more opportunities to reach their targets while maintaining the biological advantages of EVs as delivery vehicles. We hypothesized that engineering EVs with biopolymers, specifically mucins, would reduce their nonspecific uptake and increase their circulation time. We envision this technique may represent a tunable handle for modulating EV uptake to fit a desired application.

Methods

In vitro, we isolated EVs from mScarlet‐expressing FreeStyle 293‐F cells, stably engineered to display varying densities of GFP‐tagged mucin on their plasma membranes, using differential centrifugation. Measurement of mucin loading on EV surfaces was determined via GFP fluorescence, and mucin EV morphology was evaluated using cryo‐EM. Changes in nonspecific uptake conferred by mucins were determined by flow cytometry using an in vitro uptake assay. RAW 264.7 mouse macrophage‐like cells were utilized in this assay, as they provide an indication of the potential effects on nonspecific uptake by the MPS in vivo.

Results

Cryo‐EM images confirm the presence of mucin‐like projections from the EV surface, as well as changes in EV morphology leading to non‐spherical structures. Fluorescent protein gels indicate that EVs isolated from cells that express different amounts of mucin display different levels of mucin on the EV. Preliminary uptake study results indicate a reduction in non‐specific uptake compared to control conditions when EVs are loaded with high amounts of mucin biopolymer.

Summary/Conclusions

We theorize that EV nonspecific uptake can be modulated by tuning the amount of mucin biopolymer displayed on the EV surface. Further investigation into balancing mucin‐conferred EV shielding with targeting is ongoing. Studies investigating membrane property changes due to mucin loading which affect uptake will elucidate the underlying mechanisms, and preclinical studies investigating circulation time will advance therapeutic treatments. We anticipate that this study will expand the field of engineered EV therapeutics and accelerate their development to combat diseases.

PT03.17. Multi‐omics characterization of highly enriched human plasma small extracellular vesicles

Ms Huaqi Su, Assoc. Prof. Kevin Barnham, Prof. Gavin Reid, Dr. Laura Vella

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Cellular processes such as endocytosis, autophagy, phagocytosis and mitochondrial OXPHOS, among others, influence small extracellular vesicles (sEVs) biogenesis and cargo packaging, and these processes have been implicated in various diseases. The ability to measure alterations in these pathways in humans would be valuable for understanding disease mechanisms and potentially offer biomarkers for the early detection of disease. However, directly measuring changes in these pathways is challenging. The biogenesis of sEVs, including cargo sorting, happens at the convergence of these pathways, offering an opportunity to examine plasma sEVs, which can provide insight of cellular conditions in diseases. Moreover, sEVs are enriched in lipids, providing a window into diseases involving lipid dysregulation. To explore potential of sEVs‐based biomarkers in blood, it is essential to enrich and characterize blood sEVs with minimal blood contaminants (blood proteins and lipoprotein particles), that are easily co‐isolated. The co‐isolated blood contaminants pose challenges for mass spectrometry in determining sEVs molecular composition (proteins and lipids), making the value of plasma sEVs to report on cellular pathway changes or lipid alteration unclear.

Methods: Human plasma sEVs were isolated by density fractionation and size exclusion chromatography. The highly enriched sEVs were characterised by western blot, transmission electron microscopy, and quantitative mass spectrometry‐based proteomics and lipidomics.

Results: Lipidomic analysis showed that sEVs are depleted of cholesteryl esters but rich in sphingomyelin (SM), glycerophosphoethanoamine (PE), and glycerophosphoserine (PS), which have been implicated in various diseases. Proteomic analysis revealed that plasma sEVs harbour protein networks associated with endosomal, autophagic and lysosomal (EAL) pathways and mitochondria that have not been reported previously.

Conclusion: Highly enriched plasma sEVs harbour protein networks associated with EAL and mitochondrial pathways, and subsequently, can provide a platform for identifying disease associated changes in these pathways outside the cell. The functional lipids, such as SM, PE, and PS, that enriched in plasma sEVs, provide the opportunity to investigate diseases associated with lipid dyshomeostasis.

PT03.20. Proteomic analysis of extracellular vesicles secreted by human umbilical cord mesenchymal stem/stromal cells under stimulated conditions

Dr. Chaiyong Koaykul, Dr. Kunthika Mokdarta, Dr. Poorichaya Somparn, Dr. Jiradej Makjaroen, Dr. Chatikorn Boonkrai, Dr. Trairak Pisitkun

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Protein cargos inside the extracellular vesicles (EVs) are signaling molecules that affect several functions in the receiving cells. Mesenchymal Stem/Stromal Cell (MSC)‐derived Extracellular Vesicles have been growing interest in drug development for many clinical applications. MSC is the cell that produces EVs by responding to the surrounding environment. However, little is known about the cargo content of MSC‐EVs secreted by MSCs from different culture conditions. Therefore, this study focuses on analyzing the protein cargo profile in EVs from MSCs cultured under normal and stimulating conditions.

Methods

Human umbilical cord‐derived MSCs were grown under xeno‐free culture media, and the cells at cumulative population doubling levels (PDLs) between 16 – 20 were used in this study. MSCs at 70‐80 % confluence were cultured under several EV harvesting conditions: (1) condition media (control condition), (2) condition media with inflammatory cytokines (cytokine condition), and (3) condition media with hypoxia condition (hypoxia condition). After that, EVs from each condition were isolated by ultrafiltration and size‐exclusion chromatography process. Then, the protein cargo in EVs was investigated by mass spectrometry technique.

Results

The size and physical characteristics of EVs from all harvesting conditions showed no significant difference. Based on mass spectrometry analysis, we found a total of 700‐900 proteins in EV cargo in each condition. The EVs from cytokines stimulating condition showed the upregulation of anti‐inflammatory function‐related proteins, and the EVs from hypoxia stimulating condition presented upregulation of redox proteins and angiogenic function‐related proteins than control conditions.

Conclusion

This study provides the proteomic information of EV cargos from MSCs collected from controlled and stimulated culture conditions.

PT03.22. Sizing and visualization of single EVs using a super‐resolution based workflow to characterize EV populations

Dr James Rhodes, Dr Kathleen M Lennon, Dr Colbie Chinowsky, Dr Abigail Neininger‐Castro, Ms Chloe Celingant‐Copie, Dr Daniel Zollinger, Dr Grace DeSantis

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Extracellular vesicles (EVs) are a group of heterogeneous membranous particles ranging in biogenesis, size, and biomarker content. The characterization of EVs remains challenging due to the heterogeneity of EV samples and the limited sensitivity and specificity of current EV detection methods. Single‐molecule localization microscopy, such as dSTORM imaging, enables researchers to visualize and characterize EV populations with 20 nm resolution.

Methods

Purified EVs from three different cell lines were immobilized on the surface of functionalized coverslips using ONI's EV Profiler 2, either by individual or by pooled antibodies against CD9, CD63 and CD81. Affinity‐isolated EVs were then fluorescently labeled with a pan‐EV marker and selected antibodies against CD9, CD63, and CD81. EVs were imaged with dSTORM on a Nanoimager, and vesicle characterization was performed using cluster‐based analysis to extract key features, including circularity, size, density, and biomarker content inside and outside of the EV. EV size from each sample was compared to data obtained using Transmission Electron Microscopy (TEM).

Results

EV Profiler 2 showed high efficiency of capturing purified EVs using either biotinylated antibodies or ONI's PS capture method. EV profiling with an anti‐CD9, CD63 and CD81 antibody co*cktail or 2 tetraspanin antibodies and the pan‐EV stain enabled precise characterization of EV size across 3 different populations. In addition, differences in biomarker positivity fractions were observed across the 3 populations, showing the power of dSTORM imaging at quantifying biomarkers at a single EV level.

Summary/Conclusions

Single‐molecule localization microscopy, such as dSTORM imaging, allows researchers to determine EV size across populations with a higher throughput than TEM. Additionally, multicolor dSTORM imaging allows for the detection of biomarkers on single EVs, opening up a range of possibilities for EV research.

PT03.23. The modulating effect of short, cationic peptides on EV's protein corona

PhD Imola Cs. Szigyarto, Priyanka Singh, Tasvilla Sonallya, PhD Aniko Gaal, PhD Lilla Turiak, PhD Laszlo Drahos, PhD Zoltan Varga, PhD Tamas Beke‐Somfai

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Identifying the spontaneously formed protein corona on the surface of nanoscale materials is of critical importance for the therapeutic and diagnostic development of nanoparticles. In order to explore the potential role in manipulating the surface of nanoparticles and also to progress towards surface engineering, a few short, cationic peptides with well‐characterized action mechanism were selected. The interactions of selected peptides with EV protein corona create new opportunities in wound healing, tissue repair or biofilm inhibition, as both could be present in the complex microenvironment.

Methods

Nanoscale liquid chromatography coupled to tandem mass spectrometry was performed for characterizing protein corona composition. Red blood cell‐derived extracellular vesicles (RBCEVs) were isolated, as they are easily produced in high concentrations, and have several unique advantages, such as the lack of DNA, prevention of immune clearance and extended circulation due to the presence of several integral membrane proteins. As a first step, the effective peptide concentration for treatment of RBCEVs was determined using spectroscopy, flow cytometry and microfluidic resistive pulse sensing techniques. Size exclusion chromatography measurements were performed to separate intact vesicles from protein rich soluble fractions after peptide treatment.

Results

Based on results a total of 125 proteins were identified in the control sample, consisting of membrane‐bound and cytosolic proteins and metabolic enzymes. The protein content of control and peptide‐treated samples were compared, which resulted in high number of common proteins, supporting the good selection strategy of the peptides. Furthermore, the relative abundance of characteristic membrane and subcellular proteins remained mainly constant after treatment. The list of proteins found in treated sample was subtracted from the control protein list, which resulted in seventeen proteins, identified as protein corona. Interestingly, some applied peptides also caused the appearance of several new proteins, not identified in the control sample. These may originate from the electrostatic attraction of partially negatively charged EV surfaces and cationic peptides, which acts as membrane anchors in the attachment of these proteins to EV surfaces.

Conclusions

Our results envisage the applicability of cationic peptides in modulating protein corona content, as a possible tool in nanoparticles surface engineering.

PT03.24. Vesiclepedia and ExoCarta: A web‐based compendiums of extracellular vesicles cargo and extracellular particles

Mr Sriram Gummadi

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Extracellular vesicles (EVs) are nanosized membrane bound vesicles that are shed by cells into extracellular space. In addition to EVs, some of the mammalian cells are also reported to secrete extracellular particles (EPs). EVs have a key role in multiple pathophysiological processes due to their signalling capabilities and are classified into various subtypes based on their biogenesis and origin. Exosomes are small EVs that range in 30–150 nm diameter and they originate due to the fusion of multivesicular bodies with the plasma membrane.

In terms of the molecular composition, EVs sequester a diverse and rich cargo of proteins, lipids, and nucleic acids, and the ensuing cargo is altered according to the pathophysiological conditions of the host cells that secrete them. Hence, a catalogue of the EV cargo in various conditions could aid in identifying an EV fingerprint that are specific to a tissue, cell type, and/or pathology.

Methodology: Vesiclepedia and ExoCarta are built using a combination of MySQL for the background database, Zope content management system and Python as the programming language that connects the database to the content management system and the data is manually annotated and curated from published and unpublished studies.

Results: We report an update of Vesiclepedia (http://www.microvesicles.org) an online database that contains a list of RNA, proteins, lipids and metabolites that are identified in EVs and EPs and ExoCarta (http://www.exocarta.org), a manually curated web‐based compendium of exosomal cargo and it features dynamic protein‐protein interaction networks and biological pathways of exosomal proteins.

Currently, Vesiclepedia contains data obtained from 3,533 EV studies, 50,550 RNA entries, 566,911 protein entries, 3,839 lipid entries, 192 metabolite and 167 DNA entries. In the latest update, a new feature EV‐QUANT is added where it allows for relative quantification between EV protein/RNA/lipid cargo samples within one specific study. Furthermore, Vesiclepedia also contains EV and EP‐associated DNA data that were obtained from 52 studies including 167 data entries for oncogenes and mutated genes.

Conclusion: A catalogue of EV cargo will immensely benefit the research community in identifying an EV fingerprint that are specific to a tissue, cell type, and/or pathology.

PT03.26. Establishment of an immunocapture method for the separation of a rheumatoid arthritis‐related CD90+ subpopulation of extracellular vesicles

M.Sc. Stefanie Kurth, PhD André Tiaden, M.Sc. Edveena Hanser, Ute Heider, PhD Stefan Wild, Professor Diego Kyburz

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Extracellular vesicles (EVs) were previously reported to contribute to the pathogenesis of various diseases including rheumatoid arthritis (RA) – a chronic inflammatory autoimmune disease with high global socioeconomic burden. However, most studies are based on bulk analysis of EVs, neglecting the complexity of different EV subsets present in biofluids of patients. To investigate and characterize distinct EV subpopulations within a heterogeneous mix of EVs in RA we established an immunocapture separation method based on magnetic nano‐sized beads targeting a relevant marker in RA synovial tissue. In this context CD90/THY1 was selected due to the reported contribution of CD90+ fibroblasts in RA synovitis.

Methods: EVs secreted from cultured synovial fibroblasts of arthritis patients (RA and Osteoarthritis) were isolated via size exclusion chromatography (SEC) and subsequently characterized via Transmission Electron Microscopy (TEM), Nano Tracking Analyzer (NTA), NanoFCM and WesternBlot (WB). The separation of a CD90+ population from the heterogenous EV population was performed with anti‐CD90 EV Isolation MicroBeads (Miltenyi). WB and mass‐spectrometry based proteomics were applied to identify characteristics and differences between the two populations. Spiking of fibroblast‐derived EVs in RA patient‐derived synovial fluid was used to test the separation approach in more complex source material.

Results: WB and NanoFCM results confirmed the presence of CD90 on in vitro fibroblast‐derived EVs. The magnetic bead‐based separation of heterogenous fibroblast‐derived EVs resulted in a CD90+ and a CD90‐ EV subpopulation. WB and proteomics confirmed the enrichment of CD90 in the CD90+ population. Furthermore, both populations exhibited distinct EV marker patterns with enriched Annexin‐1 in CD90+ EVs, and higher expression of CD63 and Flotillin‐1 in the CD90‐ population. Additional EV markers like CD9 and Syntenin were similarly expressed. Finally, spiked CD90+ EVs were successfully retrieved from synovial fluid demonstrating the feasibility of the separation approach in complex biofluids.

Summary/Conclusion: Using a magnetic immunocapture approach we were able to separate a CD90+ population from a heterogeneous mixture of in vitro fibroblast‐derived EVs and also from synovial fluid spiked with CD90+ EVs. The ability to investigate distinct EV subsets present in biofluids of patients with various diseases may offer novel insights into their role in pathogenesis.

PT03.27. ExoPAS: numerous and pure isolation of exosomes using cationic material and PEG

Wonjae Kim, Student Kangmin Lee

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1) Introduction

Extracellular vesicle‐derived microRNAs (EV‐miRNAs) are promising biomarkers for early cancer diagnosis. Nevertheless, existing EV‐miRNA extraction technologies involve a complex two‐step process that results in low extraction efficiency and inconsistent outcomes. This study introduces miRQuick, a novel single‐step extraction method designed for the efficient and high‐recovery extraction of EV‐miRNAs from various samples.

2) Methods

The miRQuick method involves adding positively charged substances to the sample, causing negatively charged EVs to quickly aggregate and precipitate. Subsequently, EV sediment is then lysed, followed by miRNA extraction utilizing the spin column extraction method. This entire process, achievable with standard laboratory equipment, can be completed within an hour.

3) Results

We validated the miRQuick method using diverse analytical techniques with a comparative assessment against existing methods for plasma, urine, and saliva samples. The miRQuick method demonstrated significantly higher performance than other methods, not only for blood plasma but also for urine and saliva samples. Furthermore, we successfully extracted and detected 9 biomarker candidate miRNAs in the plasma of breast cancer patients using miRQuick.

4) Summary/Conclusion

Our results demonstrate that miRQuick stands out as a revolutionary single‐step method, addressing the limitations of current extracellular vesicle‐derived microRNA (EV‐miRNA) extraction techniques across diverse sample types with excellent repeatability. Its implementation holds promise for groundbreaking early cancer diagnosis, opening avenues for further research and clinical applications in the evolving landscape of biomarker discovery and diagnostics.

PT03.28. A biomimetic vortex tangential flow filtration (VTFF) system for efficient isolation and purification of extracellular vesicles

Ph.D. Candidate Yuxin Qu^{School of Biomedical Engineering, Tsinghua University, Beijing, China}, Assistant professor Han Wang^{School of Biomedical Engineering, Tsinghua University, Beijing, China}, Lan Xie^{School of Basic Medical Sciences, Tsinghua University, Beijing, China}

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

According to MISEV2018, EV isolation protocols with both high recovery and high purity have not been achievable yet. This is mainly attributed to the dilemma of effectively retaining EVs while simultaneously removing contaminants. To address this issue, we drew inspiration from nature and took cues from the unique gill arch structure of suspension‐feeding fishes. These fishes (e.g., paddlefishes) benefit from rib‐and‐groove gill rakers that allow them to efficiently filtrate zooplanktons for food without clogging. Mimicking this process, we designed a vortex tangential flow filtration (VTFF) microfluidic system for effectively recovering and purifying of EVs.

Methods

Inspired by the suspension‐feeding mechanism, we have developed microfluidic chips and auxiliary systems for efficient filtration of EVs. First, the generation of vortex profile was evaluated through numerical simulations and fluidic experiments. Subsequently, scanning electron microscopy (SEM) was employed to verify the prevention of clotting. After optimizing experimental parameters, the VTFF system was utilized for the isolation of EVs from human plasma and urine, with a comparative analysis against ultracentrifugation and PEG‐based approaches. The isolated EVs were characterized using transmission electron microscope (TEM), nanoparticle tracking analysis (NTA), enzyme‐linked immunosorbent assay (ELISA), and nanoparticle flow cytometry (NanoFCM). Moreover, this system was utilized to isolate EVs from urine of mice with and without myocardial infarction for miRNA sequencing.

Results

During chip operation, EVs undergo continuous flow and separate without adhering to the filter membranes. Baffles in the microchannels induce vortex where samples continuously scour the membranes and prevents clogging during continuous tangential flow through the membranes. The recovery of EVs from human plasma was >88% while the removal rate of free proteins was >99%, which outperformed ultracentrifugation and PEG‐based approaches. TEM images showed typical round and cup‐shaped structures, suggesting high integrity of isolated EVs. Besides, miRNA sequencing from isolated urine EVs has discovered novel differentially expressed miRNAs for myocardial infarction with biological significance.

Summary

In summary, The VTFF systems can generate vortex streaming during tangential flow filtration, and effectively purify and recover EVs from complex body fluids. Based on this system, novel urinary EV markers for myocardial infarction diagnosis were discovered.

PT03.29. A protocol to differentiate the chondrogenic ATDC5 cell‐line for the collection of chondrocyte‐derived extracellular vesicles

Mr Jose Marchan‐Alvarez, Miss Loes Teeuwen, Mr Doste Mamand, Prof Susanne Gabrielsson, Prof Klas Blomgren, Dr Oscar Wiklander, Dr Phillip Newton

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Endochondral ossification leads to longitudinal bone growth and fracture healing through the actions of chondrocytes, which synthesize and modify cartilage by the release of a variety of particles including extracellular vesicles (EVs). However, our understanding of the role of EVs in endochondral ossification is yet to be fully uncovered. Particularly, it remains a challenge to isolate typical populations of chondrocyte‐derived EVs due to the difficulties both in preserving the morphology and functional capability of primary chondrocytes in culture as well as in applying the serum‐free conditions required for EV production. Here, we used the ATDC5 cell line ‐ one of the most well‐characterized tools used to study chondrocytes in vitro ‐ to recover chondrocyte‐derived EVs from early‐ and late‐differentiation stages, representing chondrocytes before and during cartilage mineralization.

Methods: To select a suitable EV‐collection medium, we compared custom EV‐depleted media with normal growth conditions, at an early (day 10) and a late (day 18) differentiation stage, in terms of phenotype, conditioned media pH, histochemical staining, gene expression and cell viability. Using the selected medium, we then scaled‐up culture volumes and studied the functionality and viability of the chondrocytes at a cellular and ultrastructural level. Thereafter, EVs were isolated from the conditioned medium using size‐exclusion chromatography and EV characteristics, including particle size, ultrastructure and protein: particle ratio, were assessed.

Results: After screening different culture conditions, our data indicate that a serum‐free Opti‐MEM‐based culture medium preserves chondrocyte identity (expression of Sox9, Col2a1 and Col10a1 genes) as well as proteoglycan synthesis, matrix mineralization, and cell viability at both time‐points. A scaled‐up production was used to obtain EVs from early‐ and late‐stage chondrocytes. The chondrocyte‐derived EVs had typical cup‐shaped ultrastructure and expression of classical EV markers (CD63 and ALIX), at quantities suitable (3.27E+7 particles/cm2 at day 10 and 1.12E+8 particles/cm2) for downstream experiments.

Conclusion: We established a method to obtain chondrocyte‐derived EVs before and during cartilage mineralization that may aid the further understanding of their roles in endochondral bone growth and fracture healing.

Funding: This work was supported financially by the Novo Nordisk foundation, Swedish Research Council, Region Stockholm and Karolinska Institutet.

PT03.30. A quick, cost‐free, and user‐friendly cleanup protocol for dye and drug removal from small extracellular vesicle solution

Ioannis Isaioglou, Gloria Lopez‐Madrigal, Jasmeen Merzaban

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Labeling of extracellular vesicles, such as exosomes, is a key component in studies detecting their uptake rate and their organotropic biodistribution. The most common way of labeling is through lipophilic dyes, such as DIO. Removal of the excess dye from the exosome solution is necessary to proceed in downstream assays. However, current cleaning‐up methods require either a time‐consuming, extra ultracentrifugation step, usage of specific equipment, such as size exclusion chromatography system, or single‐use cleaning‐up columns.

Here, we present a novel, rapid, user‐friendly, and free‐of‐cost protocol for eliminating the excess dye from the exosome solution. Introducing the exosome parental cells into the solution containing the excess lipophilic dye leads to the trapping of the latter into the cells. Afterward, by using a simple centrifugation step, the parental cells form a pellet, resulting in a supernatant containing labeled exosomes free of excess dye. By performing the necessary controls using the Nanoparticle Analysis System and Dynamic Light Scattering assays, we confirmed that the introduction of the parental cells into the exosome solution did not cause any qualitative or quantitative alterations in the isolated exosomes.

In addition, given the role of the exosomes as a next‐generation drug delivery system, we evaluated the efficiency of the proposed protocol in eliminating the excess daunorubicin, an anti‐tumor drug. Indeed, the suggested protocol successfully removed the excess drug from the exosome solution.

In conclusion, here we present a novel, fast, user‐friendly, and free‐of‐cost methodology to clean up the excess dye or drug from an exosome solution, without affecting the quality or the quantity of the isolated particles.

PT03.31. A standardized multi‐stage purification process and comprehensive characterization of extracellular vesicles derived from HEK293F cells

Research associate Nhan Vo, Research associate Chau Tran, Research associate HB Nam Tran, Scientist T Nhat Nguyen, Research associate Thieu Nguyen, Scientist DN Diem Nguyen, Research associate Tran Pham, R&D lead Hoai‐Nghia Nguyen, R&D specialist Lan‐N Tu

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Background: Small extracellular vesicles (sEVs) with the size of less than 200 nm are gaining attention as a promising drug nanocarrier. However, the clinical translation of sEVs remains challenging due to the lack of robust manufacturing protocols to produce sEVs with high yield and high purity. The heterogenous nature of sEVs leading to unknown composition of biocargos causes further pushback due to safety concerns.

Methods: We established a quality‐controlled multi‐stage procedure to produce and isolate sEVs from human embryonic kidney (HEK293F) cells. We compared several 2‐step and 3‐step purification procedures for the particle yield and purity. Subsequently, the purified sEVs were fully characterized for identity including sub‐population analysis, content profiling including proteomics and miRNA sequencing, and preclinical safety profile in both in‐vitro and in‐vivo testing.

Results: Our findings demonstrated that culturing HEK293F cells at a high‐density saturatory phase in a semi‐continuous culture provided the optimal yield of sEVs. A novel 3‐step purification procedure combining tangential flow filtration, sucrose‐cushion ultracentrifugation, and bind‐elute size‐exclusion chromatography outperformed all other procedures in terms of sEV purity without affecting particle yield and integrity. Upon characterization, about 50% of the HEK293F‐derived sEVs were positive for all three tetraspanin markers CD81, CD9, CD63. The highly abudant proteins and miRNAs in the sEVs were mostly involved in cell cycle, protein folding and organelle organization processes. Purified sEVs further demonstrated excellent preclinical safety profile after 6 repeated intravenous injections in mice.

Conclusions: Our developed multi‐stage workflow allowed robust and reproducible production of ultrapure sEVs with stable characteristics and highly adaptable to mass‐scale production. The well‐characterized sEVs derived from HEK293F cells could be safe and reliable drug carriers for future therapeutic applications.

PT03.32. A survey study on the status of extracellular vesicle (EV) research in malaysia: current updates

Ts. Dr. Norhayati Liaqat Ali Khan, Dr. Nadiah Abu, Dr. Wai Leng Lee, Dr. Muhammad Farid Nazer Muhammad Faruqu, Dr. Jia Xian Law, Associate Professor Dr. Norshariza Nordin, Dr. Maryam Azlan, Associate Professor Dr. Rajesh Ramasamy, Dr. Sik Loo Tan, Associate Professor Dr. Wan Nazatul Shima Shahidan, Mr. See Nguan Ng, Dr. Kok Lun Pang, Dr. Vijayendran Govindasamy, Mr. Benson Koh, Dr. Pan Pan Chong, Miss Yoong Yi Chong, Mrs. Nur Hidayah Hassan, Mr. Nazmul Huda Syed, Mrs. Maimonah Eissa Sheikh Al‐Masawa

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Malaysia is witnessing a surge in the study of extracellular vesicles (EVs) providing valuable insights into intercellular communication and potential applications in diagnostics and therapeutics. Recognizing the pivotal role of collaborative efforts in advancing the field, The Malaysian Society for Extracellular Vesicles (MySEV) was established. This study aims to provide a comprehensive overview of the current status of EV research in Malaysia, including assessing the depth of understanding, the stage of research development, the encountered challenges, the awareness and adherence to existing guidelines (MISEV2018).

Methods: A targeted participant approach was done by disseminating a link to questionnaires (Google‐form) through close contacts of the MySEV Committee members to government and private institutions as well as industrial partners.

Results: 54 complete responses were gathered from participants from public and private higher education institutions and industrial partners. The majority (85.2%) of survey respondents were actively involved in EV research, reporting 1 to 3 years of experience. The remaining respondents refrained from involvement, citing challenges related to grant applications, inadequate facilities, and lack of collaboration. The primary focus was on exploring EVs as therapeutics (48.1%), with 46.3% of respondents working on EVs from stem cells. Other areas of focus include targeting EVs as biomarkers and drug carriers. The majority were at the stage of experimenting the role/application of EVs in their field of interest (vaccine/diagnostic/therapeutic). Ultracentrifugation was the most commonly used isolation process, followed by ultrafiltration and Tangential Flow Filtration. The participants showed high awareness about the MISEV2018 guidelines whereby 90% of respondents were informed about and adhered to it. The key challenges in EV research in Malaysia included the lack of analysis instruments and incomplete availability of protocols.

Conclusion: This study sheds light on the dynamic landscape of EV research in Malaysia, evidenced through respondents active engagement, primarily on its therapeutic potential. The establishment of MySEV signifies a collaborative effort to advance this field. Challenges such as securing grants, inadequate facilities and limited collaborations need to be addressed. We believe, EV research in Malaysia is heading in the right direction and has great potential to contribute to the overall EV ecosystem internationally.

PT03.33. Advancing scalable production of purified adipose‐derived stem cell extracellular vesicles

Jing Zhou, Ph.D candidate Jiajia Dai, Ph.D candidate Haonan Di, Ph.D candidate Yunyun Hu, Ph.D candidate Niangui Cai, professor Xiaomei Yan

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Extracellular vesicles derived from adipose‐derived stem cells (ADSC‐EVs) demonstrate remarkable therapeutic potential in degenerative and inflammatory disease. However, prevailing methods for ADSC‐EV generation are resource‐intensive, time‐consuming, and often lack the necessary assurance of high purity and mass production capability. In this study, we introduce a scalable workflow designed for the efficient cultivation, isolation, enrichment, and purification of ADSC‐EVs. This innovative approach integrates three‐dimensional (3D) cultivation using microcarriers with a custom‐built tangential flow filtration (TFF) system linked to chromatography. Employing a laboratory‐built nano‐flow cytometer (nFCM) enables the real‐time assessment of particle concentration and size distribution of ADSC‐EVs at various stages, providing immediate insights into their yield. Additionally, nFCM enhances the efficiency and precision of evaluating ADSC‐EV purity by integrating Gaussian Mixture Model (GMM) analysis with the size distribution data.

Methods

ADSCs underwent cultivation in a microcarrier‐based three‐dimensional (3D‐M) culture system to promote growth. A specialized tangential flow filtration (TFF) system, uniquely designed for highly‐efficiency isolation and enrichment of EVs from culture supernatants, was constructed and employed. This was followed by chromatography separation to further remove impurity components. Three chromatography columns with different resins were examined for their performance increasing ADSC‐EV purifty. The nFCM was pivotal in assessing particle concentrations, purity, and size distribution of ADSC‐EVs at each stage to optimize the preparation method. GMM analysis integrated into nFCM data provided a rapid purity assessment.

Results

The TFF system significantly boosted EV yield by approximately 10‐fold compared to classical ultracentrifugation (UC) while maintaining EV integrity. However, the purity of EVs upon TFF separation (TFF‐EVs) was approximately 45%, half that of UC‐EVs. Incorporating a preferred chromatography column elevated ADSC‐EV purity to 90%, increasing total yield 4‐fold compared to UC‐EVs. Validation against Triton‐X100 lysis data affirmed the accuracy of GMM analysis in the rapid determination of ADSC‐EV purity.

Conclusions

The as‐developed scalable workflow achieves high‐quality production of ADSC‐EVs suitable for potential large‐scale clinical therapy. Its integration of 3D cultivation, TFF, chromatography, and nFCM analysis offers a promising avenue for efficient, high‐purity ADSC‐EV generation at scale, while acknowledging the cost‐intensive nature of traditional methods.

PT03.35. Benchmarking surface functionalization strategies for marker independent EV capture and profiling

Mr. Hugues Martin, Dr. Andreas Wallucks, Dr. Andy Ng, Ms. Molly Shen, Dr. David Juncker

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Various extracellular vesicle (EV) analysis techniques often rely on surfaces to capture the EVs, notably planar surfaces in immunofluorescence imaging and beads in flow cytometry. Commonly, affinity binders enrich EVs positive for one protein and measure other targets’ presence with detection antibodies. This inherently restricts analysis and fails to establish a protein expression baseline in the total population, convoluting data interpretation. Marker independent capture surfaces can be used to establish an unbiased expression profile or to classify subpopulations in single EV analysis. Most marker independent surfaces capture EVs by their physical properties such as surface charge or hydrophobicity. The resulting biases in EV proteomic composition, however, are so far poorly characterized. Here, we provide a systematic comparison between common and newly proposed marker independent surfaces.

Methods: Glass coverslips were functionalized with silanes terminated with various functional groups including aldehyde, amino, and methoxy. These surfaces were studied for physisorption, electrostatic capture, or hydrophobic capture. Furthermore, aldehyde functionalized coverslips were functionalized with either membrane curvature‐sensing peptides, lactadherin, an affinity binder of common EV lipids, or a tetraspanin antibody co*cktail. We used size photometry and fluorescence imaging (SPFI), an in‐house single EV imaging platform that allows characterization of both size and protein expression, to characterize EVs captured on the different surfaces. The expression profiles were compared with flow cytometry, and the size distribution, with nanoparticle tracking analysis, both capture‐free methods. We used SEC purified EVs from HT29 human adenocarcinoma and 293T human embryonic kidney cells.

Results: Electrostatic capture using silanes with an aldehyde functional group had similar protein expression profile, while electrostatic capture‐based amine‐functionalized surfaces had higher expression of CD9 but lower expression of CD63 and CD81. Hydrophobic capture based methoxy‐functionalized surfaces had lower EV protein expression of all markers and, furthermore, tend to capture smaller EVs. Peptide, lactadherin and tetraspanins capture surfaces will be presented at the conference.

Conclusion: Target‐independent EV capture mechanisms impact the observed EV populations. While surfaces that capture based on physisorption, electrostatic capture or hydrophobic capture are marker independent, they aren't bias‐free. These results should be considered when used with capture surface techniques.

PT03.36. Beyond the boundaries of conventional isolation techniques: Functional self‐assembled coordination polymer nanoparticles for instant one‐step selective and efficient enrichment of exosomes – ExoFlocs™

Mr Mohamed Sallam, Mr Cong‐Minh Nguyen, Dr Amandeep Singh Pannu, Dr Indira Prasadam, Mr Yezhou Yu, Professor Serge Muyldermans, Dr Frank Sainsbury, Professor Nam‐Trung Nguyen, Professor Nobuo Kimizuka

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

The advancement of innovative isolation techniques for extracellular vesicles (EVs) has garnered significant attention owing to their pivotal roles in intercellular communication and potential as disease biomarkers. This study introduces an original approach utilising self‐assembled functional coordination polymer nanoparticles (CPNs) for the rapid and efficient isolation and enrichment of small extracellular vesicles (sEVs), particularly exosomes, from complex biological fluids. Our pioneering nanostructured material called ExoFlocs™ is employed in conjunction with anti‐PLAP monoclonal antibodies for selective sEVs enrichment, followed by characterisation and biomarker analysis. The investigation employs a one‐pot synthesis method with adaptive antibody inclusion, yielding ExoFlocs™ nanostructures with adjustable size and composition. Comprehensive characterisations, encompassing dynamic light scattering, zeta potential analysis, high‐resolution scanning electron microscopy, and energy‐dispersive X‐ray spectroscopy, validate the stability and attributes of ExoFlocs™. The study successfully isolates placental sEVs from cell culture supernatant using anti‐PLAP@ExoFlocs particles, achieving a notable capturing efficiency of 97.5%. These isolated sEVs are characterised by their expression of endosomal markers. Notably, a distinctive approach is introduced to rescue sEVs from ExoFlocs™ nanoparticles, utilising inorganic phosphate ions and simplifying downstream processes. Furthermore, the utility of isolated sEVs for subsequent research is validated through RT‐qPCR analysis of a specific mRNA biomarker, KiSS1. The study demonstrates that the isolated sEVs maintain their RNA content and integrity. The innovative one‐step, chelation‐free adaptive inclusion technique holds promise for diverse applications in the realms of targeted drug delivery, biomarker discovery, and enhanced diagnostic tools.

PT03.38. Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy

Jie Gong, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Meng Han, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Bairen Pang, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Qi Wang, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Haotian Chen, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Zhihan Liu, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Cheng Zhou, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Yong Li, Comparative study of urine small extracellular vesicles isolation methods in prostate cancer liquid biopsy Junhui Jiang

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Prostate cancer (PCa) is a major man's health challenge. Current diagnostic tools in PCa such as PSA test and biopsy are inaccurate and not specific. Liquid biopsy is a non‐invasive approach and can overview all pictures of tumor development. Urine is an excellent source for PCa liquid biopsy using extracellular vehicles (EVs), which play important roles in cancer communications. However, a standardized and reliable method for urine EVs separation remains elusive. In this study, we aimed to compare EXODUS (a novel automated exosome separation system), with ultracentrifugation (UC) and two commercial kits to find which approach is the best one for small EVs (sEVs) isolation applied for PCa liquid biopsy isolation

Methods: Urine sEVs were isolated from the urine samples of six healthy individuals (n=6) using four distinct methods including EXODUS, UC and two commercial kits (exoEasy™ Maxi Kit and Total Exosome Isolation‐TEI from urine). sEVs isolated were characterized by, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blotting (WB). The purity indexes of sEV isolated were evaluated using particle number and protein concentration ratio. Liquid Chromatography‐Tandem Mass Spectrometry (LC‐MS/MS) was utilized to profile urine sEVs isolated from 4 different separation techniques.

Results: We demonstrate that UC and EXODUS isolations show more consistent repeatability and the particle concentration of EXODUS isolates was significantly higher compared to UC and exoEasy, but not significantly different compared to TEI. The purity of UC and EXODUS isolates was significantly higher than that exoEasy. Downstream proteomic analysis is ongoing.

Conclusion: This study provides a comprehensive evaluation of four urine sEVs isolation techniques, laying the groundwork for selecting appropriate methods tailored to specific research objectives. EXODUS demonstrates a notable enhancement in urine sEVs extraction efficiency and purity compared to other methods, showing promise for its integration into clinical practice for the rapid isolation of urine EVs from human urine and the exploration of potential protein markers in PCa patients. This advancement holds potential in revolutionizing PCa early diagnosis and risk stratification in liquid biopsy.

PT03.39. Comparison of asymmetric depth filtration and ultrafiltration combined with size‐exclusion chromatography for EV isolation from cell culture media

Dr. Vasiliy Chernyshev, Dr. Elena Svirshchevskaya, Mr. Mikhail Ivanov, Dr. Denis Silachev

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1) Introduction

It is challenging to isolate EVs from complex biofluids such as plasma, urine, saliva, amniotic fluid, and the growth medium of cultured cells. A complex biomolecular milieu of biofluids, such as protein agglomerates and lipid nanoparticles, which overlap with EVs in size and other physicochemical features, are frequently contaminants of EV samples. In a recent study we developed and presented an innovative method of EV isolation that is based on asymmetric depth filtration (DF). The procedure is low‐cost and straightforward. In 3‐4 h it reproducibly allows to isolate EVs with a high yield and purity from complicated biological fluids using just simple equipment, e.g. traditional centrifuge. In this study we performed a comparison between DF and ultrafiltration coupled with size‐exclusion chromatography (UF‐SEC) method to isolate EVs from cell culture media which was not done previously but has significance in future studies where isolation may be critical in the experimental pipeline.

2) Methods

HCT116 and HT29 colorectal cancer cell lines were used in this study. Cell culture growth medium was split into two equal volumes (50 mL each), one used for DF and the other for UF‐SEC to isolate EVs. Obtained EV samples underwent complete characterization which included nanoparticle tracking analysis, western‐blotting, electron microscopy and proteomics. Data was then compared between the two types of EV isolation techniques.

3) Results

Hydrodynamic and geometric size distribution of EVs was comparable between the two isolation methods. The number of EVs isolated from equal sample volume and purity which was determined by evaluating the amount of EVs present per microgram of protein was nearly the same. Proteomics of EVs isolated by DF from both cell lines showed more protein types that were identified when compared to UF‐SEC.

4) Summary/Conclusion

In this side‐by‐side comparison of two techniques of EV isolation from cell culture media, DF and UF‐SEC, it was determined that both provide high purity samples. When taking into account the initial sample volume it was also found that both provide nearly the same yield from cell culture media. However, DF showed advantage over UF‐SEC in proteomics which should be taken into account.

PT03.40. Comprehensive evaluation of extracellular vesicle markers through diverse isolation strategies

Dr. Kaiping Burrows, Dr. Leandra Figueroa‐Hall, Dr. Ahlam Alarbi, Dr. Bethany Hannafon, Cole Hladik, Dr. Rajagopal Ramesh, Dr. Victoria Risbrough, Dr. T. Kent Teague, Dr. Martin Paulus

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1)Introduction. Extracellular vesicles (EVs) play pivotal roles in intercellular communication, influencing various physiological and pathological processes. With increasing interest in EVs for therapeutic applications, establishing standardized methodologies for their isolation is crucial. The heterogeneous nature of EVs and contaminants in biological fluids present challenges, necessitating a comprehensive comparison of isolation methods and their impact on well‐established EV markers—CD9, CD63, and CD81. Understanding the variability in isolation methods is vital for enhancing the reproducibility and reliability of EV research.

2)Methods. Seven isolation methods were employed: 1) polymer‐based precipitation, 2) low‐molecular‐weight electrolytes‐based precipitation, 3) Size Exclusion Chromatography (SEC), 4) SEC with different fraction collections, 5) Precipitation followed by SEC, 6) Silicon‐Carbide methods, and 7) Differential Ultracentrifugation. EVs were isolated from human plasma, plasma with proteinase K (PK) treatment, human serum, and serum with PK treatment. Blood collected in BD Vacutainer tubes was processed, and plasma and serum aliquots were stored at ‐80°C until EV isolation. EV isolation was performed from 250µL ‐ 500µL of each sample type using the seven methods. EV specificity, size, and concentration were assessed through western blot, immunoassays, and microfluidic resistive pulse sensing. EV markers (CD9, CD63, and CD81) concentrations were evaluated using immunoassays.

3)Results. The SEC method on serum samples yielded the highest CD9 concentrations(6.4AU/mL), while both SEC and polymer‐based precipitation from serum+PK showed the highest CD63 yield(0.55AU/mL). Polymer‐based precipitation from plasma or serum yielded the highest CD81 concentrations (0.27AU/mL). Evaluating purity (EV markers to total protein ratio) revealed the SEC method on serum EVs had the best CD9 purity, and the SEC method on serum+PK EVs had the best CD63 purity. Both SEC methods and Precipitation + SEC demonstrated good CD81 purity in plasma and serum.

4)Summary/conclusion. This comprehensive study highlights the superior performance of the Size Exclusion Chromatography (SEC) method among seven isolation techniques for extracellular vesicles across various sample types. The method consistently demonstrated high yields and purity for CD9, CD63, and CD81, emphasizing the importance of selecting appropriate isolation methods for specific EV markers. These findings contribute valuable insights toward standardizing EV research methodologies, ultimately enhancing reproducibility and reliability in the field.

PT03.41. Confident isolation and proteomics of bacterial extracellular vesicles by size exclusion chromatography

Ms Haekang Yang, Ms Shinwon Chae, Mr Chul Won Seo, Ms Seoyeon Kim, Professor Yoon‐Jin Lee, Professor Dongsic Choi

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Bacteria release the extracellular vesicles (EVs), known as outer membrane vesicles from Gram negative and membrane vesicles from Gram positive. These lipid bilayer particles are ranged from 20 nm to 100 nm which play diverse roles in bacteria pathogenesis such as antibiotics resistance, transfer of materials, and quorum sensing. Bacterial EVs have a unique density with 1.15 g/mL and thus ultracentrifuge or density gradient ultracentrifuge have been widely used to enrich the EVs. However, these methods are time‐consuming and often suffer from contamination issues. Therefore, we established the confident procedure of bacteria EV isolation using optimized size exclusion chromatography (SEC) method while minimizing contamination and optimizing time utilization.

Methods: Limosilactobacillus reuteri and Lactiplantibacillus paraplantarum were obtained from Korean Collection for Type Cultures (KCTC). Bacteria were inoculated into the MRS broth and anaerobic cultured at 37°C and 30°C upto O.D. 1.2 for L. reuteri and L. paraplantarum, respectively. After preclearing of bacteria, the supernatant was concentrated by 100 kDa centrifugal filter. For ultracentrifuge procedure, the concentrated medium (CM) was 50‐fold diluted and then subjected to ultracentrifugation at 150,000g. For SEC procedure, CM was loaded on the column and fractionated. We compared the yield and purity using NTA, microBCA assay, SDS‐PAGE, Coomassie blue staining, TEM and Western blotting for each of the experimental steps. Mass spectrometry‐based quantitative proteomics were conducted to reveal unique proteome of bacterial EVs.

Results: Coomassie blue staining, TEM and Western blotting showed the enriched bacterial EVs from SEC fractions. To compare the purity of each methods, particles (by NTA) per protein amount was applied and SEC method showed the better purity of EV isolations than ultracentrifuge method. Furthermore, proteomic analyses represented that the proteins from membrane and cytosol are enriched in EVs implying unique sorting of selective proteins into EVs form parental bacteria.

Summary/Conclusion: Taken together, we established the efficient isolation method of bacterial EVs by SEC with validation of their proteome by mass spectrometry. This procedure would provide the information of more reliable EV cargos and practical protocol for large scale isolation of bacterial EVs.

PT03.42. Development of a method for large‐scale purification of extracellular vesicles using the PS affinity method

Dr. Afshin Iram, Shotaro Masuda, Hana Onizuka, PhD. Ryo Ukekawa, PhD. Takahiro Nishibu

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1) Introduction

Extracellular vesicles (EVs) contain various bioactive substances such as lipids, nucleic acids, and proteins. Since EVs derived from mesenchymal stem cells (MSCs), have therapeutic potential for various diseases, the use of EVs as therapeutic agents has been thought to be promising. Consequently, multiple methods, such as tangential flow filtration (TFF), size‐exclusion chromatography (SEC), anion exchange chromatography (AEX), and their combination have been developed for a larger scale EV purification. Here, we have developed a scalable and reproducible method for affinity purification of EVs by using Tim4 protein, which specifically binds phosphatidylserine displayed on the surface of EVs. Because the binding is Ca²+‐dependent, intact EVs can be easily released from Tim4 by adding Ca²+ chelators. In this study, we demonstrated purification of EVs from bone marrow‐derived MSC culture supernatant.

2) Methods

Recombinant Tim4 protein was covalently immobilized on the surface of agarose regin and the Tim4‐resin was packed into a 1 mL column. 1000 mL of bone marrow‐derived MSC culture supernatant was filtrated through ⌀0.22 µm filter to remove cell debris and 200 mL of the resulted supernatant was directly applied into the Tim4 column. After washing the column, 4 mL of PBS containing Ca²+ chelators was applied to elute the captured EVs. The resulted EVs were analyzed by using NTA, ELISA and BCA methods. In parallel, EVs were also purified from the 200 mL of the same culture supernatant by using TFF, TFF + SEC, or TFF + AEX, and the resulted EVs were analyzed for comparison.

3) Results

From 200 mL of MSC supernatant, Tim4 column, TFF, TFF + SEC, and TFF + AEX methods gave 1.68 x 10¹¹, 2,74 x 10¹¹, 1,08 x 10¹¹, and 0.7 x 10¹¹ particles, respectively. The highest purity of the purified EVs was obtained by the Tim4 column method (0.5 x 10¹⁰ particles/µg protein) compared to the other methods (0.10 ‐ 0.14 particles/µg protein). ELISA assays using anti‐tetraspanin antibodies also showed a similar trend.

4) Summary/Conclusion

We propose that our affinity purification method has the potential for use in the manufacturing process of therapeutic EVs in fewer purification steps.

PT03.43. Development of an applicable method for bacterial extracellular vesicle isolation from mouse stool supernatant

Shujin Wei, Professor Wanli Xing

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

The animal intestinal tract is colonized by a huge number of microorganisms, which establish a dynamic and bidirectional interaction with the host. Bacterial extracellular vesicles (BEVs), which contain bioactive substances from their parental cells, can undertake diverse biological functions. Therefore, analyzing BEVs in stools may provide more information for disease research. However, the isolation of BEVs is still facing challenges, particularly in distinguishing BEVs from EVs derived from the host. There is an urgent need to develop approaches to separate BEVs conveniently. We aim to develop a new method based on epsilon‐poly‐L‐lysine (ϵ‐PL), which is a broad‐spectrum antimicrobial peptide targeting the surface of bacteria, to enrich BEVs from mouse stool supernatant rapidly.

Methods

We first precipitated BEVs from bacterial culture media using our in‐house ϵ‐PL‐based method (PL). The isolated BEVs were identified by transmission electron microscopy, nanoparticle tracking analysis, and LC‐MS/MS analysis. The results were compared with those of the commonly used ultracentrifugation method (UC). Next, the cell selectivity of ϵ‐PL was validated in a simulated sample containing EVs derived from both mammalian cells and bacteria using western blotting analysis. Thirdly, a protocol to enrich BEVs from mouse stool supernatants was established. Using 16S rRNA gene sequencing, the microbial compositions of BEVs enriched by UC and PL were analyzed and compared with stool samples.

Results

By binding to the surface of BEVs, ϵ‐PL can facilitate precipitation of BEVs at a relatively low centrifugal speed (10,000 × g). BEVs isolated by PL from bacterial culture media were comparable to those isolated by UC in size distribution, morphology, and protein profile. Moreover, ϵ‐PL shows a higher affinity for BEVs relative to EVs derived from mammalian cells, which makes it possible to selectively enrich BEVs from stool supernatants in a single step. The microbial composition analysis of BEVs isolated from mouse stools conveyed unique profiles and may identify novel biomarkers for disease.

Summary/Conclusion

This study provides a new method to enrich BEVs from mouse stool supernatant. As a cost‐effective, simple, and scalable method, the PL method could be used as a powerful tool for therapeutic or diagnostic purposes requiring BEV enrichment.

PT03.44. Does EV purity affect downstream functionality?

Research Officer Janice Tan, Principal Investigator Ivy Ho

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

Extracellular vesicles (EVs) are small membranous structures released by all cell types into the extracellular environment. EVs play a pivotal role in intercellular communication and have garnered interest as biomarkers of various diseases. In addition, transfer of EVs between cell types was shown to affect the functionality of the recipient cells. The purity of EV is crucial in their function and potential clinical application. In this study, we employed two different methods to isolate EVs from conditioned media (CM) and compared their size, purity and functionality on microglia/macrophage.

Methods:

EVs were isolated from the same batch of human glioma cells U251MG cultured in serum‐free DMEM. The CM was divided equally and subjected to ultracentrifugation (UC) or size exclusion chromatography (SEC). Isolated EVs were characterised based on MISEV2018 guidelines (western blot, transmission electron microscopy, nano‐flow cytometry). The immune‐modulation effect of the EVs was tested in microglia/macrophages (migration, phagocytosis and cytokine expression).

Results:

To avoid deleterious effect from contaminating proteins co‐purified during UC or SEC, the purity of the EVs were assessed. Protein measurement against particles number evaluation showed higher protein concentration in UC‐isolated EVs compared with SEC. Using immunoblotting, both UC and SEC‐EVs were found to express CD81, CD63, and TSG101. CD81 was significantly increased in UC‐isolated EVs. Interestingly, Syntenin‐1 was only present in SEC‐EVs. Data from Nano‐flow showed minimal size difference despite a difference in concentration. EV‐exposed microglial cells were evaluated and the effect on migration, phagocytosis, filopodia formation and cytokine expression was tested. Net migration of microglial cells was quantified after 24 hours. The data showed that both EVs isolated using UC and SEC exert similar effects on the microglia/macrophages in vitro.

Summary/conclusion:

Both methods isolated EVs with minimal variations which may indicate a difference in cargo. This difference could potentially influence downstream functions. The absence of syntenin‐1 in UC‐isolated EVs, despite their similar size, suggests that the two isolation methods may have captured distinct EV populations of the same size. Additional investigations will be conducted to elucidate the reasons for the exclusive presence of syntenin‐1 in SEC‐EVs and to explore its potential involvement in subsequent cellular functions.

PT03.46. Evolution of an EV enrichment protocol: from minimal information to proteomics

Dr Felicity Dunlop, Dr Shaun Mason, Dr Taeyoung Kang, Professor Suresh Mathivanan, Professor Aaron Russell

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Extracellular vesicles (EVs) and major plasma protein constituents have several overlapping biophysical properties. This provides considerable challenges for EV enrichment, accurate quantification and downstream analysis. The objective of this project was to combine multiple purification approaches, including size exclusion chromatography (SEC), strong anion exchange (SAX) magnetic bead enrichment and single‐pot, solid‐phase‐enhanced sample preparation (SP3) to develop a method to enrich EVs from 500 µL of human plasma for proteomics analysis.

Methods

Blood was collected in EDTA tubes and centrifuged to remove red blood cells and platelets. Plasma stored at ‐80°C was thawed and centrifuged (precleared plasma). Precleared plasma was subjected to SEC. Fractions were collected for SAX magnetic bead enrichment, with or without concentration by centrifugal filtration. After overnight binding with SAX magnetic beads and washing, EVs were lysed and EV proteins solubilised using an SDS‐based lysis buffer and heating at 95⁰C. Reduction and alkylation was conducted simultaneously with TCEP and IAA. Proteins were then prepared using a standard SP3 protocol with STAGE tip desalting prior to injection on an LC‐MS (Orbitrap). To establish a more robust reproducible approach, 500 µL plasma samples were split into three 150 µL aliquots after preclearing. Each aliquot was individually processed using SEC/SAX then analysed via LC/MS. Triplicate proteomics data was combined.

Results

The minimal information for EV studies were met using electron microscopy, TRPS and Western blotting; the latter indicating the presence of EV markers CD63 and syntenin, absence of calnexin and a reduction in albumin and ApoA1 in SEC‐enriched plasma EVs, compared with precleared plasma. Syntenin, CD9 and CD81 were detected by proteomics analysis in SEC‐enriched plasma. However, ApoB100 lipoprotein was also enriched. Therefore, the SEC output was subsequently subjected to SAX to further enrich for EVs based on surface charge. The addition of the SAX purification step doubled the number of quantifiable EV‐associated markers, measure by LC/MS. This protocol consistently detected ∼500 proteins with 80‐100 of these, quantifiable EV markers.

Summary/Conclusion

Our SEC/SAX enrichment protocol doubles the amount of quantifiable EV markers, compared with SEC alone, as measure by LC/MS from 150 µL of human plasma analysed in triplicate.

PT03.47. ExoCAS‐2: rapid and pure isolation of exosomes by anionic exchange using magnetic beads

Student Jaeeun Lee

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1) Introduction

Extracellular vesicles (EVs) are emerging as crucial biomarkers in liquid biopsies. However, despite extensive efforts to translate EVs into clinical practice, the use of EVs is currently hindered by the limitations of existing EV‐isolation technologies, which remain in their early stages of development.

2) Methods

This study introduces a novel magnetic bead‐based ion exchange platform, termed ExoCAS‐2 (exosome clustering and scattering), for efficient EV isolation. This platform leverages the inherent negative charge of exosomes to facilitate their binding to magnetic beads coated with a polycationic polymer. The magnetic properties of the beads enable facile manipulation through washing and elution steps, allowing for the isolation of highly pure and high‐yield exosomes within 40 minutes.

3) Results

The proposed method successfully isolates exosomes, as evidenced by analyses of their size distribution, morphology, surface and internal protein markers, and exosomal RNA. Compared to commercially available methods, ExoCAS‐2 demonstrates superior performance in terms of key aspects such as operation time, purity, and recovery rate.

4) Summary/Conclusion

ExoCAS‐2 highlights the significant potential of the magnetic bead‐based ion exchange platform, ExoCAS‐2, for efficient and high‐quality isolation of exosomes from blood plasma, paving the way for their further translation into clinical applications.

PT03.48. ExoFilter: large capacity extraction of EVs using a positive charge mesh filter in continuous flow

Student Yongwoo Kim

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1) Introduction

The clinical potential of extracellular vesicles (EVs) as therapeutic agents and drug delivery vehicles becomes more and more evident. So, many techniques for EVs isolation have been developed. However, most of the exosome isolation technologies developed so far have been customized in small quantities in a laboratory environment. Therefore, large‐scale processing technology is required.

2) Methods

We have proposed a novel method (ExoFilter) for isolating EVs with a charge‐based filter in continuous flow mode. The cationic materials were bound to a porous nylon mesh (pore size 1 µm) installed on a spin column (d = 6 mm) and then passed through the plasma 1mL. NTA, RT‐qPCR, and Western blot analyses were used to evaluate the efficiency of exosome isolation. SEM image analysis confirmed EVs captured on the mesh surface.

3) Results

The Exofilter method successfully extracted EVs from plasma and serum. EV isolation efficiency increased exponentially as the number of stacked meshes increased. These results demonstrated that the positively charged mesh filter can rapidly and effectively capture and extract exosomes from continuously flowing samples.

4) Summary/Conclusion

Based on the experimental results of this study, we scaled up the ExoFilter, which can accommodate 1 mL of sample, to a large‐capacity ExoFilter which can accommodate 1 L of sample. We also demonstrated that the current continuous ExoFilter method can be used to effectively isolate exosomes from large‐scale samples (up to 1 L) such as cell culture media.

PT03.49. miRQuick: An innovative charge‐based EV isolation method for highly efficient extraction of EV‐miRNAs from liquid samples

Student Lee Kangmin

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1) Introduction

Extracellular vesicles (EVs) are minute membrane‐bound particles released by cells, capable of carrying crucial biological information. While scientists recognize their potential as biomarkers for various diseases, the technology to isolate and purify them remains in its early stages.

2) Methods

This study presents a novel method for isolating EVs, termed ExoPAS (exosome precipitation and scattering), which utilizes a cationic material and polyethylene glycol (PEG). The cationic material attracts the negative charge of EVs in biofluids, resulting in the formation of EV clusters. Subsequently, PEG's mesh‐like structure binds to these clusters, offering stabilization and preventing undesired aggregation with other molecules. This study meticulously analyzes the isolated exosomes from blood plasma, examining their size, morphology, protein content, and RNA composition.

3) Results

The ExoPAS method successfully extracted EVs from diverse biofluids, including plasma, serum, saliva, and urine. Notably, it yielded significantly higher quantities and purity of EVs compared to existing precipitation‐based extraction techniques.

4) Summary/Conclusion

The proposed ExoPAS method outperformed commercially available methods in terms of both purity and recovery rate, indicating that the combination of the cationic material and PEG holds significant promise for robust EV isolation from various biofluids.

PT03.50. High‐throughput isolation and sorting of nanoparticle loaded exosomes

Dr. Hye Sun Park, Taewoong Son, Mi Young Cho, Hyunseung Lee, Eun Hee Han, Dr. Kwan Soo Hong

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1. Introduction

Extracellular vesicles (EVs) have been known to play an important role in intercellular communication. Depending on their origin, EVs can be classified exosomes, which are produced by the endosomal pathway, and microvesicles, which are originated from the plasma membrane. Although various technologies for isolating and purifying exosomes from EVs have been continuously reported, the diversity of characteristics depending on the methods and the difficulty in standardization still remain challenging issues.

2. Methods

Here, we propose a method to isolate and sort exosomes from various cell lines, such as cancer, immune, and stem cells, using nanoparticles. The optimal purification methods were applied according to the characteristics of the nanoparticles used, the purified exosomes were qualitatively analyzed using exosome markers, and the purification process was optimized through quantitative analysis according to unique properties of the nanoparticles.

3. Results

The morphological analysis of nanoparticle containing exosomes were confirmed through TEM images. And Western blot and protein quantitative analysis were conducted to confirm exosome characteristics. As the concentration of nanoparticles increased, the amount of purified exosomes increased, and the luminescence properties of the nanoparticles were quantitatively analyzed with various parameters. Also, it was observed that the isolation of exosomes using nanoparticles could give different yield depending on the type of the cells.

4. Summary

We showed that exosomes containing nanoparticles can be isolated from cell culture media via particle characteristics. We demonstrate collection of exosomes derived from various types of cells sorted from different EVs without differential centrifugation. We suggest that this method opens a new way to investigate EVs based on nanomaterials.

PT03.52. Impact of hyaluronidase on tetraspanin expression of extracellular vesicles (EVs) in synovial fluid from patients with rheumatoid arthritis and osteoarthritis using the Exoview platform

Mrs. Edveena Hanser, Prof. Dr. Diego Kyburz

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Synovial fluid (SF) is a complex biofluid that is produced in excess in patients with joint diseases and is used for diagnostic purposes. SF is highly viscous due to its content of hyaluronic acid, which contributes to a poor inter‐study reproducibility of EV analysis in SF. In the present study we have assessed the impact of hyaluronidase treatment on tetraspanins expression profile in SF and in enriched EVs using a single‐particle interferometric reflectance imaging sensor (SP‐IRIS)‐based ExoView R200 platform.

Method: SF was obtained from knee joints of 4 rheumatoid arthritis (RA) and 4 osteoarthritis (OA) patients, with informed consent. SF was centrifuged, aliquoted and kept at ‐80° until further use. SF aliquots were thawed and treated with 30U/ml of hyaluronidase (Sigma) at 37°C for 45 minutes, treated and untreated aliquots were centrifuged and diluted with PBS. EVs were isolated from treated SF using size exclusion chromatography (SEC). The characterization was done by nanoparticle tracking analyzer and transmission electron microscopy. The concentration of treated, untreated SF and of isolated EVs was adjusted to 1.00E+09 particles/ml for Exoview R200, NanoView Biosciences.

Result: The expression profile of tetraspanins in terms of the number of events on capture spots CD63, CD81 and CD9 was higher in hyaluronidase treated SF from patients with RA compared to untreated SF. In contrast, the tetraspanin expression of treated and untreated OA SF samples was similar. This may be attributed to the fact that OA SF is more viscous and might need higher concentrations of hyaluronidase. The colocalization profile of tetraspanins differed between EVs from treated and non‐treated SF. When EVs were measured directly in SF by Exoview, a higher number of EVs was detected as compared to measurement after EV isolation by SEC.

Conclusion: Hyaluronidase treatment of SF in RA resulted in a higher number of events compared to untreated SF, suggesting removal of hyaluronic acid increased EV yield. Thus, hyaluronic acid treatment of SF should be considered to optimize EV analysis. In addition, enrichment of EVs by SEC resulted in a lower count, suggesting that SF can be used directly without prior EV isolation.

PT03.54. Isolation and enrichment of extracellular vesicles with double‐positive membrane protein for subsequent biological studies

Dr. Huixian Lin, Dr. Chunchen Liu, Prof. Bo Li, Prof. Lei Zheng

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: The isolation and enrichment of specific extracellular vesicle (EV) subpopulations are essential in the context of precision medicine. However, the current methods predominantly rely on a single‐positive marker and are susceptible to interference from soluble proteins or impurities. This limitation represents a significant obstacle to the widespread application of EVs in biological research.

Methods: Herein, we propose a novel approach that utilizes proximity ligation assay (PLA) and DNA‐RNA hybridization to facilitate the binding of two proteins on the EV membrane in advance enabling the isolation and enrichment of intact EVs with double‐positive membrane proteins followed by using functionalized magnetic beads for capture and enzymatic cleavage for isolated EVs release. The TEM, SEM, NTA were used for the isolated EVs characterization and the NanoFCM was used for capture efficiency assessment. For further exploring its application, RNA sequencing was conducted and a breast cancer cohort was collected.

Results: The capture efficiency of this method reaches approximately 83.87%, with each magnetic bead providing an average of 1425 EVs captured. The method we constructed facilitates the isolation and enrichment of large‐scale subpopulations of EVs with a double‐positive membrane protein. Additionally, this method demonstrates excellent specificity, avoiding interference from soluble proteins to a certain extent. Furthermore, our study highlights the potential application of isolating and enriching EVs with a double‐positive membrane protein in biological function studies and biomarker screening. Notably, this technology shows promise in isolating and enriching EVs from heterogeneous plasma‐derived EVs for cancer diagnosis, thus promoting the clinical application of specific EV subpopulations.

Conclusion: In conclusion, our study presents a simple, effective, and novel strategy for isolating and enriching EVs with a double‐positive membrane protein without complex equipment. This strategy has the potential to drive forward EVs research.

PT03.55. Molecular imprinted polymer‐based artificial peptide (MIPap) enables isolation of astrocyte‐specific extracellular vesicles (asEV) in serum

Yong Shin, Professor Eun Jae Lee

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1) Introduction

Exosomes are small vesicles enclosed by a membrane that serve as essential mediators of intercellular communication and are also valuable biomarkers for various diseases, including cancer. However, traditional methods for isolation exosomes, including cell type specific exosomes, are labor‐intensive and require complicated pretreatment techniques.

2) Methods

In this study, molecular imprinted polymers‐based articial peptide (MIPaps) for exosome isolation utilizing protein and peptide from the tetraspanin protein family (CD63/CD9 and CD81) were developed. The technique was confirmed by size distribution by NTA, morphology by SEM/TEM, miRNA level by RT‐qPCR, and protein content of exosomes. Additionally, the capture percentage and recovery rate were calculated.

3) Results

Our findings suggest that peptide templates can be used as an alternative to protein templates for synthesizing MIP‐based MNPs. Lastly, MIP‐based MNPs made with GLAST templates were employed to capture astrocyte‐derived exosomes. The results indicate that MIP‐based MNPs created with GLAST peptides as templates can be utilized to isolate serum exosomes and analyze astrocyte protein markers and miRNA, which may be useful in the diagnosis and monitoring of Neuromyelitis Optica Spectrum Disorder (NMOSD).

4) Summary/Conclusion

The MIP‐based MNPs demonstrate the potential to capture exosomes from biofluids and provide a rapid, simple, and high‐yield approach for clinical applications of human excreta analysis.

PT03.56. Novel strategy for affinity capture and release sEV

Professor Wei Duan, Mr Rajindra Napit, Dr Rocky Chowdhury, Mr Satendra Jaysawal

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

CD63/CD81 and CD9 are well‐established sEV biomarkers. However, the existing methods for isolating sEVs have several significant limitations that hinder their widespread adoption in research laboratories and clinical settings. Aptamers are single‐stranded DNA/RNA molecules that adopt a specific three‐dimensional structure, enabling them to bind with high affinity and specificity to target molecules. Through a peptide‐based SELEX approach, we isolated DNA aptamers against a specific epitope on one of extracellular domains of human CD9. With several rounds of molecular engineering, we have evolved our CD9 aptamer to a ligand that binds to native CD9 proteins on the plasma membrane of both human cells and sEVs with high specificity. Utilising this newly developed CD9 aptamer and CD81/CD83 aptamers, we developed a novel tetraspanin aptamer‐based affinity isolation methods for the scalable isolation of cancer biomarker‐positive sEVs. Both the EV capture and release are carried out in physiological buffer and pH. Further refinement of our novel aptamer‐guided EV isolation strategy will pave the way for future personalized precision oncology.

PT03.57. Optimization of separation methodologies for obtaining high yield‐high purity urinary extracellular vesicles

Beatriz Martín‐Gracia, Optimization of separation methodologies for obtaining high yield‐high purity urinary extracellular vesicles Håkon Flaten, Optimization of separation methodologies for obtaining high yield‐high purity urinary extracellular vesicles Krizia Sagini, Optimization of separation methodologies for obtaining high yield‐high purity urinary extracellular vesicles Alicia Llorente

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1) Introduction

According to the Global Cancer Observatory, approximately 1.4 million new cases of prostate cancer (PCa) and 375,000 related‐deaths were registered worldwide in 2020. Even if the detection PSA is a widely used tool for the diagnosis of PCa, many studies agree that its use leads to overdiagnosis and overtreatment because this protein is not cancer‐specific. For this reason, the search for new biomarkers represents an urgent need in the management of PCa. In the last decade, extracellular vesicles (EVs) have emerged as a very promising source of biomarkers for PCa in liquid biopsies because they are a molecular fingerprint of the cells of origin and are found in biofluids. Particularly, several studies have focused on urinary EVs because urine is easily collected and is in close contact with the prostate. However, as with other biofluids, it is important to optimize the methodology around urinary EV separation and analysis to promote the use of EV‐associated molecules as PCa biomarkers.

2) Methods

In this study two EV separation methods, differential centrifugation (DC) and ultrafiltration coupled to size exclusion chromatography (SEC), were optimized and compared to each other in terms of small EV yield and purity by analyzing both the number of EVs by nanoparticle tracking analysis and the expression of EV markers by Western blot.

3) Results

When performing DC, results showed that the addition of a 200 nm filtration step largely removed from the EV pellet uromodulin, a very abundant protein in urine that co‐isolates with EVs. On the other hand, concentration of urine samples with a 10 kDa ultrafiltration device prior to SEC gave a higher EV yield compared to 30 kDa and 100 kDa devices. After concentration, the isolation of EVs by SEC also avoided the contamination with uromodulin.

4) Summary

Preliminary studies resulted in SEC providing a slightly higher yield of EV recovery with a similar purity to DC. Considering that DC is a more laborious and time‐consuming technique than SEC, the use of ultrafiltration coupled to SEC is a good EV isolation alternative that can help in the implementation of EV‐based biomarkers in the clinic.

PT03.58. Optimizing high‐throughput isolation of extracellular vesicles from primary cells in small to medium‐scale 3‐dementional bioreactors with serial purification methods

Dr. Zheng Zhao

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1) Introduction: In the burgeoning field of extracellular vesicle (EV) research, efficient isolation from primary cells at small to medium scales (15 mL to 2 L) remains a critical challenge. Addressing this, our study explores a novel approach utilizing Mesenchymal stem cells (MSCs) with 3‐demtional bioreactors. Tangential Flow Filtration (TFF), diafiltration, and ion‐exchange purification are applied to achieve high‐throughput isolation. The isolated EV were validated by Nanoparticle Tracking Analysis (NTA), western blot, uptaking assay, wound healing assay, to show the therapeutic potential of MSC EVs.

2) Methods: MSCs were sustained in 2 L bioreactor, and EV depleted media was exchanged in day 5, and conditioned media was collected on day 8. A 0.8 um filter was used for clarification, and 750 kDa hallow fiber was used for TFF and diafiltration. A 4 mL volume of ion exchange column was used to removal rest of protein impurities. The isolated EV were store under 4C and validated by western blot, uptaking assay, wound healing assay, and proteomic analysis to show the therapeutic potential of MSC EVs

3) Result: Particle concentration analysis revealed consistently robust concentrations throughout the entire process, ranging from 1.2E9 particles/mL to 3.02E10 particles/mL, with a final volume reduction from 2 L to 45 mL. The overall particle yield reached approximately 1.36E12 particles, demonstrating a commendable total recovery rate of 85%. Western blot results corroborated the presence of essential EV markers, including CD9, CD63, ALEX, and TSG101. Cellular uptake studies demonstrated rapid internalization within the first hour, while wound healing assays underscored the therapeutic potential of isolated MSC EVs by significantly enhancing the cell healing process compared to the control.

4) Summary/Conclusion: By focusing on this innovative method, we enhanced the EV purification processes from MSC with small to medium scales range up to 2 L and also validated the integrity and therapeutic potentials of these EV. With proper upstream equipment's, the process can be easily upscaled to 50 L.

PT03.59. Pillared interdigitated electrodes for small extracellular vesicle capture

Miss Emma Morris, Associate Professor Karl Hassan, Professor Craig Priest, Dr Bin Guan, Dr Renee Goreham

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths annually. Currently, the only way to diagnose lung cancer is to undertake a biopsy, which is usually done after symptoms are present. At this point, the patient survival rate is low. For this reason, a screening process that is non‐invasive and portable is needed to increase patient survival rates. With each person exhaling 10,000 L of breath each day, breath offers an alternative sampling source that is accessible and does not cause the patient discomfort. This project aims to develop a breathalyser to target biomarkers found within breath, namely extracellular vesicles. Extracellular vesicles, released by various cell types, have recently been discovered in exhaled breath. Since exhaled breath condensate holds a spectrum of molecules and biomarkers, it's crucial to employ a detection method that can isolate small extracellular vesicles from impurities. The integration of pillared interdigitated electrodes facilitates the organic isolation of these nanoparticles. Leveraging thiol‐modified DNA aptamers with high specificity and affinity to surface proteins, such as the common biomarker CD63 and CD44 associated with lung cancer, the micropillar device adeptly captures small extracellular vesicles on its gold surface. The thiol group of the modified aptamer will interact with the gold surface creating a stable bond, following incubation withextracellular vesicles derived from A549 lung adenocarcinoma cell line and exhaled breath condensate from healthy patients within a concentration curve of 3.10 x 10⁷ – 7.94 x 10⁷ particles per mL, a monolayer will form upon the gold surface. The monolayer can be characterised, as the gold surface acts as an electrode, by electrochemical impedance spectroscopy. This response serves as a measurable indicator of successful small extracellular vesicle capture. Future adaptation of this work presents the incorporation of a polyclonal aptamer library to offer increase specificity to an extracellular vesicle target. The envisioned outcome is the creation of a portable screening device for lung cancer, promising advancements in prognosis and facilitating effective monitoring of cancer progression.

PT03.60. Protocol optimisation for extracellular vesicle isolation and characterisation: evaluation of ultracentrifugation, size exclusion chromatography and charged core bead chromatography methods

Dr Farha Ramzan, Hui Hui Phua, Dr Vidit Satokar, Dr Shikha Pundir, Dr Anastasia Artuyants, Dr Cherie Blenkiron, Dr Chris Pook, Prof Mark Vickers, Dr Ben Albert

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Extracellular vesicles (EVs) are reported to play a key role in intercellular communication and are present in abundance across a range of body fluids. Emerging evidence has suggested that EVs in breast milk may impact the health and well‐being of infants. Various methodologies have effectively isolated EVs from breastmilk, including ultracentrifugation, size exclusion chromatography (SEC), and density‐based separation. However, each approach has strengths and limitations, often involving complex and time‐consuming approaches. Therefore, there is an urgent need to establish an efficient isolation technique that optimally balances ease of use and affordability while maximising yield and purity. This is key for identifying the functional components within EVs that may mediate infant health outcomes.

Methods: Using banked breastmilk samples from the Fish Oil in Pregnancy Trial (ACTRN12617001078347; 1mL), a comprehensive evaluation of EV isolation methods, including ultracentrifugation, SEC, and charged core bead chromatography using Capto™ Core (CC) was examined. Isolated EVs from different methods were compared by nanoparticle tracking analysis (NTA) to determine particle size and quantity, transmission electron microscopy (TEM) to visualise EV integrity and morphology, and western blot to investigate selected EV‐associated protein cargo.

Results: Following TEM analysis, all EV isolation methods were shown to contain 40‐150 nm sized EVs. However, CC isolation visibly enhanced EV integrity with less contaminant debris and lipid‐type droplets. As evidenced by western blotting, all isolates were positive for small EV markers (CD9, CD81 and CD63). However, CC isolation resulted in cleaner EV isolates with less lactoferrin (milk contaminant) and Calnexin (cellular marker). Interestingly, both UC (9.04*1010 particles/ml) and SEC (1.16*1011 particles/ml) resulted in higher concentrations of particles as compared with CC isolation (2.45*109 particles/ml).

Conclusion: Based on our findings, the CC isolation method (Capto™ Core) serves as a viable option for isolating an EV‐enriched subset, with the removal of larger quantities of contaminant material than alternatives whilst maintaining the integrity of the EVs. The next step involves conducting a comprehensive molecular characterisation of the isolated subset and evaluating the functionality of the EV cargo in cell models.

PT03.62. Rapid and efficient isolation platform for plasma extracellular

vesicles: EV‐FISHER

Dr Weilun Pan, Prof Lei Zheng, Prof Jinxiang Chen, Prof Bo Li

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Extracellular vesicles (EVs) have found diverse applications in clinical theranostics. However, the current techniques to isolate plasma EVs suffer from burdensome procedures and limited yield. Herein, we report a rapid and efficient EV isolation platform, namely EV‐FISHER, constructed from the metal‐organic framework featuring cleavable lipid probes (PO43−‐spacer‐DNA‐cholesterol, PSDC). The EV‐FISHER baits EVs from plasma by cholesterol and separates them with an ordinary centrifuge. The captured EVs could be released and collected upon subsequent cleavage of PSDC by deoxyribonuclease I. We conclude that EV‐FISHER dramatically outperforms the ultracentrifugation (UC) in terms of time (∼40 min vs. 240 min), isolation efficiency (74.2% vs. 18.1%), and isolation requirement (12 800 g vs. 135 000 g). In addition to the stable performance in plasma, EV‐FISHER also exhibited excellent compatibility with downstream single‐EV flow cytometry, enabling the identification of glypican‐1 (GPC‐1) EVs for early diagnosis, clinical stages differentiation, and therapeutic efficacy evaluation in breast cancer cohorts. This work portrays an efficient strategy to isolate EVs from complicated biological fluids with promising potential to facilitate EVs‐based theranostics.

PT03.63. Salivary extracellular vesicles isolation methods impact the robustness of biomarkers detection

Dr Jérémy Boulestreau^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4,France}, Dr Laurence Molina^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184, Montpellier,CEDEX 4, France}, Alimata Ouedraogo^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184,Montpellier, CEDEX 4, France}, Louen Laramy^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184,Montpellier, CEDEX 4, France}, Ines Grich^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182, 34184,Montpellier, CEDEX 4, France}, Dr Thi Nhu Ngoc Van^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 40182,34184, Montpellier, CEDEX 4, France; SkillCell, Montpellier, France}, Dr Franck Molina^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine,1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4, France}, Dr Malik Kahli^{Sys2Diag UMR9005 CNRS/ALCEN, Cap Gamma, Parc Euromédecine,1682 rue de la Valsière, CS 40182, 34184, Montpellier, CEDEX 4, France}

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Extracellular vesicles (EVs) are lipid bound nanoparticles known to play a key role in cell‐to‐cell communication, including homeostatic and pathological processes. Their presence in the peripheral fluids including saliva, a biofluid easy to collect in a non‐invasive way, makes EVs potent tools for discovery of diagnosis or prognosis biomarkers. Several methods to isolate and characterize salivary EVs (sEVs) have been described but require further optimization and standardization to foster the translation to clinical application. The aim of this research was to rigorously characterize and directly compare salivary EVs isolated from two different fast and scalable technics (co‐precipitation and immuno‐affinity) to the current time‐consuming ultracentrifugation method.

Methods: In this work, we collected saliva samples from nine healthy volunteers. sEVs were isolated using three different methods: ultracentrifugation (UC), co‐precipitation (Q) and immunoaffinity (M). The EVs size and distribution were analyzed using NTA and their morphology by cryo‐EM. Protein and miRNA cargos were also determined by western blot, proteomics and RT‐qPCR. We also systematically assessed the impact of saliva filtration on 0.22 and 0.45 µm filters before EV isolation as this was never clearly investigated.

Results: Our findings reveal that: sEVs are abundant and can be isolated from small volumes (1ml) of saliva. UC and Q EVs have the same size distribution, but twice less particles for Q samples. M EVs are significantly smaller (84 nm vs 264 and 227 nm for UC and Q respectively). Protein and small RNA cargos greatly varies depending on the isolation method chosen and we show that EVs isolation allows detecting specific biomarkers undetected in the whole saliva. We confirm that miRNAs are principally contained in EVs and not in free circulating form in saliva. We also have determined that filtration is detrimental for EVs isolation. Finally, our results suggest that co‐precipitation method is compliant, suitable for biomarker discovery, and diagnostic.

Summary/Conclusion: This work characterizes a neglected source of EVs and provide evidence that co‐precipitation method is efficient, suitable for analysis of clinical samples and cost‐effective for isolation of salivary EVs from small volumes of saliva.

PT03.64. Single‐particle multiplex analysis of EV‐biophysical properties of fractionated particle populations by ion exchange chromatography

Professsor Takanori Ichiki, Chiharu Mizoi, Kento Toyoda, Professor Naohiro Seo

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

Recent efforts have been directed towards the development of methods for isolating and purifying exosomes from extracellular vesicles (EVs). While techniques utilizing surface molecules such as tetraspanins and phospholipids have been considered highly specific, there is emerging evidence suggesting that leveraging physical properties like size and charge differences, in combination with appropriate separation methods, may offer a highly efficient means of exosome isolation. Looking ahead to clinical applications of exosomes, it is imperative to explore platform technologies for the scalable and quality‐assured preparation of exosomes.

Methods:

Culture supernatant of human dental pulp stem cells (DPSC) was subjected to ultrafiltration to concentrate EVs. Subsequently, ion exchange column chromatography was employed to fractionate EVs based on their charge differences. This method, following the high‐efficiency exosome recovery protocol developed by Seo etal., utilized a DEAE Sepharose column with a linear gradient of NaCl aqueous solution [N. Seo etal., J. Extracellular Vesicles 11, e12205 (2022)]. The isolated particle populations were then subjected to single‐particle multiplex measurements using a microfluidic device developed by Ichiki‘s group, evaluating the size and zeta potential of each particle [T. Ichiki etal., Microcapillary chip‐based extracellular vesicle profiling system. Extracellular vesicles: methods and protocols, 209‐217 (2017)].

Results:

Fractions extracted in NaCl solutions at specific salt concentrations (200 mM) and (400 mM) exhibited maximal particle number density. Multiplex measurements using the microfluidic device revealed distinct particle populations: one showing a size distribution in the range of 60‐300 nm and a relatively narrow distributions of zeta potential peak at approximately ‐16 mV, and the other displaying a size distribution of 30‐450 nm and zeta potential peak at approximately ‐20 mV, respectively. These results support the successful recovery of particle populations resembling exosomes and microvesicles.

Summary:

By employing ion exchange chromatography, distinct particle populations resembling exosomes and microvesicles were obtained. The results of single‐particle multiplex measurements using the microchip‐based nano‐particle analysis system clarified the biophysical differences in isolated EV particles. This achievement is anticipated to contribute to the development of a technological platform for the convenient and quality‐assured preparation of exosomes in large quantities, fostering their potential clinical applications.

PT03.65. Tailored cellulose nanofiber sheets capture and preserve small extracellular vesicles from micro‐volume body fluids and reveal the unknown profiles of extracellular vesicles

M.D., Ph.D. Akira Yokoi, M.D., Ph.D. Kosuke Yoshida, B.Sc. Masami Kitagawa, Ph.D. Takao Yasui, M.D., Ph.D. Hiroaki Kajiyama

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

Extracellular vesicle (EV) isolation is the fundamental and critical factor for EV research, and one of the limitations of EV recovery is the requirement for a certain volume of biofluids. We recently invented tailored cellulose nanofiber (CNF) sheets (Yokoi A etal., Nat Commun. 2023), and here, we aimed to test the CNF sheets' performance toward EV isolation and explore their utility in clinical applications.

Methods:

To test the quality of EVs, which were captured by CNF sheets, we performed nanoparticle tracking (NTA) assays, western blotting (WB) analyses for EV markers, and a cryo‐electron microscope. We applied small RNA sequencings to assess the profiles of EVs. For functional analyses, we use an ovarian cancer mouse model using ID8 cells, a murine epithelial ovarian cancer cell line. To validate the utility of CNF sheets in the human body, we obtained EVs from 210 samples of seven perioperative patients.

Results:

Using 10 µL of human serum, small‐size EVs were successfully isolated by CNF sheets, and purity and yield were significantly higher than with serial‐ultracentrifugation methods. Moreover, sEVs were preserved in the sheet for a week in a dry condition. By attaching the EV sheet to the moistened organs in vivo, the sEVs from trace ascites were collected. In the mouse model, CNF sheets revealed that cancer‐related miRNAs were detected in the very early phase. In cancer patients, the direct EV sheet attaching method during the surgery identified the location‐based unique sEV miRNA profile, and the tumor surface sEVs had distinct profiles from tissues or whole ascites. Trajectory analyses revealed that the connection pattern differed in patients with localized or advanced disease. Tumor‐associated sEV‐miRNAs on tumor surfaces were also detectable in serum, urine, or saliva and decreased in post‐operation.

Summary/Conclusion:

CNF sheets provide a whole new concept of EV analyses, including EV isolation from micro‐volume body fluids and EV preservation in dry conditions for a week. Furthermore, CNF sheet attachment methods enable the capture of ascites sEVs on organ surfaces, revealing the location‐based EV heterogeneity of cancer patients. This tool has broad potential applications contributing to basic and translational EV research.

PT03.66. Targeting EV enriched lipids for non‐biased capture and analysis

Dr Bradley Whitehead, PhD Litten S Sørensen, Anders T Boysen, Prof Peter Nejsum

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1) Introduction

Single vesicle imaging has advanced significantly, allowing multiplex analysis of EV markers to determine EV heterogeneity and phenotyping of vesicles. However, many systems require EV immobilisation and rely upon antibodies targeting EV markers, such as CD63 or CD81 for capture that introduces positive selection bias that can obscure true phenotypic analysis. Therefore, the aim of this study was to assess a mechanism of capture via targeting ubiquitously expressed EV lipids and to determine the level of sample pre‐processing required to enable selective capture to enable a “clinic friendly” method of EV isolation and characterisation

2) Methods

Cholera toxin subunit B (CTxB) with known binding to GM1 sphingomyelin was selected for assessment. Extracellular vesicles were harvested from the cell culture supernatant of RAW macrophage cell lines and human plasma from anonymous donors from Danish blood bank, Aarhus Hospital. EVs were isolated using ultracentrifugation (UC), size exclusion chromatography (SEC) or a combination of SEC and UC. Binding of biotinylated CTxB to EVs and non‐EV fractions was determined using dot‐blot arrays and contrasted to known markers of EVs (CD63) and markers of LDL (ApoB) and HDL (ApoA). Binding of CTxB to purified LDL and HDL was assessed. Removal of LDL and HDL by immunoprecipitation, anionic polymer precipitation, UC, SEC or SEC plus UC was assessed using dot blot. Finally, EV capture was mediated by immobilisation of biotinylated CTxB capture ligand on streptavidin coated slides and binding of fluorescently labelled EVs was assessed.

3) Results

Lipid mediated detection of EVs from conditioned cell culture media was selective and efficient, however, EVs isolated from human plasma using UC or SEC alone showed significant contamination with HDL or LDL, respectively. Furthermore, the CTxB showed cross‐reactivity with non‐vesicle SEC fractions and it was determined the ligand also bound with high affinity to LDL/HDL. The removal of lipoprotein contamination was assessed and a combination of SEC followed by UC was sufficient to remove lipoprotein and enable selective EV capture by GM1 lipid binding ligand.

4) Conclusion

EVs can be selectively analysed using unbiased capture via lipids, however this is dependent on efficient removal of lipoprotein contamination.

PT03.68. Xeno‐free human platelet lysate depleted of exosomes for enhanced extracellular vesicle yield from stem cells, immune cells, and cancer cells

Mr. Yee‐Hsien Lin, Mr. Han‐Tse Lin, Mr. William Milligan, Dr. Min‐Chang Huang

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Background and Aim

The therapeutic potential of extracellular vesicles (EVs) has emerged as a prominent focus in biomedical research. EVs play a crucial role in cell‐to‐cell communication, influencing various disease processes, cancer development, and tissue regeneration. Exosomes, a subcategory distinguished by their size, contain a plethora of bioactive molecules and genetic information, sparking interest in novel therapeutic and diagnostic applications. Leveraging their distinctive biological properties, lipid bilayer exosomes can be engineered as an effective drug delivery system for tissue regeneration and cancer treatment. Therefore, meticulous methodology is imperative to generate high‐quality cell derived EVs, particularly in the context of cancer research and therapeutic applications.

However, conventional cell culture supplements like FBS or human platelet lysate (hPL) pose challenges, potentially influencing target cell physiology due to supplement‐derived EVs. This not only raises concerns of misinterpreted results but also complicates downstream isolation and analysis. Hence, the use of qualified ancillary materials and a controlled culture environment is paramount to ensure consistent EV expression from cells, both in research and clinical settings.

In this context, a novel gamma‐irradiated hPL (ED hPL Gi), characterized by its xeno‐free nature and high EV depletion, is employed in the culture system for producing cell‐derived EVs. ED hPL Gi creates an effective environment, supporting the long‐term secretion of EVs from MSC, immune cells, as well as cancer cells.

Methods, Results & Conclusion

When cells reached 30–50% confluency in a culture plate or reached a cell density of 1x106/ml, the culture medium was changed to 0‐5% ED hPL Gi medium to initiate EV production. Our results indicate that ED hPL Gi is viable for all the target cells in the study, producing a substantial amount of EVs while extending cell activity. Moreover, cells exhibited continuous growth throughout the experiment, maintaining over 85% cell viability. Therefore, ED hPL Gi emerges as a promising supplement for the production of MSC‐, immune cell‐ and cancer cell‐derived EVs, suitable for both exosome research and GMP manufacturing for clinical applications.

PT03.70. Real‐time Label‐free platforms for size determination and cell interaction studies of extracellular vesicles

Msc In Medicinal Chemistry, doctoral researcher in Pharmacy Elena Scurti¹, PhD Martina Hànzlikova¹, MSc Johanna Puutio², PhD Fadak Howaili³, PhD Kai Härkönen⁴, Professor Pia Siljander², PhD Saara Laitinen⁴, Professor Tapani Viitala^1,3

¹Division of Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland, ²EVcore facility, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland, ³Åbo Akademy University, Turku, Finland, ⁴Finnish Red Cross Blood Service, Helsinki, Finland

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Proper characterization of extracellular vesicles (EVs), specifically subpopulations of EVs, is a challenging task. Also, cell assays for assessing EV cell uptake are mainly static, which omits the dynamic nature of biological interactions, and require labels, which may alter EV properties and cell behavior. The aim of this work was to develop unique real‐time label‐free surface plasmon resonance (SPR)‐based analysis platforms for EV size determination, subpopulation identification and EV‐cell interaction studies.

Methods

Red blood cell (RBC)‐ and platelet (PLT)‐derived EVs, and commercial reference EVs were captured by general EV (i.e., CD9, CD63, CD81) and EV specific antibodies (i.e., CD41 (PLT EVs), CD235ab (RBC EVs)) immobilized on the SPR sensor surface. The ratio between the SPR signal responses measured at two different wavelengths (i.e., 670nm and 785nm) was used to determine the size of the EVs through mathematical calculations. DLS and NTA were used as comparative size values. Different concentrations of EVs were allowed to interact with HeLa and PC3 cell lines seeded on fibronectin coated SPR sensor, and kinetic data for the cell interactions were extrapolated from the SPR signal responses.

Results

EV antibodies were successfully immobilized on the SPR sensor and demonstrated effective capturing of EVs, which enabled size determination of EVs. The EV sizes obtained from the SPR analyses ranged from 150 nm for RBC EVs, between 30‐200 for PLT EVs and their subpopulations, and 150‐200 nm for commercial reference EVs, largely agreeing with DLS and NTA data. EV cell interactions with HeLa cells showed concentration dependent SPR responses. In case of PC3 cells the interaction kinetics measured with the cell‐based SPR platform revealed that PLT EVs are taken up by the cells more readily than RBC EVs.

Conclusions

Real‐time label‐free SPR analysis platforms for EV characterization were successfully developed. The size of EVs determined with the SPR analysis platform proved to be accurate. The cell‐based SPR analysis platform proved the selectivity of cell layers towards a specific type of EV, and the cellular responses, which were dependent on the EV concentration, provided information about the interaction kinetics between EVs and cells under dynamic conditions.

PT03.71. How Centrifugation Can Improve Your EV Workflow

Ms. Amy Henrickson¹, Dr. Lutz Ehrhardt, Dr. Shawn sternisha

¹Beckman Coulter, Indianapolis, United States

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: With the progress in EV research, an understanding of the different workflows is essential. As different production and purification methods typically lead to different purities, while different characterization techniques highlight different perspectives of the purified vesicles, it is essential that an in‐depth understanding of these methods is available. Here, we will discuss several different workflows that center on centrifugation purification and analysis to help researchers understand the benefits and limitations of the different methods.

Methods: Preparative ultracentrifugation offers many different purification options. The current need to improve EV purification might require re‐thinking the currently used purification strategy to further remove contaminations, which might interfere with your workflow. Centrifugation offers multiple purification strategies, including density gradient ultracentrifugation, rate‐zonal centrifugation, and differential centrifugation. Each method has its benefits and limitations, which will be discussed.

Centrifugation can be used for both the preparation of EVs and the characterization of samples using the analytical ultracentrifuge (AUC). AUC works by monitoring the sedimentation and diffusion patterns of particles under centrifugal force. From this data, several sample properties can be determined, including sample purity and size distribution, both of which are important aspects of EV research. Additional biophysical properties, including sedimentation and diffusion coefficients, anisotropy, hydrodynamic radius, and partial specific volume, can also be determined by AUC.

Results: Here, we demonstrate several different preparation methods and discuss their benefits and limitations in regard to use and contaminant removal. While demonstrating the characterization that can be achieved with AUC.

Summary/Conclusion: Understanding the different purification methods will enable researchers to choose the best method for their EVs. AUC has long been established as a strong characterization of viral vectors; it is a first principal characterization technique that enables researchers to study their samples in their final formulation. Collectively, these findings will help researchers with sample purification and highlight the advantages of adding AUC as an orthogonal characterization technique for EVs.

PT03.72. Optimizing a workflow for the analysis of extracellular vesicles

Dr. Anis Larbi¹

¹Beckman Coulter Life Sciences, France

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Several methods are employed for extracellular vesicles (EVs) analysis. Isolation methods typically involve ultracentrifugation, size‐exclusion chromatography, or precipitation‐based approaches. Characterization techniques, such as electron microscopy, nanoparticle tracking analysis, flow cytometry, and Western blotting, provide information about EV size, morphology, surface markers, and protein content. Here we sought to present a typical workflow for the integration of flow cytometry in EV characterization.

Methods

A comprehensive workflow for the characterization of EVs is proposed, with two distinct models depending on the purpose: biomarker/heterogeneity analysis (commonly used in R&D) and specific EV production (for manufacturing purposes). The workflow includes several key steps such as EV preparation, purification, quality control, and characterization. Flow cytometry is a widely used method in this process and is often complemented with orthogonal methods for comparison.

Results

We highlight the significant benefits of automation in EV preparation, which effectively reduces variability introduced by human operators. This automation ensures consistent and reliable results throughout the process. Additionally, flow cytometry stands out as a superior method for EV characterization due to its ability to sensitively detect and count single EVs using fluorescence. The ability to analyze EVs individually is particularly important in biomarker/heterogeneity analysis. Moreover, the user‐friendly nature and robustness of the tested cytometer offer significant advantages, especially in a manufacturing setting. Lastly, the seamless integration of the flow cytometry into an EV workflow analysis further proves its advantages.

Conclusion

In this work, we demonstrate the seamless integration of flow cytometry into EV research. The utilization of the flow cytometry allows for the acquisition of high‐quality and reproducible data, owing to its exceptional sensitivity. Collectively, these findings strongly suggest the advantages of incorporating flow cytometry as a complement to, or even a replacement for, orthogonal methods in EV analysis.

PT03.73. MISEV 2023: the Beckman Coulter Life Sciences approach for Extracellular Particles

Dr. Anis Larbi¹

¹Beckman Coulter Life Sciences, France

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

The recent release of MISEV2023 marks a significant milestone for the EV research community, aiming to establish standardized guidelines and recommendations for the analysis and reporting of extracellular vesicle studies. The pursuit of knowledge in this field involves utilizing various solutions to generate data, while ensuring quality and reproducibility. This report focuses on how current practices in centrifugation and flow cytometry align with MISEV2023, along with potential gaps. Detailed protocols are provided for centrifugation methods like differential ultracentrifugation and analytical ultracentrifugation, as well as flow cytometry protocols for particle counting, characterization, and sorting. Specifically, the ability to detect and characterize small EVs by flow cytometry is highlighted for its exceptional fluorescence sensitivity (scatters and fluorescences). We also delved into the analysis and reporting aspects of the different techniques, offering a critical review of the current status quo. By examining the needs for proper analysis and reporting of EV‐derived data scientists can gain valuable insights to enhance their EV research endeavors.

PT03.75. EV Quant: A quantitative web‐based compendium of extracellular vesicles cargo for studies in vesiclepedia

Mr SRIRAM GUMMADI¹

¹Latrobe University, Australia

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

EV Quant: A quantitative web‐based compendium of extracellular vesicles cargo for studies in vesiclepedia

Introduction: Extracellular vesicles (EVs) are nanosized membrane bound vesicles that are shed by cells into extracellular space. EVs have a key role in multiple pathophysiological processes due to their signalling capabilities and are classified into various subtypes based on their biogenesis and origin. Exosomes are small EVs that range in 30–150 nm diameter and they originate due to the fusion of multivesicular bodies with the plasma membrane.

In terms of the molecular composition, EVs sequester a diverse and rich cargo of proteins, lipids, and nucleic acids, and the ensuing cargo is altered according to the pathophysiological conditions of the host cells that secrete them. Hence, a catalogue of the EV cargo in various conditions could aid in identifying an EV fingerprint that are specific to a tissue, cell type, and/or pathology.

Methodology: EV Quant is a quantitative plugin added to Vesiclepedia and are built using a combination of MySQL for the background database, Zope content management system and Python as the programming language that connects the database to the content management system and the data is from both published and unpublished studies.

Results: We describe an update of Vesiclepedia (http://www.microvesicles.org) an online database that contains RNA, proteins, lipids and metabolites that are identified in EVs and Eps.

EV Quant, a plugin added to vesiclepedia which contains the quantified EV cargo for the experiments in vesiclepedia. Users can search for a biomolecule and compare the quantification data between treatments in a specific experiment and in addition to that, all of the EV cargo identified in that experiment is summarised as a data table and a heatmap showcasing the top 100 differentially regulated candidates will be plotted in real time.

Currently, Vesiclepedia contains data obtained from 3,533 EV studies, 50,550 RNA entries, 566,911 protein entries, 3,839 lipid entries, 192 metabolites and EV Quant has 62,822 quantification entries.

Conclusion: A catalogue of EV cargo will immensely benefit the research community in identifying an EV fingerprint that are specific to a tissue, cell type, and/or pathology.

PT03.76. Optimized protocol for isolation of extracellular vesicles (EV) ‐ carried microRNAs from platelet‐free plasma using size‐exclusion chromatography (SEC) and phenol – guanidine extraction

MD Miłosz Majka¹, PhD Katarzyna Czarzasta², MD, PhD Małgorzata Wojciechowska², PhD Małgorzata Czystowska‐Kuźmicz¹

¹Medical University of Warsaw, Chair and Department of Biochemisrty, Warsaw, Poland, ²Medical University of Warsaw, Laboratory of Centre for Preclinical Research, Chair and Department of Experimental and Clinical Physiology, Warsaw, Poland

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

MicroRNAs (miRNAs) have been identified as potent EV‐derived molecules with promising clinical implementation. Packaging of miRNAs into vesicles increases their stability but also complicates their specific isolation. There are several methods for the extraction of circulating miRNAs from plasma with following quantification by RT‐qPCR. We optimized a protocol for RNA isolation from EV‐rich SEC fractions and determined how it influences EV‐miRNA characteristics in a set of platelet‐free plasma (PFP) from clinically characterised individuals.

Methods:

EVs were isolated from 1ml of human PFP using SEC and nanoparticle tracking analysis (NTA, ZetaView) was used to analyze the particle size and number. The EV‐protein markers were then characterized by Western blotting. MiRNeasy Serum/Plasma Kit (Qiagen) was used to isolate RNA from PFP and EV‐rich samples. RNase and detergent sensitivity assay was performed prior to isolation. A Nanodrop spectrophotometer was used to evaluate the total RNA concentration and purity and Agilent Bioanalyzer small RNA chips were used to evaluate the size range of isolated RNA. Extraction efficiency was evaluated by Taqman advanced miRNA assay in RT‐qPCR using array cards and singe‐tube assay.

Results:

EV‐rich fractions isolated by SEC consisted of RNA of higher purity than the protein‐rich fractions. RNA content of those fractions was well protected against RNAse‐proteinase treatment while RNA concentration significantly decreased after addition of detergent. The modified protocol rendered significantly more RNA of higher purity compared to the standard protocol. Analysis of clinical samples showed that the modified protocol enables to isolate microRNAs from EV‐rich fractions of different profile compared to PFP.

Summary:

This study succeeded in providing conditions to extract a good‐quality EV‐RNA from PFP for the RT‐PCR miRNA assays. The optimized protocol increases yield and purity of obtained small RNAs allowing to more sensitive and reproducible analysis of microRNA content of extracellular vesicles.

PT03.77. Comparative Analysis of Plasma and Serum Exosomal Small RNA Sequencing Profiles

Dr. Alex Chauhan¹, Hinal Zala¹, Simone Yamasaki¹, Enaam Merchant¹, Dr. Mohamed El‐Mogy¹, Dr. Songsong Geng¹, Dr. Taha Haj‐Ahmad¹, Dr. Yousef Haj‐Ahmad¹

¹Norgen Biotek Corp., Thorold, Canada

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Peripheral blood is commonly collected using various devices, with potassium EDTA and serum tubes being prevalent. Plasma and serum serve as primary samples for monitoring disease progression. Exosomes in these samples offer real‐time surveillance of prevalent diseases. Extensively studied, exosomal RNA extracted from extracellular vesicles plays a role in regulating transcriptional expression. Due to their low abundance, next‐generation sequencing is optimal for characterizing these RNAs. Despite being primary in exosome studies, there's limited data comparing exosomal small RNA sequencing profiles in plasma and serum. This study highlights differences in these profiles from the same donors. Blood was collected from 4 donors in EDTA and Serum tubes. Plasma and Serum were separated and were stored at ‐80°C until further use. Intact Exosomes were purified from 0.2, 0.5 and 1.0 mL plasma and serum volumes. Extracted exosomes were further processed to extract exosomal RNA. Small RNA library was constructed from all the purified exosomal RNA and sequenced using Illumina's NextSeq 550 platform. A pattern showing an increase in the reads mapped to genome was observed with the increase in plasma (p=0.0122) and serum (p=0.0141) volumes. Similar trend was observed in total small RNA species for plasma (p=0.0037) and serum (p=0.0396) samples. This trend was further reflected in the percentage of reads assigned to miRNA (p=0.0031), piRNA (p=0.042) and rest of the small RNA species combined (gencode; p=0.0473) in plasma samples. Serum samples showed higher percentage of reads mapped to genome as compared to plasma sample, however this was significant only for 1.0 mL serum volume. Among the reads that were mapped to the genome, there was a significant difference in the percentage of miRNA, piRNA and circularRNA reads, between plasma and serum samples. This study reveals a significant impact of sample volume on exosomal small RNA sequencing profiles in plasma and serum. Both plasma and serum exhibited comparable proportions of miRNA, piRNA and circularRNA reads for a 1.0 mL sample volume. At volumes below 1.0 mL (0.2 mL and 0.5 mL), serum demonstrates a higher percentage of small RNA species compared to plasma, suggesting its preference in such cases.

PT03.80. Innovative Ultrapure Exosome Extraction Using Hybrid Charge‐Based Filtration and Tangential Flow Filtration

Mr. Yoing‐woo Kim¹, Mr. Kang‐Min Lee¹, Professor Sehyun Shin¹

¹Korea University, Seoul, South Korea

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: To utilize extracellular vesicles (EVs) as therapeutic agents and drug delivery vehicles, it is crucial to address the issue of minimizing impurities present in the extracts of extracellular vesicles. Unfortunately, most of the currently developed exosome isolation techniques have failed to address this purity issue, demanding urgent development of innovative technologies.

Methods: Here, we propose a novel method for isolating EVs using a recently developed charge‐based filter by our research group, along with hybridizing the conventional tangential flow filtration (TFF) method. The ExoFilter consists of a multi‐layered structure composed of a positively charged porous filter (pore size 1 µm). In this study, three methods were employed: 1) TFF followed by ExoFilter, 2) ExoFilter followed by TFF, and 3) repeated application of ExoFilter‐TFF. The results were analyzed using techniques such as NTA, BCA, RT‐PCR, etc., to evaluate the efficiency of exosome separation

Results:All three methods, which involved the combination of ExoFilter and TFF, yielded unexpected outcomes by significantly decreasing impurities while maintaining a consistent exosome extraction yield. Particularly, the pre‐ExoFilter followed by TFF method, which initially captures positively charged nanoparticles and subsequently removes particles smaller than 50nm, exhibited the highest purity, achieving a performance level approximately one‐tenth of the impurity level of TFF. The results of the TFF followed by ExoFilter and repeated ExoFilter‐TFF processes showed impurity levels approximately one‐fourth of those of TFF with comparable yields.

Summary/Conclusion: These findings present a novel method that combines two different extraction techniques, resulting in innovative reduction of impurity content without compromising extraction yield. It is deemed as a combination of charge‐based extraction and size‐based filtration techniques, which complement each other's drawbacks without conflict. Moreover, the proposed method offers practicality for large‐scale implementation, achieving over a tenfold increase in purity without compromising yield.

PT03.81. Use of advanced aptamer technology in EV research

Mr. Rajindra Napit¹, Mr. Satendra Jyasawal¹, Ms. Jasmine Catague¹, Mr. Haben Melke¹, Dr. Rocky Chowdhury¹, Dr. Lingxue Kong¹, Dr. Wei Duan¹

¹Deakin University, Warun Ponds, Geelong, Australia

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

Extracellular Vesicles (EVs) play a vital role in intercellular communication by transporting genetic material and proteins. Despite their significance, current purification methods such as ultracentrifugation and chromatography are time‐consuming and can damage EVs, hindering their potential clinical applications. Given their importance in diagnostics and therapeutics, there's a pressing need for alternative purification techniques. This project aims to address this need by exploring new methods to isolate and enrich EVs effectively.

Methodology:

The method involves utilizing predictive tools aid in optimizing aptamer design, ensuring spacer arm addition minimally impacts aptamer affinity. Followed by use of denaturing agents to disrupt the 3D structure of aptamers, enabling effective capture of extracellular vesicles (EVs) without the need for harsh reagents like detergent, high salt, or heat. Aptamers targeting universal EV biomarkers like CD63, CD81, and CD9 are employed for small EV capture, with a focus on the CD63 aptamer due to its commonality in small EVs. Various matrix immoboolization strategies for the conjugation of aptamers will be explored.

Results:

The CD63 aptamer was modified with the help of computational prediction tools to enable its immobilization without apparent affecting its affinity. The aptamer affinity was then tested on antibody captured EVs to assess the binding capacity of aptamer. The result showed modified aptamer is capable of binding to EVs in PBS buffer. Next the aptamer will be immobilized on the solid surface to capture and purify the EVs.

Conclusion:

By leveraging predictive tools for aptamer design and employing denaturing agents, this research demonstrates a promising approach for efficient EV capture under mild or physiological conditions. The successful modification of the CD63 aptamer and its demonstrated binding capacity to EVs pave the way for further development of novel purification strategies with potential clinical applications.

PT03.82. Purifying exosomes to meet manufacturing demand using a gentle, size‐based, and scalable purification solution

Dr Jagan Billakanti¹, Dr Jon Lundqvist, Dr Peter Guterstam

¹Cytiva, Brisbane, Australia

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

A scalable workflow for the purification of exosomes, a type of extracellular vesicle (EV), is a major challenge for therapeutic‐grade exosome manufacture. Exosomes are large ― between 40 and 150 nanometres (nm) in diameter ― and downstream processing includes the removal of much smaller size contaminants such as host cell proteins and DNA. Established technologies for research, R&D, and diagnostic purposes include ultracentrifugation, density gradient separation, gravity separation, or a combination of these methods. Although these technologies generate enriched exosomes suitable for characterization, they can damage the exosome structure, suffer from poor yields and scalability, and require long preparation times, which could reduce biological function. Therefore, these methods are not suitable for manufacturing large quantities of therapeutic exosomes.

Methods:

We describe a workflow for both research and clinical scales of manufacturing. The EV enrichment includes depth filtration to remove cells, Benzonase treatment to degrade DNA/RNA, tangential flow filtration using 750 kDa to concentrate exosomes, followed by either gentle size‐exclusion chromatography (SEC) with Cytiva™ superSEC resin or chromatography (MMC) with Capto™ Core 700 resin; the latter combines size exclusion and binding mode for separation different species from the load material.

Results:

During the primary tangential flow filtration (TFF), a 750 kDa hollow fiber produced 100% recovery of EV while different chromatography steps produced different recovery profiles. For example, superSEC resin showed 7% more EV recovery and 3.3 times faster separation over Sepharose™ CL‐2B. superSEC resin demonstrated similar performance at two different scales with three different feed materials.

Summary:

In this presentation, we will demonstrate a start‐to‐finish exosome production process suitable for clinal scale manufacturing of exosomes harvested from three different cell lines. Both chromatography options mentioned above are amenable to scale‐up and packing in large‐scale columns for clinical‐grade EV manufacturing. Depending on the EV dose requirements, a secondary TFF with 750 kDa is proposed for further concentration of EV before sterile filtration.

PT03.83. Isolation of extracellular vesicles in aqueous two‐phase systems for cancer diagnosis

Ph.D. Candidate Minyeob Lim¹

¹POSTECH, Pohang, South Korea

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1) Introduction

Extracellular vesicles (EVs) have significant diagnostic potential, yet their efficient isolation remains challenging. The aqueous two‐phase systems (ATPSs) offer a high‐purity and high‐yield method for EV separation, but the underlying mechanism needs to be better understood, limiting its application. In order to elucidate the principle of separation, we apply Kramers’ theory to the system. Our results show that the liquid‐liquid interface acts as a size‐selective filter, improving the efficiency of EV isolation and providing a pathway to effective diagnostics.

2) Methods

A theoretical and simulation model for nanoparticle transport in ATPS was validated using beads of various sizes. Plasma samples from prostate cancer patients with metastasis (n = 23) and without metastasis (n = 31) were separated using ATPS. The efficiency of separating EVs, low‐density lipoprotein (LDL), high‐density lipoprotein (HDL), and albumin was evaluated using total internal reflection fluorescence microscopy, western blot, and transmission electron microscopy. EV markers (CD9, CD63) and cancer markers (PD‐L1, PSMA) were quantified via enzyme‐linked immunosorbent assay.

3) Results

The study discovered a negative correlation between interfacial tension and the diameter of particles that can pass through the interface. Using this mechanism, over 80% of EVs were isolated from plasma, with only 10% of impurities (LDL, HDL, and albumin) retrieved. Applying this technique to prostate cancer diagnosis, PD‐L1 and PSMA levels in exosomes turned out to have a strong correlation with metastasis. Metastasis detection through EV showed high efficiency (AUC = 0.88), in contrast to the traditional liquid biopsy marker, serum PSA, which demonstrated poor detection capability (AUC = 0.62).

4) Summary/Conclusion

This study demonstrated that a potential barrier developed by low interfacial tension (10 ∼ 60 µJ/m²) acts as a filter with pore sizes of 40 ∼ 100 nm, enabling efficient isolation of EVs from plasma. We expect that the potential barrier will facilitate further research using nanoparticles.

PT04.01. 25HC depleted accessible cholesterol to restrict SFTSV infection and infectious‐EVs mediated tramsmission

Postdoctor Rui Zhang

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by a novel tick‐borne phlebitis virus of Bunyaviridae, characterized by high fever, thrombocytopenia and leukopenia with high case fatality. Currently, no specific antiviral drugs available to treat patients. Extracellular vesicles are lipid bilayer vesicles that carry various contents (protein, genetic information,), communicate and regulate neighboring cells. We found that EVs derived from SFTSV‐infected cells contained infectious virions that were efficiently transported by these secreted vesicles into neighboring cells. Cholesterol in the PMs plays an important role in regulating the entry of viruses into cells. 25HC is an endogenous oxidized sterol involved in various metabolic pathways, and it is generally considered to be a soluble factor that involves in antiviral activity.

Methords: The large extracellular vesicles secreted by SFTSV infected cells were isolated and purified by gradient centrifugation, and the virions were found in MVs‐SFTSV by NTA analysis, electron microscopy analysis and content identification.

Results: We reported a natural lipid metabolite 25HC that inhibited SFTSV entry by activating the activity of lipid metabolism enzyme‐ACAT, affecting the cholesterol translocation from the cytoplasm to plasma membranes. At the same time, through this way, 25HC also inhibit the extracellular vesicles carried virions into target cells, prevented the MVs‐SFTSV mediated virus‐transmission and spread.

Summary: Our data showed the large vesicles form SFTSV infected cells contained viral particles and mediated virus transmission. 25HC simultaneously inhibited the entry of SFTSV and infectious MVs‐SFTSV, which could be regarded as an antiviral strategy to kill two birds with one stone.

PT04.04. Bacterial extracellular vesicles contain metabolites that could contribute to the pathological hallmarks of Alzheimer's disease

Samuel Wachamo^{Department of Neuroscience, Neuroscience Graduate Program, Center for Brain Immunology and Glia, Medical Scientist Training Program,University of Virginia, Charlottesville, VA, USA}, Alisha Thakur, Mallarie Broadway^{Department of Neuroscience, Neuroscience Graduate Program, Centerfor Brain Immunology and Glia, Medical Scientist Training Program, University of Virginia, Charlottesville, VA, USA}, Dr. Alban Gaultier^{Department ofNeuroscience, Neuroscience Graduate Program, Center for Brain Immunology and Glia, Medical Scientist Training Program, University of Virginia,Charlottesville, VA, USA}

Poster Pitches (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:45 PM ‐ 1:00 PM

Introduction: Alzheimer's disease (AD) is the most common form of dementia affecting 50 million people worldwide, and this number is projected to be 152 million by 2050. AD is characterized by a progressive decline in cognitive function associated with the formation of amyloid beta (Aβ) plaques, neurofibrillary tangles (NFTs), and neuroinflammation resulting in neuronal dysfunction and death. It is still not well understood what triggers the pathological hallmarks of AD, and thus there are currently few clinically efficacious disease modifying therapies for AD. However, emerging evidence confirms a crucial role of the gut microbiota through microbially produced metabolites, but the precise mechanisms remain to be elucidated. Based on previous studies that showed altered microbiota profile in AD, we hypothesized that the gut microbiota regulates microbiota‐gut‐brain axis and affects the pathological hallmarks of AD through release of bacterial extracellular vesicles (BEVs) that contain microbial metabolites that have been implicated in AD.

Methods: BEVs from intestinal contents of C57BL/6J mice were isolated by ultracentrifugation followed by size exclusion chromatography. BEVs were characterized by BCA assay, nanoparticle tracking analysis, transmission electron microscopy, ELISA, western blot, and metabolomics.

Results: Our preliminary results, for the first time, show a significant presence of metabolites, that have been associated with AD, within BEVs derived from the intestinal contents of mice. These metabolites are among the most biologically active bacterial metabolites, and their functional relevance in health and disease, especially in AD, will be investigated using 5XFAD mice, specific strains of bacteria that are known to produce the specific metabolites, and Cre‐LoxP system in future studies.

Summary/Conclusion: We discovered that BEVs contain metabolites, which can potentially regulate local and systemic immune response in AD. Ongoing studies in our lab will determine if BEVs play a functional role in the brain and mediate onset and progression of AD. The new insights into mechanisms in these studies may lead to new, effective therapeutic strategies for AD.

Funding: National Institutes of Health (T32 GM007267), Owens Family Foundation, and the Miller Family.

Keywords: Bacterial Extracellular Vesicles (BEVs), Alzheimer's disease (AD)

PT04.05. Bacterial outer membrane vesicles trigger mitochondrial stress in macrophages

Ms Chantelle Blyth, Dr Michael Lazarou, Dr Thomas Naderer

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Neisseria gonorrhoeae‐derived outer membrane vesicles (OMVs) target mitochondria and cause mitochondrial dysfunction and apoptosis [1, 2]. How mitochondria sense OMV‐mediated damage to activate apoptosis remains unclear. Here, we focused on the role of mitochondrial quality control systems, including organelle‐specific stress pathways and PINK1/Parkin mitophagy, to determine their role in regulating OMV levels, macrophage survival and immune responses.

Methods: Neisseria gonorrhoeae OMVs were isolated from Neisseria gonorrhoeae (MS11A) grown to mid‐log phase (OD600 < 1) in gonococcal‐specific broth media and then harvested via ultracentrifugation. Protein and particle concentrations were assessed using BCA and NTA, respectively. Primary bone marrow‐derived macrophages (BMDMs) from wild‐type and Parkin‐/‐ mice were differentiated and exposed to N. gonorrhoeae OMVs at 20 µg/ml. Cell viability was assessed over a 48 hours via live cell imaging with mitochondrial stress confirmed via immunoblot. The cytokine profile of OMV‐treated cells asses assessed via flow cytometry using the legend plex assay.

Results: Preliminary data show a reduction in cell death at 48 hours after OMV treatment in the absence of Parkin and a delayed mitochondrial stress response, indicated by PGAM5 cleavage assessed by immunoblot. Parkin‐/‐ BMDMs also produced an altered cytokine profile, with reduced expression of proinflammatory cytokines.

Summary: This data highlights key differences between wild‐type and knockout genotypes in response to N. gonorrhoeae OMV exposure, suggesting a potential role of mitochondrial quality control systems in regulating macrophage survival and innate immune responses. 

References:

1. Deo, P., etal., Mitochondrial dysfunction caused by outer membrane vesicles from Gram‐negative bacteria activates intrinsic apoptosis and inflammation. Nat Microbiol, 2020. 5(11): p. 1418‐1427. 

2. Deo, P., etal., Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis. PLoS Pathog, 2018. 14(3): p. e1006945. 

PT04.06. Comparative analysis of intestinal microbiota‐derived extracellular vesicles in newborns from vagin*l and cesarean section delivery: implications for modulation of immune system cells

Ms. Catalina Adasme‐Vidal, Mr. Aliosha I. Figueroa‐Valdés, Ms. Camila Fuentes, Ms. Patricia Valdebenito, Mr. Sebastián Illanes, Ms. Francisca Alcayaga‐Miranda

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

The type of birth ‐vagin*l delivery (VD) or cesarean section (CS)‐ determines the composition of commensal intestinal microbiota in newborns (CIMN). CIMN has been associated with pediatric diseases, including those related to the immune system. The interaction between gut microbiota and the immune system remains poorly understood, yet it may be mediated by bacterial extracellular vesicles (bEVs) derived from the gut microbiota. This study aims to determine whether VD/CS influences the characteristics and immunomodulatory potential of microbiota‐derived bEVs in newborns.

Methods:

bEVs were isolated from fecal samples collected from VD/CS newborns and characterized in terms of concentration and size using nanoparticle tracking analysis, morphology via transmission electron microscopy, and gram‐negative/gram‐positive bacterial markers via western blot. The immunomodulatory potential of bEVs was assessed in vitro through a peripheral blood mononuclear cell immunosuppression assay using human and mouse cells, along with phytohemagglutinin and anti‐CD3/CD28 activating beads and determining lymphocyte subpopulations by flow cytometry.

Results:

A protocol for bEVs isolation was established. No differences were observed in the concentration and size of bEVs isolated from CIMN. However, bEVs' shapes varied depending on their VD/CS origin. Likewise, bEVs derived from VD exhibited greater immunomodulation capacity than those obtained from CS, inhibiting lymphocyte proliferation.

Summary/Conclusions:

The established protocol allowed for obtaining a concentrate enriched in bEVs, whose morphology and immunosuppressive effect depend on their VD/CS origin, perhaps influenced by the Gram‐positive/negative bacterial composition of CIMN.

PT04.08. Exosomal miRNAs as markers of the biological effects of plant extracts

Doctor Alisa Petkevich, Doctor Aleksandr Abramov, Professor Vadim Pospelov

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction. According to the data of NCHS among U.S. adults aged 20 and over, 57.6% used any dietary supplement in the past 30 days. Although among the most used dietary supplements are vitamin D3 and omega‐3 fatty acids, there is a huge variety of plant extracts based dietary supplements. Different extract forms of the same plant may have completely different biological effects. With growing interest to the dietary supplements, these plant extracts become one of the important environmental epigenetics factors. Exosomes, as one of the main tools of the cell messenger system, may provide a valuable data regarding epigenetic changes in the cells being exposed to these plant extracts.

Methods. Several breast cancer cell lines (including MCF7, MCF10F, SK‐BR‐3, MDA‐MB‐231 from ATCC) were incubated for 24 hours with different plant extracts including water and isobutanol extracts of Laminaria Angustata. After incubation exosomal miRNAs (exoEasy Maxi kit, Qiagen, Germany) and supernatant miRNAs were isolated with following miRNA sequencing (Illumina 5 Mio read pairs 2*150).

Results. Exosomal miRNAs showed more significant difference in the levels of expression compare to supernatant miRNAs, what may be partly explained by their specificity to the changes in regulation of signal pathways. There were miRNAs with mostly significant changes in expression levels, which are associated with regulation of the genes, associated with cytoskeletal proteins, proliferation and apoptosis. It is interesting to note, that many miRNAs with most significant changes in expression level bind directly to DNA as the main mechanism of gene regulation.

Conclusion. Exosomes and exosomal miRNAs in regard to study of epigenetic influence of different environmental factors, including food, may be a valuable tool, providing the relevant information on the changes in the gene regulation.

PT04.09. Exploration of Purified Extracellular Vesicles (EVs) from Trypanosoma cruzi Y and G Strains on Host Cell Interaction

PhD student Paula Meneghetti, Ana Claudia Torrecilhas

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction Extracellular vesicles (EVs) are naturally released into the culture medium by Trypanosoma cruzi, the causative agent of Chagas disease is a Neglected Tropical Diseases (NTDs). Previous research has shown that EVs release is important in facilitating communication between host cells and the protozoan parasite. The characterization of EVs released during metacyclogenesis in different strains of T. cruzi (Y and G) metacyclic trypomastigotes, as well as their role in interactions with human host cells will be the focus of our research.

Methods: T. cruzi metacyclic forms were cultured in Liver infusion tryptose (LIT) medium to isolated EVs (Y and G strains), then purified using ultracentrifugation and size exclusion chromatography (SEC). THP‐1 cells were used in uptake assays with EVs labeled with PHK26 and was analyzed using flow cytometry and confocal microscopy at 30 and 60 minutes, 24 and 48 hours. In parallel, qRT‐PCR was also used to perform TNF‐alpha, CCL5, IL‐1, IL‐6, IP‐10 and IL‐10 detection assays in the THP‐1 cells incubation with EVs. Simultaneously, EVs isolated from Y and G strains were characterized using mass spectrometry (MS).

Results: We observed the interaction of EVs with THP‐1 cells within the first 30 minutes and maximum internalization occurring after 48 hs. qRT‐PCR analyses revealed the presence of cytokines and chemokines in both strains, with higher concentrations observed in the Y strain and a significant increase in the G strain, particularly in the detection of IL‐6. The MS revealed 167 proteins, 37 of which were found exclusively in the Y strain and 6 in the G strain. Among the proteins highlighted in this study were tropomyosin and guanylate binding proteins, which are known for their roles in cellular regulation and pathogen replication within host cells.

Summary/Conclusion: The active interaction observed between host and EVs derived from T. cruzi strongly suggests that these EVs may play a mediating role in facilitating interactions with host. This discovery highlights the dynamic interaction between T cruzi and its host vector and human environment, shedding light on the intricate mechanisms involved in communication as well as the potential influence of EVs during the interaction process.

PT04.10. Explore food microbes with preservative tolerance to spread of antimicrobial resistance from perspective of extracellular vesicles

Dr. Bao‐Hong Lee, MS. Yi‐Tsen Chang, Mr. You‐Zuo Chen, Mr. Hui‐Chun Lin, Dr. Wei‐hsuan Hsu

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: The widespread and inappropriate use of antibiotics lead to the generation and spread of antimicrobial resistance (AMR) microorganisms, limiting the therapeutic benefits of medicine. Preservative food additives are used to prevent the growth of microorganisms in food products, which may also lead to the emergence and spread of AMR microorganisms. However, their risks and mechanisms of action have not been paid attention to and explored. The aim of this study is to investigate the spread of AMR microorganisms to preservatives in fermented food product. Methods: This study focuses on understanding benzoic acid (BA) resistance in microorganisms from pickled vegetables. Microbes were isolated from pickled cucumbers and made resistant to BA. BA‐resistant microorganisms were identified through Sanger sequencing. EVs were isolated using ultracentrifugation and characterized for shape and size via transmission electron microscopy (TEM). EVs concentrations were measured using nanoparticle tracking analysis (NTA). Results: EVs from BA‐resistant strains were co‐cultured with other microorganisms to assess potential BA resistance transmission. Proteomic analysis identified the protein responsible for generating and spreading BA resistance, while fluorescence staining confirmed EVs uptake by microbes. The isolated microorganism from pickled cucumbers, identified as Pichia krudriavzevii (PK), produced round‐shaped EVs with lipid bilayers observed by TEM. Co‐culturing PK with these EVs allowed it to adapt to high BA concentrations. The protein SNQ2, an ABC transporter known for drug resistance, was highly expressed in BA‐resistant EVs, likely contributing to BA resistance development. Summary: Taken together, microorganisms in fermented products can develop BA resistance, undergo vertical transmission, and potentially spread resistance through secreted EVs.

PT04.11. Exploring the extracellular vesicles derived from food spoilage microorganisms in the transmission of antibiotic resistance and potential impact on the gut environment

Dr. Bao‐Hong Lee, MS. Yi‐Tsen Chang, Mr. Hui‐Chun Lin, Mr. You‐Zuo Chen, Dr. Tang‐Long Shen, Dr. Wei‐hsuan Hsu

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Antibiotics are frequently employed to treat bacterial infections, contributing to the rise of antibiotic resistance. The exchange and transfer of microbial DNA through extracellular vesicles (EVs) facilitates the horizontal dissemination of these resistant genes. These nano‐scale vesicles, containing DNA, RNA, and proteins, play a crucial role in intercellular communication. However, a limited body of research exists exploring the correlation between microbial EVs, drug resistance, and food safety. Pickled vegetables, such as cucumbers preserved with salt, organic acid, or sugar, often incorporate benzoic acid (BA) as a food preservative, effectively inhibiting the growth of yeast and mold. This study seeks to examine whether microorganisms within pickled vegetables can develop resistance to BA, elucidate the underlying mechanisms, and assess if this phenomenon adversely affects the gut environment. Methods: Microorganisms were isolated from pickled cucumbers, and BA was employed to induce resistance, with microbial species identified through Sanger sequencing. The transmission of BA resistance through EVs was evaluated by co‐culturing EVs with microorganisms, gut microbiota, and gut cells separately. Proteomic analysis aimed to identify proteins responsible for generating and spreading BA resistance, while flow cytometry was employed to identify microorganisms capable of uptaking EVs. Results: Pichia krudriavzevii (PK) was successfully isolated from pickled cucumbers, demonstrating an ability to adapt to higher concentrations of BA when co‐cultured with EVs. Furthermore, candidate proteins linked to the spread of BA resistance were analyzed. Flow cytometry was used to determine the proportion of gut microbiota that uptakes EVs, and sorted bacteria were identified through sequencing. Fluorescence staining confirmed the absorption of EVs by PK and gut microbes, as well as gut cells. Conclusion: Microorganisms in fermented products can develop resistance to BA, undergo vertical transmission, and potentially spread resistance through secreted EVs. This process may impact gut environment, posing risks to human health.

PT04.13. Extracellular vesicles derived from Akkermansia muciniphila

Promote placentation and mitigate preeclampsia

Ph.d Zihao Ou

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Preeclampsia (PE) is a multisystem disorder with high maternal morbidity and mortality rates. Currently, no practical therapeutic approach is available to prevent PE progression, except for early delivery. Gut dysbiosis is associated with PE development. Previous data showed that the abundance of Akkermansia muciniphila (Am) was lower in patients with PE than in normotensive pregnant women. Here, in this study, decreased abundance of Am was observed in a PE mouse model. Also, we found that administration with Am could significantly attenuate systolic blood pressure, promote foetal growth and improve the placental pathology in mice with PE. Moreover, Am‐derived extracellular vesicles (AmEVs) were transferred from the gastrointestinal (GI) tract to the placenta and mitigated pre‐eclamptic symptoms in PE mice. These beneficial effects of AmEVs were mediated by enhanced trophoblast invasion of the spiral artery (SpA) and SpA remodelling through activation of the epidermal growth factor receptor (EGFR)–phosphatidylinositol‐3‐kinase (PI3K)–protein kinase B (AKT) signalling pathway. Collectively, our findings revealed the potential benefit of using AmEVs for PE treatment and highlighted important host–microbiota interactions.

PT04.14. Extracellular vesicles derived from Candida albicans promote lung injury through inducing ferroptosis of macrophages

Miss Yiyi Huang, Doctor Kening Zhao, Miss Yuneng Hua, Miss Mei Huang, Doctor Ruyi Zhang, Doctor Jingyu Wang, Mr Fan Bu, Miss Junhui Wang, Professor Lei Zheng, Professor Qian Wang, Professor Xiumei Hu

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Candida albicans‐induced lung injury is one of the most prevalent diseases in patients with invasive candidiasis. However, the mechanism by which C. albicans causes invasion and lung injury in the host remains unclear. Recently, studies have reported that C. albicans secrete extracellular vesicles (EVs), a group of nanostructures carried bioactive components and participated in the pathogenic processes. In the present study, we aim to explore the pathogenic mechanism of C. albicans EVs damage to the host lung, and to provide theoretical basis and new therapeutic strategies for the clinical treatment of severe invasive candidiasis.

Methods

1. To explore whether C. albicans promote host lung injury through EVs, GW4869 was used to inhibit the EVs secretion of Candida albicans. Subsequently, C. albicans /GW4869 pretreated C. albicans infection models were constructed respectively.

2. To explore whether EVs derived from C. albicans induce host lung injury, EVs secreted by Candida albicans were extracted by ultracentrifuge, and then C. albicans EVs/ S. cerevisiae EVs infection models were constructed respectively.

3. To explore whether C. albicans EVs promote lung injury through inducing ferroptosis of macrophages, the expression of ferroptosis related indicators and molecules were detected.

Results

1. In vivo experiments showed that C. albicans and C. albicans EVs both induce acute lung injury in mice, while GW4869 pretreated C. albicans has a weakened damage effect. C. albicans and C. albicans EVs triggered decreased frequency of lung macrophages, increased MDA level and PTGS2 expression in lung.

2. In vitro experiments showed that C. albicans and C. albicans EVs induce decreased viability and increased cytotoxicity of macrophages.

3. In vitro experiments showed that C. albicans EVs increase the intracellular total iron level, MDA level and mitochondrial superoxide in macrophages, while decrease GSH level. C. albicans EVs also increased the expression of PTGS2 while decreased the expression of GPX4, which are consistent with the main features of ferroptosis in cells.

Conclusion

In the present study, we found that C. albicans was able to induce ferroptosis in macrophages by secreting EVs, thus promoting acute lung injury in host.

PT04.15. Extracellular vesicles derived from Naegleria fowleri stimulate cytokine production by innate immune cells

Asst. Prof. Sakaorat Lertjuthap*rn, Ms Narinee Srimark, Mrs Hathai Sawasdipokin, Ms Kasama Sukapirom, Ms Jinjuta Somkird, Prof. Kovit Pattanapanyasat, Ladawan Khowawisetsut

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Naegleria fowleri, a free‐living amoeba, is the causative agent of primary amoebic meningoencephalitis, a severe central nervous system infection with a high mortality rate. The disease's pathogenesis is linked to both the pathogenicity of the amoeba and the robust inflammatory response elicited by host immune cells, particularly recruited blood‐borne innate leukocytes. As extracellular vesicles (EVs) have been recognized as key mediators of intercellular communication between pathogenic amoebas and the hosts, the activation of the host immune response by EVs might significantly influence the outcome of infection. Therefore, this study seeks to investigate the influence of N. fowleri‐derived extracellular vesicles (Nf‐EVs) on gene expression and cytokine production in innate immune cells, with a specific focus on neutrophils and monocytes.

Methods: The pathogenic N. fowleri trophozoites were cultivated axenically in Nelson's medium with supplementation. After replacing the culture medium with buffer saline, the culture supernatant was collected following a 24‐hour incubation period. Nf‐EVs were isolated from the N. fowleri trophozoite culture supernatant using a multi‐step differential centrifugation and characterized through electron microscopy and nanoparticle tracking analysis. Primary neutrophils and peripheral blood mononuclear cells were purified via Ficoll‐Hypaque gradient density centrifugation. Subsequently, primary monocytes were further isolated using immunomagnetic bead separation. These primary cells were then incubated with Nf‐EVs, and the study explored the effects of Nf‐EVs on gene expression and cytokine production (TNF‐α, IL‐1β, IL‐6, and IL‐8) by these cells.

Results: Nf‐EVs exhibited the potential to elevate the expression levels of pro‐inflammatory molecules, specifically inducing heightened production of TNF‐α by neutrophils and IL‐6 and IL‐8 by monocytes. Notably, the activation of primary neutrophils and monocytes by Nf‐EVs varied in the timing of response.

Summary/Conclusion: These findings demonstrated the immunostimulatory properties of Nf‐EVs, emphasizing their role in orchestrating the activation of host immune cells and the subsequent induction of inflammation.

PT04.16. Extracellular Vesicles isolated from Virulent and Non‐Virulent trypomastigotes forms from Trypanosoma cruzi in Host Cell Modulation

Ana Claudia Torrecilhas, Master Nicholy Lozano, Full Professor Sergio Schenkman

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Extracellular vesicles (EVs) derived from trypomastigote forms of Trypanosoma cruzi modulating host cells invasion and immune responses. Although the parasite can survive in tissue culture‐infected cells, it decreases virulence in mice. In our study, we successfully restored virulence in mice upon 20 consecutive passages. As a result, we performed a comparison study, comparing the impact of EVs released by non‐virulent trypomastigotes maintained in vitro (P2) to virulent trypomastigotes maintained in vivo (P20). Our findings suggest a new link between T. cruzi virulence and EV release. Surprisingly, these EVs increase parasitism in the heart first, then the bladder, intestine, and lungs.

Methods: P2 and P20 EVs were isolated using a combination of ultracentrifugation and SEC column. THP‐1 cells were subjected to uptake assays using P2 and P20 EVs, labeled with PHK26, and then examined using the Image Streams system microscopy at 15‐, 30, and 60‐minutes intervals, as well as 24 and 48 hours. The quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) was used to examine TNF‐alpha, CCL5, IL‐1, IL‐6, IP‐10, and IL‐10 expression in THP‐1 cells after EVs P2 and P20 incubation.

Results: We investigated the dynamic cellular uptake of PHK26‐labeled EVs using ImageStreams system microscopy. Both P2 and P20 EVs interacted immediately with human monocytes. Concurrently, we employed qRT‐PCR to examine cytokine and chemokine expression in THP‐1 cells upon incubation with P2 and P20 EVs. Notably, cytokine production increased in P20 EVs when compared to P2 EVs.

Conclusion: This comprehensive approach allowed us to investigate the properties of EVs released by virulent (P20) and non‐virulent (P2) parasites during their interactions with host cells and assess the impact on immune response communication, providing a detailed understanding of the complex interactions between Trypanosoma cruzi and host cells. This allows us to understand how virulence factors influence different stages of Chagas disease.

Funding: FAPESP, CAPES, CNPq

PT04.17. Extracellular vesicles release from Aeromonas hydrophila: proteomic analysis and immunomodulatory activity

Professor MAHANAMA DE ZOYSA, Mr. Mawalle Kankanamge Hasitha Madhawa Dias

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Bacterial EVs (BEVs) are known for harbor various virulence factors and signaling molecules that facilitate cell communication and host‐pathogen interactions. The present study focuses on isolation, characterization of BEVs from Aeromonas hydrophila (AhEVs), and understand their immunomodulatory role in vitro and in vivo experiments.

Methods: BEVs were isolated using the ultracentrifugation method. Characterization was carried out using transmission electron microscopy (TEM), nanoparticle tracking (NTA), and analyzing bacterial EV markers. The proteomie profile was analyzed using LC‐MS/MS. Internalization of AhEVs was tested using fathead minnow (FHM) cells, followed by analysis of toxicity and reactive oxygen species (ROS) production. In vitro and in vivo immunomodulatory effects were investigated using FHM cells and adult zebrafish.

Results: From TEM imaging, ultrastructural morphology was confirmed to have a spherical shape, and NTA determined the mean particle size as 105.5 ± 2.0 nm. Proteomic analysis revealed 4825 unique peptides and 1284 proteins, associated with antioxidant function, transduction regulation, and transportation activity. No cytotoxicity was observed in AhEVs treated FHM cells and zebrafish larvae up to 50 µg/mL. Furthermore, fluorescent‐labeled AhEVs internalized into FHM cells. Cellular pro‐inflammatory cytokines, nuclear factor (NF)‐κB, interferon (Ifn), interferon regulatory factor (Irf) 7, interleukin (il) 8, and il11 were upregulated in AhEVs treated FHM cells. In vivo gene expression analysis revealed that AhEVs treated adult zebrafish‐induced toll‐like receptor (tlr) 2 and 4, tumor necrosis factor α (tnfα), heat shock protein (hsp) 70, il6, il10, and il1β. In the spleen of AhEVs treated adult zebrafish showed induced NF‐κB and TNF‐α proteins.

Summary/Conclusion: Overall results exhibit AhEVs have morphological and physicochemical characteristics of Gram (‐ve) bacteria. Furthermore, AhEVs display immunomodulatory activity in FHM and zebrafish. In‐depth analysis of the proteome of AhEV may provide its mechanisms of action associated with immunomodulatory function.

PT04.18. Gram‐positive bacterial extracellular vesicles released by Streptococcus parauberis: Proteomic profiling and anti‐inflammatory activity

Professor MAHANAMA DE ZOYSA, Mr. E.H.T. Thuslahn E.H.T. Thuslahn Jayathilaka, Mr. Mawalle Kankanamge Hasitha Madhawa Dias, Dr. Chamilani Nikapitiya Nikapitiya

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Streptococcus parauberis is a Gram‐positive pathogenic bacterium that causes Streptococcosis in fish and bovine mastitis. This study was focused on isolation, characterization, proteomie profiling, and anti‐inflammatory activity of S. parauberis derived EVs (SpEVs).

Methodology: SpEVs were obtained from S. parauberis free culture media using the ultracentrifugation method. The isolated SpEVs were characterized based on morphology through transmission electron microscopy, size determination via nano tracking analysis, identification of bacterial EV markers, and protein profile. Additionally, the proteomie profile was analyzed using LC‐MS/MS. The anti‐inflammatory activity of SpEVs was assessed in murine macrophages (Raw 264.7) cells treated with lipopolysaccharide (LPS).

Results: The isolated SpEVs exhibited oval‐shaped clear membrane‐bound particles with an average diameter of 168.3 ± 6.5 nm. Flagellin, a bacterial EV marker, was detected, and SDS‐PAGE confirmed the presence of three significant protein bands in SpEVs. Proteomic analysis identified 6209 unique peptides and 1039 proteins in SpEVs, with a predominant protein mass distribution in the range of 10–50 kDa. Gene ontology analysis revealed that expressed proteins in SpEVs were enriched for catalytic activity, with other notable terms including toxin activity and antioxidant activity. Functionally, SpEVs illustrated anti‐inflammatory activity in Raw264.7 cells upon LPS treatment.

Summary/Conclusion: SpEVs were successfully isolated from S. parauberis and they showed the characteristics of BEVs. Proteomic analysis affirmed the expression of distinctive proteins in SpEVs, associated with diverse physiological and immunological functions. In addition, the study confirmed the potent anti‐inflammatory activity of SpEVs.

PT04.20. Helicobacter pylori cytotoxin, VacA, hijacks dendritic cell extracellular vesicles

Miss Ruby Gorman‐batt¹, Associate Professor Meredith O'Keeffe, Doctor Terry Kwok‐Schuelein

¹Monash University, Clayton, Australia

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Dendritic cell (DC)‐derived extracellular vesicles (EVs) are known to regulate host immunity, however, their role during bacterial pathogenesis remains unclear. We observed that a critical Helicobacter pylori (Hp) virulence factor, the vacuolating cytotoxin A (VacA), specifically targets DC‐EVs. Hp is a gram‐negative bacterium that persistently infects 50 % of the world's population, and is the greatest risk factor for the development of gastric cancer (GC). VacA has been shown to assist in bacterial persistence by limiting dendritic cell (DC), T‐cell and macrophage functions. However, how the toxin interacts with these cell types in vivo remains poorly understood, but may be explained by VacA dissemination to distant immune centres by DC‐EVs. Here, we report VacA's influence on the physical properties of DC, DC‐EV and the interactions between VacA+‐EVs and recipient immune cells.

Methods

The impact of Hp 60190 VacA on the maturation state of murine DC cell line, MutuDC, was evaluated by flow cytometry and immunofluorescence microscopy. VacA localisation during MutuDC and human primary DC intoxication was assessed by correlative light electron microscopy. Small EVs (sEVs) were isolated by multi‐step ultracentrifugation for analysis by immunoblot, transmission electron microscopy, nanotracking analysis and mass spectrometry. Finally, the impact of VacA+‐sEVs on recipient immune cells was assessed by flow cytometry and immunofluorescence microscopy.

Results

VacA modulated the expression of cell surface markers, without being phagocytosed by the DCs. Instead, VacA localised exclusively to EVs of MutuDC and human primary DCs. VacA did not impact DC‐EV size, however VacA+‐sEVs were enriched in key markers of immune cell functions and possessed the ability to deliver VacA to naïve immune cells. Analysis concerning the functional impacts of VacA+‐sEVs is ongoing.

Conclusions

This study reveals that VacA specifically binds EV at the DC surface, altering DC and DC‐EV immune marker composition. We propose that VacA perturbs DC maturation and hijacks DC‐EVs to dysregulate immune cell signalling, assisting in the chronic persistence of Hp. Our findings highlight a previously unrecognised role of EVs in bacterial pathogenesis.

PT04.20. Helicobacter pylori cytotoxin, VacA, hijacks dendritic cell extracellular vesicles to dysregulate immune cell functions

Miss Ruby Gorman‐batt, Meredith O'Keeffe, Terry Kwok

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Dendritic cell (DC)‐derived extracellular vesicles (EVs) are known to regulate host immunity, however, their role during bacterial pathogenesis remains unclear. We observed that a critical Helicobacter pylori (Hp) virulence factor, the vacuolating cytotoxin A (VacA), specifically targets DC‐EVs. Hp is a gram‐negative bacterium that persistently infects 50 % of the world's population, and is the greatest risk factor for the development of gastric cancer (GC). VacA has been shown to assist in bacterial persistence by limiting dendritic cell (DC), T‐cell and macrophage functions. However, how the toxin interacts with these cell types in vivo remains poorly understood, but may be explained by VacA dissemination to distant immune centres by DC‐EVs. Here, we report VacA's influence on the physical properties of DC, DC‐EV and the interactions between VacA‐EVs and recipient immune cells.

Methods

The impact of Hp 60190 VacA on the maturation state of murine DC cell line, MutuDC, was evaluated by flow cytometry and immunofluorescence microscopy. VacA localisation during MutuDC and human primary DC intoxication was assessed by correlative light electron microscopy. Small EVs (sEVs) were isolated by multi‐step ultracentrifugation for analysis by immunoblot, transmission electron microscopy, nanotracking analysis and mass spectrometry. Finally, the impact of VacA‐sEVs on recipient immune cells was assessed by flow cytometry and immunofluorescence microscopy.

Results

VacA modulated MutuDC expression of the costimulatory molecule, CD86, without being phagocytosed by the DCs. Instead, VacA localised exclusively to EVs of MutuDC and human primary DCs. This interaction was dependant on cholesterol‐rich lipid rafts. VacA did not impact DC‐EV size, however VacA‐sEVs were enriched in key markers of immune cell functions and possessed the ability to deliver VacA to naïve immune cells. Analysis concerning the functional impacts of VacA‐sEVs is ongoing.

Conclusions

This study reveals that VacA specifically binds EV at the DC surface, altering DC and DC‐EV immune marker composition. We propose that VacA perturbs DC maturation and hijacks DC‐EVs to dysregulate immune cell signalling, assisting in the chronic persistence of Hp. Our findings highlight a previously unrecognised role of EVs in bacterial pathogenesis.

PT04.21. Helicobacter pylori extracellular vesicles contain functionally active enzymes promoting bacterial survival

Miss Nina Colon, Mr Liam Gubbels, Professor Richard L. Ferrero

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Extracellular vesicles (EVs) that are released during bacterial growth contain various cargo involved in pathogenesis. Proteomic studies showed that the gastric pathogen Helicobacter pylori produces EVs containing urease (UreA, UreB) and catalase (KatA), known to be essential for bacterial colonisation in the stomach. Nevertheless, it was not known whether urease and catalase in EVs retain functional activity. As these enzymes are colonisation factors for the bacterium, we hypothesised that functionally active urease and catalase within H. pylori EVs may promote bacterial survival against environmental stresses. Methods: EVs from H. pylori wild‐type (WT) bacteria and ureA, ureB and katA isogenic mutants were isolated by ultracentrifugation and characterised by Western blotting. Enzymatic activities of the EVs and sonicated preparations of the whole bacteria were determined using quantitative assays. The impact of EV‐associated enzymes on bacterial survival and host cell responses was assessed in in vitro assays. Results: Western blotting confirmed the presence of UreA, UreB and KatA subunits within EVs and whole cell preparations. EVs had similar levels of urease and catalase activity as the whole bacteria (1.31 ± 0.321 vs. 1.21 ± 0.504 µmol/min/mg protein, p=0.8741; and 784.4 ± 30.543 8 vs. 817.3 ± 27.8 µmol/min/mg protein, p=0.6979). EV‐associated urease had no effect on bacteria survival at acid pH. Conversely, WT EVs (40 µg/mL) protected katA mutant bacteria from exposure to 10 mM H₂O₂, similar to the WT bacteria, whereas katA mutant bacteria were killed. As bacterial EVs are highly adept at entering host cells, we next determined the localisation of EV‐delivered H. pylori catalase within human gastric epithelial cells. KatA was detected by Western blotting analyses on the nuclear fractions from cells exposed to WT, ΔureA or ΔureB but not ΔkatA EVs. Further, catalase activity was detected in the nuclear fractions from cells incubated with either WT, ΔureA or ΔureB EVs. Conclusions: Together, these data highlight that H. pylori EVs contain functionally active urease and catalase. Moreover, EV‐associated catalase was shown to protect the bacterium against environmental stresses and to be delivered to the nucleus of host cells, potentially leading to DNA damage.

PT04.23. Infective forms of Leishmania show different biological aspects and differ in the extracellular vesicle target mechanisms: Looking at the fundamental bases to understand differences

Dr Mauro Javier Cortez Veliz, Miss Deborah Brandt‐Almeida, Mrs Jenicer Kazumi Umada Yokoyama Yasunaka, Dr Simon Ngao Mule, Dr Giuseppe Palmisano, Dr Ana Claudia Torrecilhas

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

IINTRODUCTION The regulatory mechanism of CD200R/CD200 participates in several cellular processes mediated by the host's immune response. Cells expressing CD200 ligand regulate macrophages, which present the CD200R on their membrane. During infection with virulent Leishmania, the induction of CD200 in macrophages by extracellular vesicles (Evs) of infective amastigotes is essential to inhibit the iNOS/NO mechanism, which leads to parasite resistance and proliferation. However, little is known whether infective metacyclic promastigotes can induce CD200. OBJECTIVE Therefore, the present work evaluates the ability of the two infective forms of Leishmania to release Evs and induce CD200.OBJECTIVE Therefore, the present work evaluates the ability of the two infective forms of Leishmania to release Evs and induce CD200. MATERIALS AND METHODS Leishmania amastigotes and metacyclic promastigotes were incubated in their own medium for vesiculation by 1h, and the supernatants were filtered through a 0.45‐mm sterile cell strainer. Isolated Evs from the different infective forms were concentrated using amicon, and different assays were performed to validate the quantity and biological effect of Evs in macrophages by immunofluorescence and Immunoprecipitation/western blot (WB), both using polyclonal antibodies against Leishmania‐Evs. RESULTS Concentrated Evs from the different forms were incubated in macrophages for 1h, and then fixed for IF assays to detect Evs signals. Representative images showed that stationary‐phase promastigotes (enriched with metacyclic forms) and amastigotes presented a similar pattern of Evs‐immunofluorescence signal in macrophages, confirmed by quantifying Evs signal per cell. When the same approach for Evs detection was used to detect CD200, amastigote‐Evs induced a strong signal of the inhibitory ligand in macrophages. To confirm the specificity of amastigotes in inducing CD200, the different infective forms were incubated with macrophages, and the expression of CD200 was detected by IP/WB. Amastigotes induce CD200 in macrophages, but this molecule is not detected during infection of metacyclic forms. CONCLUSION Leishmania infective forms induce similar Evs‐quantities, but only amastigote‐Evs can induce CD200 in macrophages, suggesting a different Evs‐content from both infective forms. Further experiments will be prepared to understand better the difference between both infective forms to highlight new aspects of biogenesis and the releasing mechanism of Evs.

PT04.23. Host‐Parasite Interface: Exploring the Interaction of Trypanosoma cruzi trypomastigotes forms Y strain Extracellular Vesicles (EVs) with Human Monocytes and Macrophage

PhD student Juliana Fortes, Master student Nathani Negreiros, Ana Claudia Torrecilhas

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: The protozoan Trypanosoma cruzi causes Chagas disease (CD). CD is classified as a neglected tropical disease (NTD) and is estimated to kill 14,000 people across Latin America every year. The disease's treatment, diagnosis, and epidemiological control remain challenging. T. cruzi delivers EVs at various stages of its life cycle, which aid in communication with cells and modulation of the host's immune response. EVs may be useful as CD biomarkers and targets for new therapeutic strategies. The goal is to investigate to how T. cruzi‐released EVs influence the profile and behavior of primary monocytes and macrophages, with a focus on activation markers.

Methods: Nanoparticle Tracking Analysis (NTA) was used to determine the concentration and size of T.cruzi EVs. Following that, the EVs were labeled with PKH26 Dye Solution (SIGMA). Flow cytometry (FC) and confocal microscopy (CM) were used to assess the interaction of the EVs with THP‐1 and differentiated THP‐1 after 5, 15, 30 minutes, 1 h, 24 h, and 48 h. After isolating mononuclear cells from whole blood using Ficoll‐Paque (SIGMA), human monocytes were isolated using a negative selection strategy according to the commercial Pan Monocyte Isolation Kit, human (Miltenyi Biotec). Monocytes were incubated with EVs that contained the following markers: CD14‐V450 (BD Biosciences), CD16‐PE‐CY7 (ExBio), CD80‐FITC (BD Biosciences), CD86‐APC (BD Biosciences), CD69‐PE (ExBio), HLA‐DR‐V500 (BD Biosciences), TexasRed (BD Biosciences), and FC.

Results: Our results show that internalization of T. cruzi EVs was uptake using confocal microscopy after only 5 minutes of incubation with human THP‐1 cells. Flow cytometry (FC) also confirmed this uptakeand cells interactions. Furthermore, after 24 hours of incubation, significant changes in monocyte morphology were observed. Furthermore, we found a significant increase in the expression of key surface markers on monocytes, which could be important in their differentiation into macrophages.

Summary/Conclusion: The T.cruzi EVs interact with host cells, resulting in observable changes in cellular morphology and suggesting the potential to alter the host's immune response profile. These findings provide insight on the quick internalization dynamics of T. cruzi EVs, their effect on cellular morphology, and the implications for monocyte differentiation.

PT04.24. Investigating the impact of Pseudomonas aeruginosa outer membrane vesicles on alveolar macrophage responses

Miss Isabella Stuart, Mr Joshua Nickson, Dr Seong Hoong‐Chow, Associate professor Thomas Naderer

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Pseudomonas aeruginosa is an opportunistic pathogen, that can cause life threatening infections, particularly those who are immunocompromised. While conventional treatments target infection with antimicrobials, our research explores an alternative approach by focusing on host and virulence factors. To develop new treatment strategies, a comprehensive understanding of host factors and responses are needed. For P. aeruginosa infections we have turned to focusing on their outer membrane derived vesicles and how they affect alveolar macrophage functions.

Methods: Bone marrow‐derived alveolar macrophages (BMDAMs) were exposed to P. aeruginosa derived outer membrane vesicles (POMVs) with cytokine responses assessed by multiplex assays, immunoblotting, and flow cytometry. Host responses of cell‐death, apoptotic caspase activation, and mitochondrial health were examined through real‐time analysis using live cell imaging. To investigate the role of BCL‐2 family members in AMs, A1‐deficient AMs and BH3 mimetics, such as ABT‐737, and S63845, were used. Additionally, A1‐deficient mice were infected with intranasally administered P. aeruginosa. Colony‐forming units (CFUs) were enumerated from the lung, liver, and blood, and flow cytometry was used to assess cytokine secretion in the serum using a multiplex assay.

Results: POMVs induce pro‐inflammatory cytokine secretion in BMDAMs, however cell death levels remain low. Treatment with POMVs led to increased expression of anti‐apoptotic proteins, A1 and MCL‐1 in BMDAMs. Live cell imagining of A1‐deficient cells, combined with MCL‐1 inhibitor S63845, resulted in higher levels of cell death compared to WT. Infected A1‐deficient mice lost more weight than WT across the duration of infection. However, CFUs from both lung and liver returned equivalent numbers.

Conclusions: BMDAMs show increased survival rates and secretion of pro‐inflammatory cytokines in responses to POMVs. Anti‐apoptotic proteins A1, and MCL‐1 play crucial roles in alveolar macrophages by promoting macrophage survival when challenged with POMVs. Additionally, A1 appears to play an important role in P. aeruginosa lung infections. The absence of A1 does not impact the replication of P. aeruginosa, possibly suggesting there is another factor at play. These findings highlight the intricate interplay between macrophages and bacterial vesicles, shedding light on their role in modulating inflammation during infections.

PT04.25. Isolation and physiological characterisation of Ascochyta rabiei small extracellular vesicles

Ms Matin Ghaheri, Dr Ido Bar, Dr Prabhakaran T. Sambasivam, Dr Muhammad J. A. Shiddiky, Mr Abolfazl Jangholi, Prof Chamindie Punyadeera, Prof Rebecca Ford

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Across kingdoms, small extracellular vesicles (sEVs) are produced by pathogenic organisms that are associated with signalling for host recognition and invasion, including by fungi and when in contact with their plant hosts. Very little is known about plant pathogenic fungal sEVs, with data limited to a few early studies on Fusarium oxysporum, Botrytis cinerea and Penicillium digitatum. Meanwhile, Ascochyta rabiei is an endemic and widespread necrotrophic ascomycete fungus that causes significant impact on chickpea production. The presence of sEVs in A. rabiei invasion of chickpea have not yet been established which is the focus of this study.

Methods: A. rabiei sEVs were isolated from liquid broth cultures (with and without presence of host tissue) through optimised ultracentrifugation method. The isolated sEVs were characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and Mass Spectrometry.

Results: In this study, TEM and NTA analyses confirmed that our optimised ultracentrifugation method, successfully isolated sEVs from A. rabiei with an acceptable size range and morphology, revealing typical cup‐shaped structures with double membranes of 30‐150 nm in size with greater frequencies produced in response to the presence of powdered and concentrated host tissues (AP) compared to whole detached leaves (AFL). sEVs isolated from AFL media showed larger mean and mode sizes than the other. Protein concentration of sEVs isolated from medias with the presence of host, AFL, and AP, were higher than the isolated sEVs without the host. The mass spectrometry analysis led to the identification of proteins involved in cell wall synthesis, biological processes, ribosomal structure, oxidation/reduction‐related proteins, and stress responses.

Conclusions: This is the first time that the sEVs have been identified and characterised in an Ascochyta species. Bioinformatics analysis of sEV proteomics revealed a variety of pathogenic‐related proteins, like oxidation/reduction‐related proteins and stress response proteins. Our results will help better understanding the biological function of the sEVs during plant and A. rabiei interaction and providing the evidence for the role of sEVs in pathogenesis of the A. rabiei.

PT04.26. Leishmania extracellular vesicles genomic cargo: sharing is caring

Associate Professor David Langlais, MSc Atia Amin, PhD Ana Victoria Ibarra Meneses, Associate Professor Christopher Fernandez‐Prada

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Leishmaniasis is a neglected tropical disease causing about one million new cases and over twenty thousand deaths every year worldwide. The disease is caused by the parasite Leishmania spp. transmitted to its host by a sandfly and affecting some of the poorest populations, already afflicted by poor housing, displacement, and malnutrition. Despite its endemicity and important health and social consequences, the treatment arsenal is limited, and drug resistance is rising. We have recently shown that Leishmania can share and propagate drug resistance genes through extracellular vesicles (EVs). However, the genomic events leading to this generous propagation are still unclear. The objective of this project is to understand how Leishmania genomic plasticity support their capacity to develop drug resistance to antimony and what genomic cargo is packaged in EVs. Methods: Of the >20 species causing Leishmaniasis in humans, most still have incomplete reference nuclear and mitochondrial (kinetoplast) genome assemblies and annotations, which complicates their study. Firstly, we have performed a hybrid genome assembly for L. infantum using a combination of long‐read Nanopore sequencing (ONT) and short‐read Illumina sequencing. Secondly, to study the genomic event leading to antimony resistance, we have cultivated L. infantum and two other species under increasing drug pressure. Genomic DNA was extracted at every incremental step for ten independent clonal populations for each species and subjected to ONT sequencing. Clinical isolates of different resistance level to antimony were also sequenced. Thirdly, EVs were purified from antimony‐resistant clones, their DNA extracted and submitted to sequencing. Results: The hybrid assembly approach allowed us to obtain a high‐quality reference genome, resolving large telomeric repeats and gaps, and solving the complete kinetoplast maxicircle sequence. The ONT sequencing of lab and clinical antimony‐resistant isolates allowed the identification of new tricks used by the parasites to develop resistance, including various intra‐ and extra‐chromosomal amplifications. Moreover, the parasites can release these newly amplified regions into EVs to spread resistance. Summary/Conclusion: The newly assembled genome will be a resource for future studies and our results will help understand the drug resistance mechanisms of the parasite and develop new surveillance and/or therapeutics against this deadly disease.

PT04.27. Microbiome derived EVs regenerate intestinal stem cells against radiation injury

Dr. Payel Bhanja, Dr. Rishi Man Chugh, Dr. Kafayat Yusuf, Dr. Badal Roy, Dr. Shahid Umar, Dr. Subhrajit Saha

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Gut microbiome plays an important role in gastrointestinal homeostasis and repair. Akkermansia muciniphila (AM) is the dominant human bacterium that abundantly colonizes in nutrient‐rich mucus layer and protects against gastrointestinal damage and inflammation. Clinical trials based on anti‐inflammatory or anti‐neoplastic effect of AM are promising. Moreover, AM derived extracellular vesicles (EVs) carry important paracrine signals for intestinal homeostasis. Previous reports demonstrated that therapeutic use of bacteria derived EV to mitigate injury and inflammation. Our study suggested that anaerobic commensal microbiome such as AM are sensitive to ROS and absence of such species due to irradiation exposure contribute in intestinal injury. We hypothesized that oral supplementation of A. muciniphila, derived EV (AM‐EV) can mitigate radiation induced intestinal injury. In mice model of radiation induced intestinal injury we have demonstrated that oral supplementation of AM‐EV (50mg/mice for 3 times) at 24 hours after whole or partial body irradiation can mitigate radiation induced intestinal injury with restitution of crypt villus structure. To mitigate intestinal epithelial injury it is important to rescue intestinal stem cells (ISCs) and activate regenerative function of ISCs. AM‐EV treatment activated the regenerative function Lgr5+ve ISCs both in ex‐vivo intestinal organoid system and in Lgr5‐EGFP‐ires‐CreERT2‐TdT mice exposed to irradiation. Proteomics study of these EVs identified several key proteins including activator of folate pathway, HIF1alpha signaling pathway and TRAF6 which are important for ISC regeneration. In summary, present study demonstrated that AM‐EV based therapy can be considered as a potential mitigator of Gastrointestinal acute radiation syndrome.

PT04.29. Microbiota‐derived extracellular vesicles regulate host liver gluconeogenesis

Dr Jian Tan, Ms Jemma Taitz, Dr Duan Ni, Ms Camille Potier, Prof Ralph Nanan, Prof Laurence Macia

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: The gut microbiota regulates many host processes including immunity and metabolism. Bacteria‐derived extracellular vesicles are involved in a variety of biological processes such as quorum sensing, material exchange, antibiotics resistance and host immunomodulation. However, much less is known about how extracellular vesicles produced by the gut microbiota influence host physiology.

Methods: Whole microbiota extracellular vesicles (MEV) were isolated from faeces of control diet (AIN93G) fed animals. Faeces were resuspended in 0.02µm filtered PBS and supernatant centrifuged at 10,000g for 10min, then 18,000g for 45min before filtration through a 0.22µm filter and centrifuged at 100,000g for 2 hours. MEV were characterized by Nanoparticle Tracking Analysis using the ZetaView and visualized using transmission electron microscopy. For biodistribution study, MEV was stained with the lipophilic dye DiD and 60µg of stained MEV orally administered to mice and organs imaged using the IVIS 6 hours later. For chronic exposure study, 1x10¹⁰ MEV were administered orally by gavage to mice daily for at least 4 weeks. For in vitro studies, HEPG2 cell line was cultured in complete DMEM media and stimulated with 3x10⁹ MEV per well in a 6 well plate.

Results: Administration of MEV is biodistributed predominantly to the liver and led to upregulation of glucogenic genes Pepck and G6p in mice livers. MEV upregulated hepatic gluconeogenesis directly, as its addition to the HEPG2 human hepatocyte cell line led to an increase in PEPCK and G6P expression in vitro. This impact of MEV on gluconeogenesis was confirmed by pyruvate tolerance testing, with MEV‐treated animals having significantly elevated glucose levels compared to control mice. Sustained gluconeogenesis in MEV treated mice led to metabolic impairment with the development of mild insulin resistance and glucose intolerance. Interestingly, we also found that fasting, which induces gluconeogenesis, led to a significant increase in the production of extracellular vesicles by the gut microbiota.

Summary/Conclusion: While the gut microbiota can affect host metabolism, this work highlights a novel role for MEV as inducer of liver gluconeogenesis. Our work suggests the evolutionary role of the gut microbiota in supporting host glucose demands via their production of extracellular vesicles.

PT04.30. Presence of viral particles and origin of extracelluar vesicles isolated from patients with COVID‐19 and their association with clinical outcome

Msc Jaques Franco Novaes De Carvalho, Msc. Gabriela Rodrigues Barbosa, Msc Marina Malheiros Araújo Silvestrini, Dr. Sidneia Sousa Santos, Dr. Flávio Freitas, Dr. Nancy Cristina Junqueira Bellei, Dr. Andréa Teixeira de Carvalho, Dr. Ana Claudia Torrecilhas, Dr. Reinaldo Salomão

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

INTRODUCTION. Extracellular Vesicles (EVs) isolated from cells infected with the SARS‐CoV‐2 virus are still unknown, but they are known to be used as EVs in the transfer of viral components in other viruses. Our goal is to use flow cytometry to characterize EVs isolated from plasma samples from patients with COVID‐19 sepsis and confirm the presence of viral particles as well as SARS‐CoV‐2 variants. METHODS. Plasma samples were collected from patients with COVID‐19 and sepsis (n=42) admitted to Hospital Sepaco in So Paulo between 03/2021 and 08/2021, as well as from healthy individuals (n=09). We isolated EVs by UC at 100,000 x g for 16 hours and used NTA to select 20 patient samples with the highest concentrations of EVs (particles/mL) to evaluate the presence of SARS‐CoV‐2 viral particles using the RT‐qPCR kit targeting the RdRp, Envelope (E), and Nucleocapsid (N) genes. EVs, phosphatidylserine (Annexin V), and tetraspanins (CD9 and CD81) were identified using flow cytometry (CytoFlex S), as well as the cellular origins of neutrophils (CD66b), endothelial cells (CD144), and T lymphocytes (CD3), and platelets (CD42a).RESULTS. RT‐qPCR analysis revealed gene amplification in 14 of 20 patient EV samples. Two samples revealed a Gamma variant. Flow cytometry revealed that the average percentage expression of EVs labeled with Annexin V and platelet origin markers was (17.94% 6,357), followed by CD3 T lymphocytes (17.62% 5,834), CD66b neutrophils (10.42% 4,832), and CD144 endothelial cell (8.60% 2,151). There were no significant differences in each marker when comparing gender groups, differences in clinical stage, days of hospital stay, and EVs with PCR (+) versus PCR (‐).CONCLUSION. EVs have been identified as possible carriers of SARS‐CoV‐2 viral particles. Flow cytometry analysis of patients revealed a higher proportion originating from platelets. However, the evidence for the definitive role of EVs in differentiating between patient groups remains inconclusive. The images of EVs isolated by electron microscopy will be analyzed in the following steps. Financial Support: CAPES, FAPESP, CNPq.

PT04.31. Probiotic extracellular vesicles: Characterisation and unravelling the proteomic cargo of extracellular vesicles derived from Lactobacillus delbrueckii

Mr Kyle Bramich, Dr Rahul Sanwlani, Prof Suresh Mathivanan

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Probiotic species of bacteria are typically introduced into the body through various food sources and can confer a beneficial effect on the host. Over the past decade, extensive research has revealed the therapeutic potential of probiotic extracellular vesicles (EVs) in several disease contexts including cancer, ulcerative colitis, viral infections, and obesity. However, the current limitations in biogenesis knowledge, universal isolation methods, and understanding of intestinal epithelium uptake have prevented realisation of their clinical utility. To further understand how probiotic EVs can affect specific mechanisms, the proteomic contents of EV isolated from a probiotic strain was investigated.

Methods: Using combinational isolation techniques and omics approaches, we aimed to isolate and purify EVs from a probiotic bacterial strain and identify enriched protein cargo. Isolation was achieved using tangential flow filtration and differential centrifugation coupled with ultracentrifugation. Characterisation of isolated L. delbrueckii EVs was further confirmed by microscopy and proteomic analysis of EV samples was performed using mass spectrometry (LC‐MS/MS).

Results: LC‐MS/MS hits identified a total of 301 proteins in the EV samples and comparison to whole cell lysate revealed 285 common proteins between samples. Interestingly, gene ontology biological processes implicated these proteins in several biological processes such as biosynthetic processes (52.4%), nucleotide binding (34.1%), and metabolism (70.7%). Gene ontology molecular function further validated these findings, suggesting that proteins from L. delbrueckii EVs have functions as ribosome structural constituents and binding to RNA, metal ions, and nucleotides.

Conclusions: Proteomic analysis revealed the multiplicity of L. delbrueckii EV protein cargo and the associated diverse roles in various important biological processes. These findings also indicate that cargo from probiotic EVs have roles in a range of metabolic processes including, peptide and cellular amide metabolism, tRNA metabolic processes, and carboxylic acid metabolism.

PT04.32. Proteomic analysis of Olive flounder (Paralichthys olivaceus) plasma derived exosomes responses to Edwardsiella piscicida infection

Professor MAHANAMA DE ZOYSA, Mr. E.H.T. Thuslahn Jayathilaka, Mr. Mawalle Kankanamge Hasitha Madhawa Dias, Dr. Chamilani Nikapitiya Nikapitiya

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Etiological agent of the fish disease Edwardsiellosis, Edwardsiella piscicida is an important pathogen in various marine and fresh water animals and can transmitted via food chain to humans and other animals. Overuse of antimicrobials in the agriculture and aquaculture arouse multi‐drug resistance (MDR), thus prioritizing various types of vaccines against edwardsiellosis. To date, few commercial fish vaccines were successful, hence, searching of new control strategies are essential. This study aimed to investigate alterations of plasma‐derived exosomes proteins in olive flounder with experimentally challenged E. piscicida and to investigate the differentially expressed proteins (DEPs) that may responsible for diagnosing the infection.

Methods: We isolated and characterized exosomes from phosphate buffered saline (PBS) injected (PBS‐Exo) and E. piscicida challenged (Ep‐Exo) olive flounder plasma. For quantitatively identify the DEPs between two, isobaric tags for relative and absolute quantitation (iTRAQ)‐based high performance liquid chromatography‐tandem mass spectrometry analysis was conducted. The DEPs were subjected to Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analyses.

Results: Nanoparticle tracking analysis suggested higher plasma exosome concentration and lower diameter in length of exosomes are circulating in fish plasma that infected with E. piscicida compared to PBS‐injected controls, but was statistically non‐significant. Out of expressed 407 abundant proteins, up or downregulated 321 DEPs were identified. Among those, 75 were significantly upregulated while 24 were significantly downregulated (fold change >1.2 or < 0.83; q‐value <0.05) and 76 were at basal (fold change <1.2 and >0.83). Enrichment analysis indicated that E. piscicida mainly modulated the complement activation proteins and most DEPs were in complement activation pathways. STRING network analysis further confirmed most DEPs are mainly involved in innate immune responses.

Summary/Conclusion: This study revealed that E. piscicida infection possesses number of DEPs that could consider for promising biomarkers for diagnosis of E. piscicida infection in fish. Further, our findings give clues to understand host‐pathogen interaction mechanism, which may assist development of new therapeutics and drug delivery carriers in future.

PT04.33. Role of HIV‐associated extracellular vesicles in human papillomavirus (HPV) infection

Professor Ge Jin, Dr. Zhimin Feng

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Role of HIV‐associated extracellular vesicles in human papillomavirus (HPV) infection

Zhimin Feng and Ge Jin

Case Western Reserve University School of Medicine, Cleveland, OH, USA

Introduction: People living with HIV (PLWH) are more susceptible to co‐infections, including high‐risk human papillomavirus (HPV) in the oral cavity. This co‐infection increases the risk of HPV‐related oropharyngeal cancers. HIV‐associated extracellular vesicles (EVs) are implicated in various HIV comorbidities, including co‐infection and cancer. However, their role in promoting high‐risk HPV co‐infection in the oral cavity remains unclear. This study investigates the potential role of HIV‐associated EVs in facilitating HPV infection in the oral cavity.

Methods: EVs were isolated using the differential ultracentrifugation method from culture supernatants of HIV‐positive J1.1 and HIV‐negative Jurkat T cells grown in media supplemented with EV‐depleted FBS. Western blotting confirmed the presence of tetraspanin proteins. The ZetaView Twin (Particle Metrix Inc.) was used to quantify the EVs. HPV16 pseudoviruses (PsV) were prepared from HEK293TT cells transfected with the mCherry‐HPV16‐L1 and HPV16‐1 constructs. The 3‐dimentional (3‐D) oral mucosal cultures (MatTek Inc.) were used for HPV infection experiments.

Results: EVs isolated from culture supernatants of J1.1 T cells and the saliva of HIV‐infected individuals contained viral transcripts spanning the trans‐activation response element (TAR), Tat, and Nef regions of the HIV genome. We observed that HIV‐associated J1.1 EVs significantly enhanced HPV16 PsV infection in the 3D oral mucosal cultures compared to EVs from HIV‐negative T cells. HPV16 PsV only infected oral mucosal cultures with simulated microwounds, suggesting its dependence on the basal layer cells exposed through epithelial disruptions. Importantly, cetuximab, a monoclonal antibody to epidermal growth factor receptor (EGFR) for cancer treatment, blocked the pro‐infection effect of HIV‐associated EVs, suggesting EGFR's role in mediating HPV infection in the presence of HIV‐associated EVs in the oral cavity.

Summary/Conclusion: Our findings demonstrate that HIV‐associated EVs play a crucial role in promoting HPV acquisition and infection in the oral cavity of PLWH. Further research on HIV‐associated EVs and their interplay with HPV infection may lead to novel therapeutic strategies to prevent HPV infection in this vulnerable population.

Funding: This work was funded by NIH/NCI R01CA264910.

PT04.33. Role of extracellular vesicles in the pathogensis of Citrus exocortis viroid infection tomato plants

Professor Tang‐long Shen, Hao‐Yuan Chien, Ta‐Hsin Ku

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Viroids are naked, circular single‐stranded RNA molecules, about 250–400 nucleic acids in size, and do not translate any proteins. Citrus exocortis viroid (CEVd) can infect not only citrus but also other crops such as tomatoes (S. lycopersicum L. cv. Rutgers) and cause epinasty, necrosis, and stunning. Currently, known pathogenic mechanisms are mainly viroid‐derived small interfering RNAs (vd‐siRNAs) that target and cleave host messenger RNA and cause host mRNA silencing to produce symptoms. Extracellular vesicles (EVs) are lipid‐bilayer particles that play a role in communicating between cells or tissues by transporting substances such as proteins, lipids, or nucleic acids. Previous studies indicated that the host would transport small RNAs to the pathogen through EVs and cause low expression of Botrytis cinerea‐related pathogenic genes, thereby inhibiting its pathogenicity. Since viroids move through the plant and cause systemic diseases, it is important to study how naked and lacking protein capsids protect viroids in the plant. For this, we assume that EVs can act as a vector for the transport of viroids within the plant to cause systemic diseases. First, we inoculated CEVd onto tomatoes (S. lycopersicum L. cv. Rutgers), then purified the stem and leaf EVs six weeks post‐inoculation and confirmed the particle number, diameter, and structure by NTA (nanoparticle tracking analysis) and TEM. Second, we detected the viral content contained in EVs by RT‐qPCR and found that the CEVd amount of the inoculation group (CEVd‐EVs) was significantly higher than the mock group (mock‐EVs). In addition, the proteomic analysis revealed that CEVd‐EVs contain several proteins involved in metabolic and catabolic process. In order to understand whether the viroids could be transported by EVs or not and cause symptoms or stress on the plants, we re‐inoculated CEVd‐EVs and mock‐EVs on healthy tomato plants. After four weeks post‐inoculation, the biomass and leaf area decreased in the CEVd‐EVs inoculation group. These findings provide evidence that EVs could serve as a mode of transport for viroids, potentially contributing to the spread of infection. Additionally, the alterations in plant phenotype suggest that the interaction between viroids and EVs may have broader implications for plant health and disease resistance.

PT04.35. TcVPS23: A component of ESCRT‐I complex is a key factor in secretion of extracellular vesicles, endocytosis of Transferrin and act as important virulence factor in Trypanosoma cruzi experimental infection

Pos‐doctoral Nadjania Saraiva de Lira Silva, Ana Claudia Torrecilhas, Full Professor Sergio Schenkman

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: The ESCRT machinery (Endosomal Sorting Complex Required for Transport) is made up of four multi‐subunit complexes: ESCRT‐0, ESCRT‐I, ESCRT‐II, and ESCRT‐III. These complexes play a role in the transport of ubiquitinated cargoes to intraluminal vesicles that form from multivesicular bodies derived from endosomal compartments. These multivesicular bodies eventually fuse with lysosomes to degrade endocytosed cargoes or with the plasma membrane to secret intraluminal vesicles as a heterogeneous mixture of exosomes. Trypanosomatids are protozoan parasites that secrete extracellular vesicles and rely on endocytosis for survival. However, the EV biogenetic pathways in Trypanosoma cruzi are almost completely unknown, and no ESCRT proteins have been identified, despite their predicted existence.

Methods: We used CRISPR/Cas9 to investigate the ESCRT‐I complex, specifically the TcVPS23 protein, for its role in extracellular vesicle (EV) formation in T. cruzi trypomastigotes and epimastigotes. TcVPS23 emerged as a critical virulence factor in T. cruzi, correlating with other pathogen findings. We also performed Nanoparticle Tracking Analysis (NTA), detected the specific markers such as trans‐sialidase (TS), and functional assays (in vitro and in vivo).

Results: TcVPS23 mRNA was found to be more abundant in trypomastigotes than in epimastigotes, indicating that this system is fully functional in Kinetoplastida and can act as a virulence factor in T. cruzi. The partial deletion of the TcVPS23 gene reduced Transferrin uptake but not BSA endocytosis. TcVPS23 has also been shown to be important for the secretion of epimastigotes and trypomastigotes‐derived EV, as well as the expression of trans‐sialidase on the surface of trypomastigotes. Lower TcVPS23 expression reduced the parasite's ability to invade cells and increased the intracellular amastigotes in L6 cells at 48, 72, and 96h. TcVPS23 mutant was inoculated in vivo showed decreased parasitemia and mast cell recruitment in the heart, and it increased the percentage of infected mice that survived.

Summary/Conclusion In conclusion, these findings demonstrate the importance of TcVPS23 in EV secretion, endocytic modulation, and performance as an important virulence factor in T. cruzi, highlighting the ESCRT protein as a promising candidate for vaccine development against Chagas disease

PT04.37. The Helicobacter pylori autotransporter ImaA associates with extracellular vesicles to promote host inflammatory responses in gastric epithelial cells

Mr Angus Cramond, Ms Nina Colon, Mr Jack Emery, Dr Dongmei Tong, Dr Caroline Skene, Professor Richard L. Ferrero

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Helicobacter pylori produces several autotransporter proteins that use a conserved beta barrel to transport passenger domains through the bacterial membrane. Each H. pylori autotransporter protein has a distinct passenger domain; except for the vacuolating cytotoxin, VacA, these autotransporters remain largely unstudied. Previous work on the H. pylori autotransporter ImaA found that imaA knockout bacteria induce stronger IL 8 responses in gastric epithelial cells (AGS), suggesting that ImaA may be an immunomodulatory protein. Proteomic studies showed that H. pylori ImaA associates with extracellular vesicles (EVs). Thus, we hypothesised that EVs containing ImaA may be able to modulate host responses. Methods: We generated H. pylori imaA mutant bacteria, then isolated EVs from these and the corresponding wild‐type (WT) bacteria by ultracentrifugation. EVs were characterised by Nanoparticle tracking analysis (NTA). Interleukin‐8 (IL‐8) responses of AGS gastric epithelial cells to EVs were determined by ELISA. To investigate the immunomodulatory effects of ImaA in vivo, we stimulated splenocytes from mice that had been vaccinated on days 0 and 28 with H. pylori WT or imaA EVs, or PBS, then euthanised on day 56. Results: NTA showed that H. pylori imaA and WT EVs have similar particle sizes (median sizes 95 and 100 nm, respectively). imaA EVs induced weaker IL 8 responses than WT EVs (3.7‐ and 1.1‐fold change from unstimulated for WT and imaA EVs, respectively, p=0.0043). Splenocytes from mice that were vaccinated with either H. pylori WT or imaA EVs ex vivo showed reduced IFN γ and IL‐17 responses to ConA stimulation ex vivo, when compared with splenocytes from unvaccinated mice, suggesting EVs suppress Th1 and Th17 responses. Summary/Conclusions: Taken together, we propose that H. pylori ImaA contributes to the pro‐inflammatory effects of EVs on epithelial cells. Further work is required to understand the role of EV‐associated ImaA in vivo.

PT04.38. The Helicobacter pylori virulence factor, Tipa, is carried by bacterial extracellular vesicles to the nuclear compartment of host cells

Mr Jack Emery, Doctor Variya (Way) Nemidkanam, Ms Nina Colon, Ms Kate Friesen, Ms Georgie‐Wray McCann, Associate Professor David McGee, Doctor Natalia Castaño‐Rodríguez, Doctor Dongmei Tong, Doctor Caroline Skene, Doctor Laurent Terradot, Professor Richard L. Ferrero

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: The bacterium Helicobacter pylori tightly regulates the host immune response to modulate inflammation, but if dysregulated, this leads to cancer. H. pylori tumour necrosis factor‐α‐inducing protein (Tipα) was reported to promote tumour necrosis factor (TNF) responses and carcinogenesis. Although Tipα is secreted in a soluble form by the bacterium, it is also packaged with bacterial extracellular vesicles (EVs). We hypothesised that H. pylori EVs carry Tipα to the nucleus, resulting in perturbed nuclear processes that promote carcinogenesis.

Methods: H. pylori isogenic tipA and tipA/tipA+ mutant strains were generated and characterised by NTA and Western blotting. EVs isolated from H. pylori wild‐type (WT) and mutant bacteria were incubated with gastric epithelial cells (AGS) and THP‐1‐derived macrophages. Intracellular localisation of EV‐associated Tipα was determined by cell fraction and confocal microscopy. Proinflammatory cytokine responses were determined by ELISA.

Results: We confirmed H. pylori EVs harbour Tipα and that WT and mutant EVs shared similar particle numbers and median sizes. Also, the amounts of Tipα secreted into culture supernatants varied between H. pylori strains but was not associated with disease outcome. EV‐associated Tipα was present within the cytoplasm of AGS cells at 4‐hours post‐treatment and accumulated in the nuclear compartment by 18 hours. Consistent with previous work, the recombinant form of Tipα induced TNF production in THP‐1 cells. Similarly, tipA EVs induced significantly more TNF (p = 0.0007) and interleukin‐8 (IL‐8; p = 0.0127) than WT or tipA/tipA+ EVs in THP‐1 cells. Interestingly, however, tipA EVs induced significantly lower levels of IL‐8 than those from WT or tipA/tipA+ EVs in AGS cells (p = 0.0402).

Summary/conclusion: Taken together, we propose that EV‐associated Tipα is an immunoregulatory virulence factor which targets the nuclear compartment of host cells to promote gastric carcinogenesis.

PT04.39. The impact of maternally‐derived gut bacterial extracellular vesicles on the offspring's developing immune system

Miss Jemma Taitz, Mr Jian Tan, Mr Duan Ni, Mr Georges Grau, Mr Nicholas King, Mr Ralph Nanan, Ms Laurence Macia

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

The gut microbiota is established at birth and has a profound impact on host immunity. The stage before weaning presents a critical window where exposure to microbiota components is essential for proper immune development. It is less known, however, how exposure to maternal gut microbiota components may affect immune development at an earlier stage in utero. Bacterial extracellular vesicles (BEVs) produced by gut microbiota are novel players in the microbiota‐host relationship, however, their role, particularly during pregnancy, remains largely unexplored.

Methods

C57BL6 mice were orally administered 60ug of gut‐derived BEVs during pregnancy from E0 to E19 (PBS administered as control). Offspring were transferred to untreated foster dams at birth and culled prior to weaning at D21.

Results

Bodyweight and liver weight of BEV‐exposed offspring was significantly increased compared to offspring of PBS mice, suggesting a potential impact on offspring's metabolism. BEV‐exposed offspring had increased spleen and mesenteric lymph node cellularity, while thymus and bone marrow cellularity were unaffected, suggesting BEV exposure in utero may affect secondary lymphoid organ development. By spectral cytometry, we observed significantly increased proportions of activated CD8 and CD4 T cells in secondary lymphoid organs in the BEV‐exposed mice but did not detect changes in thymocyte profiles, suggesting BEV exposure in pregnancy does not impact offspring lymphopoiesis but rather may affect lymphocytes in the periphery. We identified that in vitro, BEVs induce induced T cell proliferation equally in both groups, suggesting BEV exposure in utero does not induce‐antigen specific proliferation.

Summary:

Our findings suggest that in utero exposure to gut derived‐BEVs alters T cells located in secondary lymphoid tissues. While the mechanism and receptors involved remain to be elucidated, this work highlights the role of MEV on the developing immune system in utero.

PT04.40. The role of Neisseria gonorrhoeae outer membrane vesicles in inducing trained immunity in Macrophages

Dr Jiaru Yang, Dr. Seong Hoong Chow, Dr. Pankaj Deo, Associate Professor Thomas Naderer

Poster Pitches (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:45 PM ‐ 1:00 PM

Introduction

Trained immunity refers to innate immune cells showing increased immune response to pathogens after stimulation with nonlethal concentration of bacterial molecules or pathogens. Gonorrhoea is sexually transmitted disease caused by Neisseria gonorrhoeae. In the absence of new antibiotics, developing a gonorrhoea vaccine is a pressing need. Outer membrane vesicles (OMVs) are a promising vaccine platform. The meningococcal vaccine (4CMenB) that is based on OMVs elicits some protection (∼30%) against Neisseria gonorrhoeae infections in humans and animal models using vagin*l challenge. Current efforts are aiming at identifying the proteins that confer cross‐protection against N. gonorrhoeae to develop a more specific OMV vaccine. How OMVs activate the innate immune system that leads to protective immunity and whether OMVs in general induce trained immunity remains unclear. We hypothesise that N. gonorrhoea OMVs induce trained immunity in monocytes and macrophages, which can be explored to develop a N. gonorrhoea vaccine.

Methods

We isolated OMVs from N. gonorrhoeae and trained human monocytes for 24 hours. Trained monocytes were differentiated to macrophages and challenged with N. gonorrhoeae, Candida albicans, E. coli or with purified molecules, such as LPS. We then measured immune responses, such as TNF‐α and IL‐6 secretion, and programmed cell death signalling in trained and control macrophages. Inhibitors that target histone deacetylases (TSA, Butyrate sodium, LMK235, RGFP966 and Tubastatin), pro‐apoptotic factors (BCL‐2 and BCL‐xL inhibitor), NLRP3 inflammasome (MCC950) and caspases (QVD) were included during the training to identify host pathways that promote immunity.

Results

OMV‐trained monocytes and macrophages produced higher concentrations of IL‐6 and TNF‐α when restimulated with N. gonorrhoeae, E. coli and LPS compared to the untrained cells. Trained cells display higher survival rate when they encounter restimulations compare to the untrained cells. Inhibitors targeting histone deacetylases, cell death signalling and NLRP3, impaired trained immunity as determined by IL‐6 and TNF‐α secretion and cells survival rate.

Conclusions

OMVs derived from N. gonorrhoeae activate trained immunity in human monocytes. OMV‐dependent training depends on histone acetylation and is regulated by the apoptosis, NLRP3 and caspase activity. We will discuss how OMV‐dependent training of innate immune cells confers protection against various pathogens.

PT04.43. Using circulating bacterial outer membrane vesicles to diagnose bacterial infections

Phd Qianbei Li, Professor Lei Zheng

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Bacterial infections pose a significant global public health concern, leading to sepsis and lethal organ dysfunctions. Traditional diagnostic approaches, however, are time‐consuming and suffer from low positivity rates, thus impeding timely treatment for bacterial infections. In essence, this stems from our limited understanding of the pathogenic mechanisms behind bacterial infections and the absence of more precise markers. Bacterial outer membrane vesicles (OMVs) serve as critical intermediaries for bacterial‐host communications, yet their roles and clinical significance in bacterial infections remain largely uncharted. Herein, we introduce a novel OMV‐specific labeling technique predicated on the selective recognition of bacteria by antibiotics, based on witch enable to distinguish and quantify OMVs from other sources of EVs when coupled with a Flow Nanoanalyzer. Leveraging this methodology, we discerned markedly elevated OMV levels in the blood samples of bacterial infection patients, particularly those with bloodstream infections (BSI). Moreover, we found that bacteria released OMVs from infection foci into the bloodstream, which subsequently instigated mitochondrial damage and triggered apoptosis in mouse macrophages. This study furnishes a pioneering quantitative approach for the specificity analysis of OMVs and unveils their diagnostic and pathogenic potential in bacterial infections.

PT04.45. Foam cell‐derived extracellular vesicles regulate the environment surrounding atherosclerotic plaques

Foam cell‐derived extracellular vesicles regulate the environment surrounding atherosclerotic plaques Akihiko Okamura¹, Dr. Yusuke Yoshioka¹, Shungo Hikoso², Takahiro Ochiya¹

¹Department of Molecular and Cellular Medicine, Tokyo Medical University, 6‐7‐1 Nishishinjuku, Shinjuku‐ku, Japan, ²Department of Cardiovascular Medicine, Nara Medical University, 840 Shijo‐cho, Kashihara, Japan

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Ischemic heart disease (IHD) is a leading cause of death worldwide, and it is essential to understand the molecular mechanisms of plaque progression and rupture to prevent the incidence of IHD. This study aims to investigate the effect of extracellular vesicles (EVs) derived from foam cells (FCs), which are considered to be the main component of plaque, on the cells surrounding the plaque.

Methods

Human monocyte‐derived cell lines differentiated into macrophage‐like cells and smooth muscle cells (SMCs) were loaded with enzyme‐modified non‐oxidizing LDL for 96 hours to form FCs. Each FC was cultured in serum‐free medium for 48 hours, and EVs were purified from the culture supernatant by ultracentrifugation. FC‐derived EVs (FC‐EVs) were analyzed for particle size and number by NTA, and their components were also analyzed for lipidome. FC‐EVs were added to macrophages, SMCs, and endothelial cells (ECs), and the expression levels of atherosclerosis‐related genes were analyzed by qPCR. Cell migration and proliferation were also measured by the live cell imaging system. Macrophages and SMCs treated with FC‐EVs were loaded with LDL, and the viability of the cells and the amount of lipid uptake were evaluated. In addition, FC‐EV‐treated ECs and macrophages were co‐cultured to evaluate the adhesive ability of macrophages to ECs.

Results

FC‐EV‐treated ECs upregulated the expression of genes related to intercellular adhesion ability and increased the number of macrophages adhering to ECs. FC‐EVs internalized more lipids than non‐FC‐EVs, and their lipid composition was influenced by the lipid profile of LDL. FC‐EV‐treated SMCs increased cell migration capacity and enhanced lipid uptake. Furthermore, they were less resistant to the cytotoxic effects of LDL and induced more cell death compared to EV‐untreated SMCs. FC‐EV‐treated macrophages increased migration capacity. All effects were more significant in the group of macrophage‐derived FC‐EVs added.

Conclusions

This study suggests that FC‐EVs may increase plaque volume by accumulating macrophages and SMCs in the surrounding plaque. Furthermore, increased lipid uptake and induction of cell death were observed, suggesting that FC‐EVs may accelerate atherosclerosis by functioning surrounding the plaque. We are investigating the effects of FC‐EV treatment on atherosclerosis model mice.

PT04.46. Hypericum perforatum‐derived exosomes‐like nanovesicles‐based photosensitizer for photodynamic therapy in adipose tissues

Professor Jianbo Wu¹, Dr. Ziyu Li

¹Southwest Medical University, Luzhou, China

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Recent investigations underscore the capacity of photodynamic therapy (PDT) to induce adipocyte apoptosis, thereby mitigating obesity. Nonetheless, extant synthetic photosensitizers manifest limitations that hinder their clinical viability.

Methods: In the current study, we used Hypericum perforatum‐derived exosomes‐like nanovesicles (HPExos) as a novel photosensitizer, and investigated its PDT effects in adipose tissue during obesity. HPExos‐were administered to high fat diet mice via intraperitoneal injection, followed by targeted irradiation with specialized LED lights.

Resutls: We here show that HPExos combined with PDT accumulated in visceral white adipose tissues results in a reduced body weight and improved insulin sensitivity. HPExos combined with PDT induced apoptosis by driving high levels of ROS. In addition, HPExos combined with PDT significantly downregulated the expression of transcription factors, PPARγ, C/EBPα, and SREBP and lipogenesis protein FABP4 both in vitro and in vivo, associated with a decreased FFA levels. Conclusion: These findings suggest that HPExos could act as an effective photosensitizer in the regulation of glucose hemostasis through inhibiting adipocyte differentiation and lipogenesis in the treatment of obesity.

PT05.03. Characterising soluble TREM2 in extracellular vesicles derived from cultured human macrophages

Miss Drishya Mainali, Mr Anjie Ge, Dr Monokesh Sen, Miss Yvonne A. Candia, Dr. Claire Goldsbury, A/Prof. Laura Piccio

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Triggering receptor expressed on myeloid cells 2 (TREM2) is an important signalling mediator for central nervous system immune modulation and repair. Ectodomain shedding of TREM2 results in the formation of soluble TREM2. sTREM2 is found elevated in the cerebrospinal fluid (CSF) of people with neuroinflammatory conditions in comparison to healthy controls (Piccio etal., 2008). However, the function of soluble TREM2 beyond its biomarker potential remains unclear to date. Extracellular vesicles (EVs) play a vital role in cell‐to‐cell communication, potentially modulating pathological events in recipient cells. The overall hypothesis of this study is that TREM2/sTREM2 is localised to myeloid cell derived extracellular vesicles (EVs) and could mediate its function.

Methods: PBMC from healthy controls were cultured for seven days with 5% autologous human serum. The cultured supernatant was spun serially at 1000g‐force and at 20,000 g‐force for 20mins at 4C. The supernatant was ultracentrifuged at 18,000 g‐force to isolate large EVs and again at 100, 000 g‐force to isolate small EVs. ELISA measured sTREM2 concentrations in the supernatant after the 18k and 100k runs. The EVs pellets were digested with 1:30 trypsin to protein ration and analysed with LC/TMS under data‐independent acquisition (DIA). The EVs were characterised via Nanoparticle Tracking Analysis (Zetaview) after cell‐mask green (CMG) labelling of the veiscles and negative stain transmission electron microscopy (TEM).

Results: We observed a significant depletion of sTREM2 by ELISA in the cell culture supernatant post EV isolation, indicating the presence of sTREM2 in the EV pellet. sTREM2 fragment (VLVEVLADPLDHR) was found in both large and small EVs as well as the supernatant. CMG labelling showed less concentration of EV in the sample (almost by a factor 1000x) in comparison to unlabelled particle analysis. Negative stain TEM confirmed the presence of EVs.

Summary: This study indicates a potential association between sTREM2 and extracellular vesicles for the first time. However, the presence of sTREM2 in the EVs needs further investigation to confirm this association. The next step will be to examine sTREM2 abundance in the plasma of individuals with Multiple Sclerosis compared to healthy controls.

PT05.04. CRISPR/Cas9, genome editing and EVs: Exogenous bacterial Cas9 expression alters small EV secretion and their protein cargo in p53 dependent manner

Professor Suresh Mathivanan

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

CRISPR‐Cas9 is a widely utilised genome editing technique that has provided an efficient means to study the function of genes in various cells and tissues. Despite this utility, there is currently a limited understanding of the impact of Cas9 expression in mammalian cells, particularly in the biogenesis and secretion of small extracellular vesicles (sEVs). Here, we investigated the changes in the protein cargo and secretion of sEVs upon expression of SpCas9 in human colorectal carcinoma HCT116 and human embryonic kidney HEK293 cells.

Methods:

A panel of cells with varying P53 status were utilised. sEVs were isolated by ultracentrifugation and characterised by Western blotting, EM, NTA and quantitative proteomics. Human and murine cancer cells releasing EVs were implanted in immune‐compromised (nude) and immune‐competent (C57BL/6) mice, respectively.

Results:

Expression of Cas9 impeded the proliferation of HCT116 cells and increased the abundance of p53 and its downstream target p21. Cas9 expression also increased the abundance of EV‐enriched proteins CD63 and CD9 in HCT116 cells and further accelerated the secretion of sEVs. Subsequent label‐free quantitative proteomics analysis of whole cell lysates highlighted small but noticeable alterations in the abundance of the few proteins upon expression of Cas9. In contrast, proteomic analysis of sEVs revealed a cell type‐dependent differential abundance of several proteins. Knockout of P53 impacted the phenotype of sEVs.

Conclusion:

Overall, the study highlighted the cell‐type dependent alterations in the protein cargo and secretion of sEVs upon expression of Cas9.

PT05.05. Cytoplasmic DNA accumulation upon the inhibition of small extracellular vesicles secretion induces cGAS and ULBP1 activation in acute myeloid leukemia blasts

Dr. Jamal Ghanam, Dr. Venkatesh Kumar Chetty, Prof. Dr. Dirk Reinhardt, PD. Dr. Basant Kumar Thakur

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Cancer cells secrete small extracellular DNA (sEVs) with higher DNA (EV‐DNA) content than their normal counterparts. However, the question remains: how is EV‐DNA selected for secretion in sEVs, and under which circ*mstances? Here, we studied the implication of inhibiting sEVs secretion on cytoplasmic DNA (cyDNA) accumulation, DNA sensing activation, and cancer immunogenicity in acute myeloid leukemia (AML).

Methods

To inhibit sEVs secretion, 2D and 3D AML cultures were treated with the farnesyltransferase (FT) and sphingomyelinase (SP) inhibitors Manomycin A and GW4869 in the presence or absence of DNA damage‐inducing agents Cytarabine (Ara‐c) or hydroxyurea (HU). Rab27a‐knockout cells were also cultured under the same conditions. sEVs isolation was done based on size exclusion chromatography, and characterized according to MISEV2018 guidelines. cyDNA was characterized by dsDNA staining and quantification after subcellular fractionation.

Results

sEVs secretion was considerably suppressed by using the combination of FT and SP inhibitors similar to Rab27a‐KO, as seen by western blot and beads‐assisted FACS analyses of sEVs markers. The relative EV‐DNA concentration has significantly increased in sEVs isolated from cells treated with Ara‐c and HU. Next, we observed a substantial accumulation of cyDNA with concomitant activation of DNA sensor cGAS and a significant increase in IRF3‐S386 phosphorylation in cells. A subtle increase in pIRF3 (S396) nuclear foci has been observed when combining FT and SP inhibitors and Ara‐c treatment. Analysis of DNA damage markers revealed a specific increase in ɣH2A.X nuclear foci and a high expression of the exonuclease TREX1. In AML cells, expression of surface ligands for natural killer (NK) cells receptor (NKG2D‐L) such as ULBPs family is upregulated upon cellular stress, including DNA damage. AML cells were stained for ULBP1 expression, which was significantly higher under FT and SP inhibitors and Ara‐c treatment. This has significantly increased NK cells toxicity against AML blasts.

Conclusion

The inhibition of sEVs secretion has resulted in a significant accumulation of cyDNA, enhancing the anti‐tumor activity of NK cells against AML blasts. EV secretion appears to be another mechanism evolved by cells to prevent cyDNA accumulation. Therefore, targeting cytoplasmic DNA excretion via sEVs opens up new possibilities for cancer treatment.

PT05.06. Defining the parameters for sorting of RNA cargo into extracellular vesicles

Associate Professor Mona Batish, Graduate Student Ahmed Abdelgawad, Assistant Professor Vijay Parashar

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: There is mounting evidence that different RNA species are packaged into extracellular vesicles (EVs). Recent work has shown that not all RNAs are equally likely to be packaged into EVs but the process of sorting and selection of RNA cargo to be preferentially packaged into EVs is not completely understood. Here, we explored the role of inherent features including RNA length, GC content, secondary structure, number of exons and presence of consensus motifs that can be attributed for preferential enrichment of RNAs into EVs.

Methods: We utilized RNA sequencing on RNA isolated from DLD‐1 cells to determine the RNAs enriched in EVs. Breifly, EVs were isolated and characterized as per MISEV guidelines. Total RNA was isolated from these EVs and cells and sent for RNA sequencing. The RNA candidates were identified by mapping to human reference genome. The raw data was analyzed for differential enrichment of different RNA sub types into EVs.

Results: We found a significant correlation between the length of RNA, GC content and structuredness to its enrichment into EVs while the number of exons did not show any effect on its enrichment. We also found presence of GC‐rich novel motif specifically in some EV bound RNA species.

Conclusions: These results indicate that the RNAs physical attributes govern its eventual fate for packaging into EVs and extracellular transport. Thoroughly validation of these features could pave way for better engineering of RNA cargo into EVs.

PT05.07. Effects of electromagnetic fields on the release and content of extracellular vesicles

Hailong Wang, Research Associate YiHua Wang, Professor Gregory Worrell

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

1) Introduction:

Extracellular vesicles (EVs) are small membrane‐bound structures that originate from various cell types and carry molecular cargo to influence the behavior of recipient cells. The use of EVs as biomarkers and delivery vehicles for diagnosis and treatment in a wide range of human disease is a rapidly growing field of research and clinical practice. Four years ago, we postulated the hypothesis that electromagnetic field (EMF) can influence the release and content of EVs.

Since then, we have optimized several technical aspects of our experimental setup.

2) Methods:

We used a bioreactor system that allows cells to grow in a three‐dimensional environment mimicking in‐vivo conditions. We designed a custom‐made EMF stimulation device that encompasses the bioreactor and delivers uniform EMFs. We established a three‐step EV purification protocol that enables high‐density production of EVs. We then performed mass spectrometry‐based proteomics analysis on EV‐related proteins and used high‐resolution nanoparticle flowcytometry for single‐vesicle analysis.

3) Results:

We demonstrated that electrical stimulations of current amplitudes at physiological level that are currently applied in therapeutic deep brain stimulation can modulate EV content in a frequency‐dependent manner, which may have important implications for basic biology and medical applications.

4) Conclusion:

First, it raises intriguing questions about how the endogenous electrical activity of neuronal and other cellular assemblies influence the production and composition of EVs. Second, it reveals an additional underlying mechanism of how therapeutic electrical stimulations can modulate EVs and treat human brain disorders. Third, it provides a novel approach of utilizing electrical stimulations in generating specific EV cargos.

PT05.10. Establishment of a method for mass production of extracellular vesicles based on microRNA regulation

Dr. Tomofumi Yamamoto, Dr. Hirotaka NIshimura, Dr. Noritaka Hashii, Dr. Akiko Ishii‐Watabe, Dr. Yusuke Yamamoto, Prof. Takahiro Ochiya

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

In recent years, therapeutic application of extracellular vesicles (EVs) has become active in the fields of cancer, inflammatory diseases, and regenerative medicine, and clinical trials on EVs derived from mesenchymal stem cells (MSCs) are also being conducted around the world. However, one of the issues that must be resolved in applying EVs as biological medicines for more general indications is establishing a manufacturing method that can stably produce EVs. Therefore, we attempted to establish a new method that highly promotes the secretion of high‐quality EVs from mesenchymal stem cells.

Methods

As reported previously (Urabe F, etal., Sci. Adv., 2020), miRNAs regulating EV were screened. ExoScreen method, a high‐sensitive EV detection system, was used for this study. Combined with miRNA‐mimic library, we identified miRNAs, which enhanced the EV secretion in many types of normal cells including MSCs. The microarray and in silico analysis were performed to examine the direct targets of the specific miRNA. Anti‐inflammatory effect of EVs was also investigated in a LPS‐stimulating inflammatory model.

Results

The screening results showed that the production of EVs from miR‐3202 transfected cells was found to be 1.5‐ to 4‐fold higher than that of the control. Nanoparticle tracking analysis showed no significant difference in particle size. In addition, ABCA1 was identified as a direct target of miR‐3202. Attenuating the ABCA1 with siRNAs and the treatment of the ABCA1 inhibitors promoted EV secretion 1.5‐ to 3.5‐fold higher than each control. These results suggested that an miR‐3202/ABCA1 axis facilitated the EV secretion in MSCs. Next, we performed functional assays of miR‐3202‐induced MSC‐derived EVs. MSC‐derived EVs usually have anti‐inflammatory effects, so we used LPS‐stimulating inflammatory model to investigate the biological function of EVs produced from miR‐3202 treated MSCs. As a result, the effect of miR‐3202‐induced MSC‐derived EVs on anti‐inflammation was comparable to that of control MSC‐derived EVs as long as the same number of particles were treated. This data indicates that the EVs induced by miR‐3202 are functional.

Conclusions

We found that miR‐3202 enhanced EV secretion by reduction of ABCA1 expression in MSCs, and miR‐3202‐induced MSC‐derived EVs had anti‐inflammatory effects.

PT05.12. Exploiting the human peptidome for discovery of EV release‐modulating agents

Dr. Ruediger Gross, Ms. Hanna Reßin, Mr. Nico Preißing, Dr. Ludger Ständker, Prof. Dr. Jan Münch

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Despite advances in the understanding of the molecular biology of EV biogenesis and release, details on how these processes are regulated are limited. Consequently, few agents with potent/selective EV‐release modulating activity are known. Considering known functions of EVs in numerous infectious, oncological and neurodegenerative conditions but also the emerging use of EV biotechnology for drug delivery, agents inhibiting or boosting EV release, respectively, would be of great interest. We here explored the human peptidome, containing millions of compounds with enormous structural and functional diversity, to identify such agents endogenously present in humans.

Methods

1000 liters of human hemofiltrate (HF) was used to prepare a peptide library by cation‐exchange chromatography followed by reverse‐phase chromatography. The resulting ∼ 500 fractions contain the entire human blood peptidome and were screened for modulating EV release using a HEK293T CD63‐Tluc reporter cell line. 48h after addition of fractions, luciferase activity was measured in the supernatant (EV release) and in lysates (retained EVs/EV biogenesis) and the quotient calculated as the EV release factor. Cytotoxic effects were evaluated by ATP levels. Fractions were ranked for magnitude of release/biogenesis modulation and compared to controls and hit fractions evaluated for dose‐dependency.

Results

In the initial screening, EV release‐boosting activity was observed for several neighboring fractions. In fractions with most pronounced effects, quantification suggested stimulation of EV release without changes in biogenesis (4.2‐fold increase in supernatant‐, 5‐fold decrease cellular luciferase activity, EV release factor 20). Distinct eluates also exerted inhibitory effects on both cellular and release EV simultaneously, hinting at a general biogenesis blockade (4‐fold reduced supernatant‐, 2‐fold reduced cellular luciferase levels, release factor 0.5). For these primary hit fractions, effects were dose‐dependent and exerted at non‐cytotoxic concentrations.

Summary/Conclusion

These results offer first insights into the diversity of EV‐modulating agents to be discovered in the human peptidome. Following further rounds of chromatography, the responsible peptide(s) will be identified using mass spectrometry and chemically synthesized to confirm the activity. In addition to potential therapeutic and biotechnological applications of such EV‐release modulating peptides, identifying their endogenous role will offer new insights into how EV‐release is physiologically fine‐tuned.

PT05.13. Gene copy numbers dictate extracellular vesicle cargo

Sumeet Poudel, Jerilyn Izac, Zhiyong He, Lili Wang

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Extracellular vesicles (EVs) are nanometer‐sized vesicles that carry heterogeneous cellular components including proteins, nucleic acids, lipids, and metabolites. The intravesicular and surface contents of EVs are a goldmine of biomarkers and have both physiological and pathological importance. Because of the heterogeneous cargo, EVs can act as mediators of local and distal cell‐cell communication that could potentially trigger cancer metastasis, for example. However, how the genome signature regulates EV cargo and an understanding of the function is not well studied. This study aims to understand how genetics impact EV cargo loading.

Methods: EVs were extracted from cultures of the vector copy number cell lines with a fluorescent reporter (green fluorescent protein, GFP) ranging from 0 – 4 copies. Extracted EVs were characterized by Spectradyne nCS1 for count and size distribution. Imaging flow cytometry was used to analyze the fluorescent intensity for GFP+ EVs and their counts from the total EVs (CD9+ EVs). RNA content of the EVs was also compared among the copy number cells along with the total RNA by ddPCR. In addition, naturally occurring copy number cells are also being analyzed to understand the role of copy numbers for EV cargo loading.

Results: Preliminary results suggest that the gene copy number dictates the protein cargo of the EVs but not the RNA content. Increasing copies of reporter GFP in genetic loci correlated with increasing count of GFP+ EV intensity and count with flow cytometry.

Conclusion: It is important to understand the role of copy numbers in EV cargo. It is especially important to understand its role in cell and gene therapy space because as an effective measure to reduce the genotoxic and tumorigenic potential caused by uncontrolled integration, the US Food and Drug Administration recommends the integrated vector copy number (VCN) must be less than 5 copies per cell. Other studies are also being conducted to understand if gene copy number plays a role in cargo loading in naturally occurring or pathological states.

PT05.16. Identification of cis‐regulatory elements involved in exosomal cargo sorting

Phd Student Gizaw Gebeyehu, Research Professor Tibor Rauch, Associate Professor Marianna Pap, Dr Geza Makkai, Dr Tibor Janosi

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Sepsis remains one of the most serious diseases with the greatest health and economic impact on societies around the world. In the context of sepsis, circulating exosome‐transported mRNAs and miRNAs can regulate the expression of immune response genes of the recipient cells. The details of exosome‐based cell‐to‐cell communication are not well known including how mRNAs are recruited and packaged into the exosomes and other extracellular vesicles we aimed to explore molecules and mechanisms related to exosomal cargo (e.g. mRNA and miRNA) loading under well‐defined conditions in cell cultures. We studied exosomal LPS‐treatment‐related RNA profile changes of THP‐1 cells and identified RNA sequences that might be implicated in cargo sorting and loading.

THP‐1 (human monocyte) cell line was treated with LPS and then exosomes were purified from the supernatant by multiple‐step ultra‐centrifugation. Total RNA was isolated from exosomal fractions using a TRISOL‐based method and subsequently sequenced by next‐generation sequencing (RNA‐Seq). The MEME program analyzed the most abundant exosomal mRNAs (i.e., the top 50 mRNAs) at their UTRs. Conventional and NEBuilder‐based cloning methodologies were employed to generate reporter gene constructs to verify the functional relevance of the MEME program‐indicated conserved sequences in UTRs. Cells were transfected by these reporter gene constructs and RNA content of exosomal fractions was monitored by qPCR.

We identified a highly conserved sequence element among the most abundant exosomal mRNAs. We have constructed Reporter genes that are suitable for investigating the function of this exosome‐associated RNA motif. Transfection studies with the reporter gene constructs are ongoing. These experiments may lead to insights into the RNA motif, protein factors, and mechanisms involved in exosomal packaging in the context of sepsis.

PT05.17. Lipid nanoparticles (LNPs) alter transcriptomic contents of extracellular vesicles (EVs) leading to functional LNP‐mRNA repackaging into EVs

Benyapa Tangruksa^{1. Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg,Gothenburg 41346, Sweden 2. Systems Biology Research Center, School of Bioscience, University of Skövde, Skövde SE‐54128, Sweden}, Doctor Muhammad Nawaz^{1. Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg 41346,Sweden}, Adjunct Professor Sepideh Heydarkhan‐Hagvall^{2. Systems Biology Research Center, School of Bioscience, University of Skövde, SkövdeSE‐54128, Sweden}, Professor Jane Synnergren^{2. Systems Biology Research Center, School of Bioscience, University of Skövde, Skövde SE‐54128, Sweden 3.Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg 41345, Sweden}, Associate Professor Hadi Valadi^{1. Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg,Gothenburg 41346, Sweden}

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction:

Lipid nanoparticles (LNPs) are widely used as effective vehicles for delivering functional mRNA, for example, in COVID‐19 mRNA vaccines. Previously, it was shown that after LNPs and its mRNA (e.g. EPO mRNA) are uptaken by cells, a fraction of the LNP‐mRNA is repacked into the cells’ own vehicles, extracellular vesicles (EVs). This process facilitates the transport of the LNP‐mRNA between cells through EVs. One emerging strategy for cardiovascular disease treatment is delivering angiogenic molecules, such as VEGF‐A mRNA, to stimulate blood vessel formation. In this study, we investigated how LNP containing VEGF‐A mRNA affects the transcriptomic content and functions of EVs.

Methods:

LNPs with encapsulated VEGF‐A mRNA were administered to lung epithelial, umbilical cord endothelial, and cardiac progenitor cells. The EVs from these cells were isolated, and their transcriptomic profiles were defined using RNA‐Seq analysis. Using either LNPs or the three EV types, equal amount of VEGF mRNA was administered to recipient cells in vitro, or in vivo via intracardiac injection. The heart tissues at and around the injected area were examined.

Results:

Following LNP administration to cells, secreted EVs contained a fraction of internalised VEGF‐A mRNA. A significant amount of the VEGF‐A mRNA was detectable in EVs from all three cell types. RNA‐Seq results demonstrate that cells respond to LNP treatment, and the RNA contents of EVs are altered, resulting in over 1000 dysregulated genes. Several pro‐angiogenic transcripts were overexpressed in EVs after LNP treatment. Additionally, EVs deliver functional VEGF‐A mRNA in vitro and in vivo, leading to protein production. Cardiac progenitor EVs promote the most angiogenesis during in vitro VEGF‐A delivery compared to other EVs. Intracardiac injections of VEGF‐A mRNA into mouse hearts produce local VEGF‐A protein without spillover to other organs. EVs from cardiac progenitor cells demonstrate the least inflammatory cytokine production in cardiac tissue compared to other EVs and LNPs.

Conclusions:

LNP treatment alters the transcriptomic contents of secreted EVs. LNPs use EVs as functional extensions to transport therapeutic mRNA between cells, but EVs do it differently by delivering a mixture of mRNAs, including the mRNA from LNPs.

PT05.18. Metalloprotease ADAM10 in secretion, composition, and targeting of extracellular vesicles

MSc Christopher C. Reimann, MSc Feizhi Song, Dr. rer. nat. Dipl. Hum.‐Biol. Hermann C. Altmeppen, Dr. Lesley Cheng, Prof. Markus Glatzel, Prof. Marina Mikhaylova, Prof. Andrew F. Hill

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Proteases are a mostly unexplored factor in the biology of extracellular vesicles (EVs). Their cellular presence may determine protein sorting into and release via EVs, while their continuous activity at the EV surface towards membrane proteins may alter the specificity of EV‐to‐cell interaction and cargo delivery. Here, we focus on the metalloprotease ADAM10 as a master regulator of signaling and adhesion proteins and a key player in brain cell communication, embryonic development, and cancer. Studying the effects of ADAM10's presence and activity on EV composition and targeting will shed new light on the factors driving EV function and could help model the consequences of ADAM10 dysregulation.

Methods: Human A549 lung carcinoma cells with wildtype (WT) or ADAM10 KO (A10KO) genotype, as well as ADAM10 inhibitor‐treated WT cells (A10inh.), were cultured in serum‐free medium. Small EVs (sEVs) were isolated from the cell supernatant by ultracentrifugation and compared by NTA, EM, western blot, and mass spectrometry. The isolated sEVs were then used for treatment of WT and A10KO cells, and cells were fixed and imaged for EV‐mediated ADAM10 transfer by confocal microscopy. ADAM10 activity in WT EV‐treated A10KO cells was determined by analyzing the conditioned media for shed fragments of the cellular prion protein (PrPc), as this substrate is solely shed by ADAM10 (i.e., no involvement of related proteases such as ADAM17/TACE) and cleavage site‐specific antibodies were available.

Results: Genetic depletion of ADAM10 (A10KO), but not inhibition, decreased the number of released sEV particles compared to WT. Focusing on PrPc as a major ADAM10 substrate, we found minor expression in WT sEVs but strong signal in sEVs from A10KO and A10inh. cells, suggesting changes in its sorting or processing. Treatment of A10KO cells with ADAM10‐positive WT sEVs caused the reappearance of mature ADAM10 in A10KO cells and partially restored PrPc shedding.

Summary/Conclusion: Our results provide new insights into ADAM10 as modulator of EV trafficking and function and suggest EVs as potential vectors for the extracellular transfer of active ADAM10, paving the way for future studies to address the functional relevance of this mechanism in health and disease.

PT05.20. The "torn bag mechanism" of small extracellular vesicle release through rupture of the limiting membrane of en bloc released large EVs

Dr. Tamás Visnovitz, Ms Dorina Lenzinger, Ms Anna Koncz, Ms Tünde Bárkai, Dr. Krisztina V Vukman, Ms Alicia Galinsoga, Dr. Krisztina Németh, Ms Kelsey Fletcher, Dr. Péter Lőrincz, Dr. Gábor Valcz, Prof. Edit I Buzás

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Recent studies have revealed that extracellular vesicle (EV) biogenesis is more complex than predicted previously. Our recent findings highlighted that colon cancer cells release large, membrane‐enclosed structures with small inner vesicles. This process is distinct from canonical exosome release via exocytosis and small ectosome discharge by plasma membrane shedding. The current study aims at investigating whether this unique large, multivesicular EV subtype was exclusively characteristic for colorectal cancer cells.

Methods

We used confocal microscopy (of both fixed and live‐cells), transmission electron microscopy (EPON‐embedded and immunogold techniques), super‐resolution live‐cell imaging and Western blot analysis. We also tested the effect of inhibiting different cellular pathways. The detection of the large EVs (lEVs) was made possible through a novel in situ fixation approach, which prevented disintegration during centrifugation and pipetting. Comprehensive characterization of the large multivesicular extracellular structures included assessment of classical EV markers such as CD63, CD81, ALIX, TSG101, as well as non‐conventional markers like TSPAN4 and LC3B.

Results

Our findings indicate that all tested cell types, including different cell lines and cells within their tissue environment, release lEVs containing intraluminal vesicles. We demonstrated that upon spontaneous rupture of the limiting membrane of these lEVs, internal small vesicles escaped into the extracellular environment through a “torn bag mechanism” and we could monitor this process by super‐resolution live‐cell imaging. Our studies suggest that the multivesicular lEVs may have an unusual intracellular origin, distinguishing them from any conventionally secreted EVs.

Conclusion

We propose that the “torn bag release” of small EVs represents a previously undescribed release mechanism of EVs of endosomal origin, potentially having roles in both homeostatic functions and cell‐cell communication. The observed “torn bag mechanism” prevalent in both in vitro and in fixed tissue samples, differ from exocytosis. It involves plasma membrane budding, ectocytosis, and subsequent extracellular rupture of the limiting membrane of the large multivesicular EVs. This novel mechanism offers valuable insights into the origin of the small EV release.

PT05.23. β‐catenin regulates the biogenesis and secretion of small extracellular vesicles by modulating senescence

Dr Taeyoung Kang

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Wnt signalling pathway has been attributed in pathogenesis of cancer and is known to regulate cellular senescence. The functional role of Wnt signalling pathway in the biogenesis and secretion of small extracellular vesicles (sEVs) is poorly understood.

Methods

sEVs secreted from cells in culture were isolated via the conventional differential‐ultracentrifugation and further characterised using Western blotting, nanoparticle tracking analysis, transmission electron microscopy. To investigate the implications cellular senescence and Wnt signalling pathway in the biogenesis and secretion of sEVs, CRISPR/Cas9‐based knockout, luciferase assay, quantitative proteomic analysis, immunofluorescence, and siRNA‐based knockdown and cellular survivability assays were conducted in isogenic cells, whose survival was either dependent or independent on the canonical Wnt signalling pathway.

Results

Knockout of CTNNB1 (β‐catenin) using CRISPR/Cas9 genome editing technique highlighted significant attenuation of Wnt signalling activity as well as reduced proliferation and induction of cellular senescence via the activation of AKT signalling pathway in human colorectal cancer cells. The induction of cellular senescence resulted in increased secretion of sEVs with altered protein cargo. Furthermore, immunofluorescence microscopy, proteomic analysis, and Western blotting collectively suggested alterations in the abundance of several regulators of sEV biogenesis and secretion, such as CD63, Cortactin and Rab27a upon induction of cellular senescence via the loss of β‐catenin. siRNA‐mediated knockdown of sEV regulators resulted in rescuing the secretion of sEVs in human colorectal cancer cells that lacked β‐catenin. Moreover, p53 and p21 knockout cells as well as exogenous expression of p21 or p16 further highlighted the implications of cellular senescence in the biogenesis and secretion of sEVs in human colorectal cancer cells that exhibit Wnt‐dependency.

Conclusion

In conclusion, the current findings of the study highlight a novel mechanism by which cells undergoing cellular senescence regulate the biogenesis and secretion of sEVs in a Wnt‐dependent manner.

PT05.24. The RNA‐Binding Protein NPM1 Is a Component of Exosomal Machinery Controlling mRNA Sorting through Binding to Specific RNA Motif

Student Kaixiang Zhang^1,2, Ying Zhang¹, Hang Yin^1,2

¹School of Pharmaceutical Sciences, Tsinghua University, Beijing, China, ²Tsinghua‐Peking Center for Life Sciences, Tsinghua University, Beijing, China

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Extracellular vesicles (EVs) carry genetic materials such as miRNA and mRNA to modulate gene expression in the cells that receive these vesicles, playing crucial roles in both physiological state homeostasis and disease development. EV mRNAs are not only promising candidates for non‐invasive diagnostic tests but also can be used for therapeutic purposes across various diseases. However, the mechanism for sorting mRNA into EVs, an important question in EV biogenesis, remains unclear. Here we have identified the typical mRNA binding protein NPM1, which facilitates the transportation process of mRNAs into EVs. This process relies on the involvement of both NPM1 can specific motifs on mRNA.

Methods: EVs from both cultured cells and serum samples were collected with ultracentrifugation. RNA binding proteins in EVs were identified by the oligo d(T) capture method. Next‐Generation Sequencing was employed to identify total mRNAs in EV. Sequence data was analyzed with MEME to discover specific motifs.

Results: In this work, we have identified NPM1 as an RNA‐binding protein that accounts for sorting of mRNA into EVs. In addition, we found that in NPM1‐depleted EVs, the mRNA level of cancerous genes decreased significantly, despite some non‐cancerous genes remaining unchanged. Utilizing computational analysis, we identified motifs in mRNA that are specifically recognized by NPM1 protein and facilitate the sorting into EVs. Moreover, when conjugating our identified motifs at 3’UTR of mRNA, enhanced level of mRNA in EVs was observed. Importantly, increased functional protein level was also found in cells taken up these EVs. Furthermore, in non‐small cancer patient serum, we found that NPM1 is highly expressed in serum EVs, also the level of EGFR mRNA in EVs, in agreement with our observation from EVs isolated from cell culture.

Summary: In conclusion, our findings that NPM1 is a binding protein for EVs RNA, which selectively transports mRNA, not only help elucidate the mechanism for mRNA sorting into EVs but also offer hints for the development of specific mRNA transport tools using EVs. These findings also offer clinical evidence supporting the association of NPM1 with cancerous mRNA shuttling via EVs.

PT05.25. Glycosylphosphatidylinositol‐anchored Proteins Promote Cellular Membrane Trafficking and Enhance Extracellular Vesicles Production

Scientist Tong Zhao¹, Associate Scientist Wei Zhao¹, Associate Researcher Shengya Xu¹, Associate Researcher Moxuan Yang¹

¹TheraXyte Bioscience, Beijing, China

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction: Extracellular vesicles (EVs) are naturally derived from all types of cells and have broadly categorized into ectosomes and exosomes according to the different plasma membrane budding orientations. EVs provide numerous advantages as drug carriers, however, large‐scale manufacturing EVs is challenging due to the limited yield in cells. Multiple Glycosylphosphotidylinositol‐anchored proteins (GPI‐APs) are organized into cholesterol‐dependent nanoscale clusters at the plasma membrane and delivered to endosomes via the CLIC/GEEC pathway. Notably, endocytosis of the cells is critical for the biogenesis of EVs, especially for exosomes. In our study, two GPI‐APs, CD55 and CD59 can increase the EVs yields in HEK293 cells significantly. Moreover, this function is highly regulated by the GPI‐anchor part, which is confirmed by various truncations and engineering.

Methods: HEK293 cells were transfected with plasmids and stably expressed target proteins after selection with antibiotics such as puromycin/hygromycin. Cells were cultured in DMEM containing 10% EV‐depleted FBS for adherent culturing or in a serum‐free medium for suspension culturing. The culture supernatant was treated with the 0.2 um filter after removing the cells and cell‐debris. EVs were then isolated by ultra‐centrifugation at 100,000 g for 85 min, followed by an SEC column. The particle number and size distribution were detected with both NTA (ZetaView) and nanoFCM (ApogeeFlow). The EV biomarkers such as ALIX, TSG101, CD9, CD81, and CD63 were detected with Western Blot or nanoFCM. The morphology of EVs was obtained by TEM or CyroEM.

Results: We initially performed the proteomics analysis, comparing EV with its cell counterpart, and found multiple GPI‐APs were sorted into EVs such as CD55 and CD59. After overexpression of CD55 or CD59, HEK293 cells increased its EVs production significantly. Various truncations of CD55 also can boost EVs production except for the GPI‐anchor deficiency. Furthermore, mCherry fusion with CD55 GPI‐anchor still increased the EVs yield. More importantly, mCherry can be loaded into EVs with high enrichment, which reveals the great potential of programmable engineering EVs for drug loading and targeting.

Summary: Here we found that GPI‐anchor can be the booster of EVs production, and simultaneously used as a scaffold for drug loading and targeting.

PT05.26. Immunomodulatory effect of AFSC conditioned media generated using Micregen's cell‐free secretome technology platform linked with neural protection

Dr Robert Mitchell¹, Mr Andrew Parnell¹, Dr Ben Mellows¹, Professor Ketan Patel¹, Dr Steve Ray¹

¹Micregen Limited, Reading, United Kingdom

Sponsor Exhibition, Poster Session and Lunch (Thursday), Exhibit hall‐Doors 14‐15, May 9, 2024, 12:00 PM ‐ 2:00 PM

Introduction

Micregen, a UK‐based biotech company investigating the regenerative capabilities of stem cell paracrine factors has developed a platform technology to manipulate stem cells to produce secretome products for use in regenerative medicine.

Here we characterise an amniotic fluid‐derived stem cell secretome (AFSC CM). This secretome has been quantified to contain over 2,000 components: miRNA, cytokines, anti‐inflammatory factors and proteins including growth factors and a major extracellular vesicle (EV) component.

This study investigated the AFSC CM immunomodulatory effects. We demonstrate the EV component to be uptaken by various cell types at a cellular and organ‐wide level and demonstrate its role in retaining neuronal plasticity in an Alzheimer's disease model.

Methods

EVs were characterised in accordance with published ISEV guidelines, size measured via TEM and NTA and purity via western blot. Immunomodulatory effects of AFSC CM were measured in vitro via ELISA and luciferase activity and the quantification of inflammatory cytokines in vivo via a Luminex panel.

EV biodistribution was quantified by fluorescent staining of isolated EV and detected via 3D confocal imaging in vitro and In Vivo Imaging System (IVIS) in vivo.

Neuronal efficacy was measured using a murine model of Alzheimer's, quantifying inflammatory markers via immunohistochemistry and behavioural effects via novel object recognition.

Results

AFSC CM improved endogenous stem cell activity, reduced stress‐induced senescence and pro‐inflammatory signalling in vitro.

The secretome contained a major EV component and these were delivered systemically to a wide range of organs in vivo and taken up by different cell types in vitro.

Systemic immunomodulation was detected by the reduction of pro‐inflammatory cytokines in two inflammatory in vivo systems. Neuronal plasticity was quantified in an Alzheimer's disease model that was linked to the recruitment of inflammatory astrocytes or microglia in the hippocampus.

Summary/Conclusions

A secretome generated from an amniotic fluid‐derived stem cell (AFSC CM) under proprietary conditions improve multiple key cellular measures that have been associated with regeneration with immunomodulation a key pathway.

EVs within AFSC CM are a key component and can be delivered systemically, reaching and effecting inflammation in a variety of organs such as the gut and brain.

OT05.O02. Interferon induced isoform of ADAR1 aids in Ewing sarcoma metastasis by fueling the pro‐inflammatory response in tumor microenvironment

Mr. Manideep Pachva, Dr. Peter Ruzanov, Dr. Valentina Evdokimova, Dr. Melanie Rouleau, Dr. Laszlo Radvanyi, Dr. Poul Sorensen

Introductory Talk and Oral Session: OT05 Cancer Metastasis, Plenary 1, May 9, 2024, 4:00 PM ‐ 5:35 PM

Ewing sarcoma(EwS) is a malignant bone cancer of childhood and adolescence, where 40% cases are present with metastasis. Our recent whole transcriptomic sequencing unveiled that EwS cells express RNA derived from various classes of repetitive elements of DNA, including but not limited to HSATs, HERVs, LINEs and SINEs. It was also unraveled that these repetitive RNAs are predominantly secreted in small bag like structures called extracellular vesicles (EVs). Interestingly, sequencing analysis revealed that the repetitive RNA secreted in EVs are highly A‐to‐I (Adenosine to Inosine) edited, compared to the cellular repetitive RNA. We hypothesized that this A‐to‐I editing is necessary for their secretion and subsequent actions in recipient tumor microenvironment cells.

We performed the knockdown of ADAR1 enzyme responsible for A‐to‐I editing using lentivirus based transduction of short‐hairpin RNA. Later, small EVs were purified from EwS ADAR1 +/‐ cells using ultrafiltration followed by ultracentrifugation. Purified small EVs were analyzed for size and small EV markers by Nanoparticle tracking analysis and Immunoblotting analysis, respectively. RNA purified from EwS ADAR1 +/‐ cells and EVs was used for whole transcriptomic sequencing. Immunofluorescence, immunoblotting, ELISA and droplet digital PCR techniques were used to monitor various functional read outs from the recipient cells (monocytes and fibroblasts) treated with EwS ADAR1 +/‐ EVs.

ADAR1 knockdown in EwS cells caused significantly reduced secretion of various types of SINE‐ALUs RNA in EVs. These various types of SINE‐ALU RNA, including but not limited to AluSz, AluJo, Alujb, AluSx, are present in mRNAs of genes belonging to stress granule assembly, anti‐viral immune response and NF‐Kβ pathway. Notably, these SINE‐ALUs are specifically edited by interferon induced isoform of ADAR1 (p150 isoform), whose expression is highly upregulated in Ewing tumors. Moreover, we noticed decreased anti‐viral immune response in recipient cells treated with ADAR1 KD EVs, compared to the control EVs. We predict that this decrease in anti‐viral immune response may be caused by the decreased levels of SINE‐ALUs in EVs. However, further experiments are warranted to confirm this.

Overall, this study unravels a novel role for secreted A‐to‐I edited repetitive RNA, specifically SINE‐ALU RNAs, in manipulating the tumor microenvironment.

OT05.O03. Tumor ‐derived extracellular vesicles endogenously released by tumors are captured by resident and non‐resident cells in the pre‐metastatic niche and activate the inflammasome in macrophages

Dr. Laurence Blavier‐Sarte, Dr. Irina Matei, Dr. David Lyden, Professor Yves DeClerck

Introductory Talk and Oral Session: OT05 Cancer Metastasis, Plenary 1, May 9, 2024, 4:00 PM ‐ 5:35 PM

Introduction: There is abundant evidence that tumor‐derived extracellular vesicles (TEV) have an important contributory role in establishing the pre‐metastatic niche (PMN). Here, we used two models of endogenous release and tracking of TEVs to examine their function in the formation of the PMN and its progression into a metastatic niche (MN).

Method: Human neuroblastoma (NB) and melanoma (MEL) cells engineered to release green fluorescent protein (GFP)‐labeled TEVs were orthotopically implanted into immunodeficient mice and their capture by host cells in the PMN and MN was monitored over time. GFP+ TEV‐capturing cells were isolated by flow cytometry and their transcriptome analyzed by TaqMan low density array and single cell RNA‐sequencing (scRNA‐Seq). TEVs purified by differential ultracentrifugation and size exclusion chromatography were incubated with mouse Kupffer cells (KC) and hepatic stellate cells (HSC) and proteins were examined by ELISA and Western blots.

Results: In the lung PMN (MEL model), scRNA‐Seq analysis of TEV‐capturing cells identified resident alveolar macrophages as the main population (78%), with small clusters of interstitial and recruited macrophages. In the lung MN, macrophages represented 43 % of TEV‐capturing cells with the remainder made of endothelial cells (EC), alveolar epithelial cells, dendritic cells (DC) and myeloid‐derived suppressive cells (MDSC). In the liver PMN (NB model), TEV‐capture was documented in KC (31%) and capsular macrophages but also in hepatocytes, HSC, and sinusoidal EC. In the MN the proportion of KC decreased (24%) and new populations were identified including DC and MDSC. A gene expression analysis in TEV‐capturing macrophages revealed a >10‐fold increase in IL‐18 expression (mRNA), confirmed by proteomics analysis. Consistently, we demonstrated cleavage of pro‐caspase‐1 and release of mature IL‐1β and IL‐18 in KC exposed to NB‐TEV which was prevented by inflammasome inhibitors Bay‐11‐7082 and MCC950.

Conclusion: Our data demonstrate that TEV‐capture in the PMN involves heterogeneous subpopulations of macrophages and non‐macrophage cells which dynamically change as the PMN evolves to a MN. The data also points to activation of the inflammasome as an early event in the establishment of the PMN. Thus, our work provides new insights into the role of TEVs in the earliest stages of metastatic progression.

OT05.O04. Extracellular vesicles derived from plasma of exercise mice attenuated aggressive breast cancer tumour growth and metastasis

Dr Pamali Fonseka, Dr Sanjay Shahi, Prof Mark Febbraio, Prof Suresh Mathivanan

Introductory Talk and Oral Session: OT05 Cancer Metastasis, Plenary 1, May 9, 2024, 4:00 PM ‐ 5:35 PM

Introduction

It has been well established that exercise is effective against tumour growth and spread of cancer. However, many cancer patients are unable to perform strenuous physical activities due to the severity of the disease. Hence, we intend to uncover the benefits of plasma extracellular vesicles (EVs) derived from mice that were subjected to exercise on breast cancer progression.

Methods

Plasma EVs were isolated using size exclusion chromatography coupled with ultracentrifugation from mice subjected to high‐intensity interval training (HIIT) exercise and sedentary mice. Characterisation of plasma EVs were performed using nanoparticle tracking analysis, transmission electron microscopy and western blotting analysis. Effects of plasma EVs derived from exercise and sedentary mice on metastatic breast cancer were investigated using quantitative proteomic analysis, proliferation, migration and PI/Annexin apoptosis assays, qPCR, IVIS imaging, orthotopic mouse models and immune cell profiling.

Results

Subjection to physical activity increased the number of EVs that were present in the plasma EVs of exercise mice (excEVs) compared to sedentary mice (sedEVs). Proteomic analysis revealed that there were significant changes to the plasma EV proteomic profile when mice were subjected to exercise compared to sedentary mice. When treated with exeEVs, a panel of breast cancer cells showed a significant reduction in migratory ability compared to the sedEVs and PBS treated cells. In order to investigate the role of plasma EVs in vivo, orthotopic mice bearing metastatic breast cancer tumours were given plasma EVs intravenously. Interestingly, there was a significant attenuation in tumour burden and metastasis in the presence of excEVs compared to sedEVs. This observation was further confirmed by qPCR and IVIS imaging of metastatic sites. Importantly, plasma EVs modulated the immune cell profiling of these mice. Moreover, quantitative proteomic analysis of tumours derived from exeEVs treated mice showed upregulation of several proteins that are known to be involved in tumour growth and metastasis suppression compared to sedEVs treated tumours.

Conclusion

For the first time, we were able to show that plasma EVs derived from physically active mice could attenuate aggressive breast cancer tumour growth and metastasis.

OT05.O05. RalA enhances hepatocellular carcinoma metastasis via upregulating protein cargos of small extracellular vesicles

Dr Lu Tian, Miss Jingyi Lu, Dr Karen Man‐Fong Sze, Dr Goofy Yu‐Man Tsui, Dr Daniel Wai‐Hung Ho, Prof Irene Oi‐Lin Ng

Introductory Talk and Oral Session: OT05 Cancer Metastasis, Plenary 1, May 9, 2024, 4:00 PM ‐ 5:35 PM

Introduction: Small extracellular vesicles (sEVs) play critical roles in mediating intercellular communication during tumor progression in the microenvironment of the primary tumor as well as distant metastatic organs. Previous research has shown that Ral regulates the biogenesis of sEVs during multivesicular body formation in C. elegans and promotes breast to lung metastasis through the upregulation of CD146. In our previous study, we demonstrated that RalA enhances hepatocellular carcinoma (HCC) progression by promoting aggressive features of tumor cells. However, whether RalA regulates tumorigenesis in an sEV‐dependent manner in HCC is unclear. Therefore, we aimed to investigate how RalA regulated sEV biogenesis and function in HCC.

Methods: We characterized sEVs from HCC cells by size, morphology, and markers using various technologies. The functions of recipient HCC cells and HUVEC cells upon sEV treatment were examined. In vivo, the role of sEVs in HCC metastasis were demonstrated using orthotopic liver injection and intrasplenic implantation models combined with sEVs injection. Besides, the protein composition of sEVs from RalA manipulated cells were profiled by mass spectrometry and the functions of targeted proteins were validated. Finally, phosphorylation microarrays were used to dissect the downstream signaling in the recipient HCC and HUVEC cells.

Results: We found that RalA upregulated the protein loading in sEVs. Functionally, upregulation of RalA in sEVs enhanced the migration and invasion in vitro and promoted intrahepatic and pulmonary metastasis in vivo. Furthermore, we identified that the surface‐associated fibronectin (FN1) was regulated by RalA; blocking the interaction using specific antibody targeting its receptor HSPG, could abolish the promoting effect mediated by sEVs. Additionally, RalA also modulated the functions of sEVs in facilitating angiogenesis, with both receptors of FN1, HSPG and integrinα5β1, contributing to this enhancement. Further analysis using phosphorylation microarrays showed activation of AKT in recipient PLC/PRF/5 HCC cells upon RalA sEVs treatment.

Conclusions: RalA enhances HCC metastasis by upregulating the surface‐associated protein FN1 in sEVs, leading to activation of p‐AKT signaling in recipient HCC cells and promoting angiogenesis. Overall, our study provides novel insights into the role of RalA/FN1 axis in sEV‐mediated tumor progression, highlighting its potential as a therapeutic target for HCC.

OT05.O06. CRISPR/Cas9‐based deletion of cortactin reduces the secretion of small extracellular vesicles, blocks cancer‐associated cachexia, and prolongs survival

Dr Sai Vara Prasad Chitti¹, Mrs Akbar L Marzan¹, Professor Suresh Mathivanan¹

¹La Trobe Institute For Molecular Science, La Trobe University, Melbourne, Victoria, 3086, Australia, Australia

Introductory Talk and Oral Session: OT05 Cancer Metastasis, Plenary 1, May 9, 2024, 4:00 PM ‐ 5:35 PM

Introduction: Cachexia, characterized by progressive wasting of muscle and fat, is a major cause of mortality in cancer patients, but clinical options against cachexia remain limited due to the multifactorial nature of the disease. Several seminal studies demonstrated that tumour‐cell‐released small extracellular vesicles (sEVs) containing key cachexins are necessary and sufficient to induce muscle and fat loss. Furthermore, it is now well known that cancer cells secrete more sEVs compared to non‐cancerous cells and interestingly, several proteins that are involved in the sEVs biogenesis and secretion are found to be upregulated in cachexia‐causing tumours. Hence, we examined whether decreasing the secretion of sEVs from tumour cells can inhibit cancer‐induced cachexia.

Methodology: Cortactin (Cttn) was knocked out (KO) using CRISPR/Cas9 technology in colon cancer cells. sEVs were isolated by differential ultracentrifugation and characterised by western blotting, TEM, and nanoparticle tracking analysis. Co‐culture and pre‐clinical studies were carried out to study the cachectic phenotype. Fluorescence‐based high‐throughput screening assay was performed to identify the drugs that decrease sEVs secretion.

Results: Loss of Cttn inhibited the release of sEVs. While C26 wild‐type (WT) derived sEVs induced atrophy in myotubes and lipolysis in adipocytes, Cttn‐KO sEVs did not induce atrophy or lipolysis. Proteomics analysis of sEVs highlighted the enrichment of cachectic proteins in WT sEVs compared to KO sEVs. Follow‐up C26 mice pre‐clinical studies highlighted that Cttn‐KO tumour‐bearing mice exhibited stable body weight, reduced tumour burden, and dramatically extended lifespan compared to mice bearing WT tumour. Remarkably, Cttn‐KO prevented tumour‐induced loss of muscle, fat, and other major organs. Consistent with this, overexpression of Cttn increased sEVs secretion and drastically decreased the lifespan of C26 mice by accelerating tumour‐induced weight loss. To use these findings for therapeutic benefit, we screened the library of FDA‐approved drugs and identified several drugs that block the release of sEVs. Administration of sEVs inhibitor to the cachexic mice resulted in the abolishment of cancer‐associated cachexia and prolonged survival.

Summary/Conclusion: Overall, these findings indicate that decreasing sEVs release from tumours might be a promising approach to treat cancer cachexia, improve quality of life, and extend the lifespan of cancer patients.

OT06.04. Spirulina EVs induce pro‐inflammatory response by targeting innate immune cells, demonstrating potential as a vaccine adjuvant

Dr. Mohammad Farouq Sharifpour, Dr. Suchandan Sikder, Dr. Yide Wong, Dr. Na'ama Koifman, Dr. Matthias Floetenmeyer, Dr. Robert Courtney, Prof. Jamie Seymour, Prof. Alex Loukas

Introductory and Oral Session: OT06 Diverse Sources of EV Therapies, Eureka, May 9, 2024, 4:00 PM ‐ 5:35 PM

Introduction

Recognized as a superfood microalga, Spirulina stands out as an exceptional cyanobacterium, famous for its many documented health benefits. Aside from its inherent nutritional and therapeutic advantages, Spirulina is increasingly gaining recognition for its untapped potential in biomanufacturing, including the production of biofuels and pharmaceuticals. Despite its acknowledged virtues, our understanding of extracellular vesicles (EVs) in cyanobacteria, including Spirulina, lags behind that of other bacterial counterparts. In an effort to bridge this gap, we successfully isolated, characterised and visualised EVs from Spirulina for the first time. Also, we explored the potential therapeutic benefits of Spirulina EVs (SPEVs) using a mouse model.

Methods

SPEVs were meticulously isolated through a dual approach involving ultracentrifugation and size exclusion chromatography. The EV size distribution and concentration were measured using nanoparticle tracking analysis and tuneable resistive pulse sensing. Purified SPEVs were visualised by TEM and CryoTEM. High‐resolution liquid chromatography‐mass spectrometry was performed on SPEVs utilising both gel‐based and in‐solution techniques for sample processing. Building on previous research indicating the immunomodulatory potential of EVs derived from other cyanobacterium, we conducted several in vivo experiments by intraperitoneally injecting SPEVs into mice to evaluate the short‐term and long‐term immune responses. Additionally, we explored the potential adjuvanticity properties of SPEVs.

Results

We purified a total of approximately 10^12 particles from a 2.25 L culture with an OD680 of 1.3. TEM and CryoTEM analyses revealed the presence of pleomorphic outer‐membrane‐vesicles (OMVs) and outer‐inner‐membrane‐vesicles (OIMVs) displaying diverse shapes, sizes, and coronation densities. In vivo immune response studies demonstrated a significant increase in neutrophils and M1 macrophages at the injection site following the intraperitoneal injection of SPEVs into mice, indicating a pro‐inflammatory effect induced by SPEVs. This finding prompted an additional experiment to investigate the potential adjuvanticity properties of SPEVs. Intriguingly, we observed that SPEVs significantly enhanced the specific IgG response in mice (by over 100‐fold) to a model vaccine antigen, Schistosoma TSP2, compared to the antigen administered with EVs from oranges or without SPEVs. Mass spectrometry identified a total of 54 proteins derived from Spirulina EVs, with the top‐scored hit belonging to the bacterial porin protein superfamily.

OT06.05. Targeting PD‐L1 in cholangiocarcinoma using milk‐derived nanovesicle‐based immunotherapy

Dr. Piyushkumar Gondaliya, Mr. Adil Ali Sayyed, Julia Driscoll, Irene K Yan, Dr. Tushar Patel

Introductory and Oral Session: OT06 Diverse Sources of EV Therapies, Eureka, May 9, 2024, 4:00 PM ‐ 5:35 PM

Introduction: Although the immune checkpoint PD‐L1 has emerged as a key target for cancer immunotherapy, the use of antibodies targeting PD‐L1 for cancers such as cholangiocarcinoma (CCA) are limited by poor tumor penetration and systemic toxicity. We have developed an RNA nano‐immunotherapeutic approach that uses milk‐derived nanovesicles (MNVs) for tumor‐cell targeted delivery of PD‐L1 siRNA or Cas9/ribonucleoprotein complexes. This strategy aims to improve treatment of cholangiocarcinoma by enhancing the anti‐tumor immune response.

Method: we engineered EpCAM aptamer‐decorated MNVs for targeted delivery of PD‐L1 siRNA (siRNA‐tMNVs) or Cas9/ribonucleoproteins (RNP‐tMNVs) to achieve targeted PD‐L1 silencing in CCA cells. The immunomodulatory effects of these nanovesicles were evaluated in 2D culture, multicellular 3D spheroids, and syngenic mouse model. In the syngenic mouse model, the use of targeted therapy was also evaluated in combination with gemcitabine therapy.

Results: The siRNA‐tMNVs and RNP‐tMNVs were efficiently taken up by CCA tumor cells and led to a significant reduction in PD‐L1 expression. In vitro studies demonstrated the potential of siRNA‐tMNVs to enhance T cell and NK cell degranulation, cytotoxicity, and cytokine production when co‐cultured with tumor cells. RNP‐tMNVs exhibited proficient PD‐L1 gene editing capabilities but required higher and repeated dosing. In multicellular CCA tumor spheroids, siRNA‐tMNVs reduced tumor cell cytotoxicity when combined with T cells or NK cells. Moreover, the combination of siRNA‐tMNVs with gemcitabine substantially reduced tumor burden in an immunocompetent orthotopic tumor model. The combination therapy further increased the infiltration of CD4+, CD8+ T cells, and NK cells compared to control groups, indicating a potentiated anti‐tumor immune response.

Summary/Conclusion: The targeted delivery of siRNA‐tMNVs and RNP‐tMNVs via nanovesicles represents a promising advance in cancer immunotherapy. Our findings show that this approach can boost anti‐tumor immune responses and reduce tumor burden, opening up new avenues for effective tumor treatment strategies. Further development of this nanovesicle based RNA nano‐immunotherapeutic approach could have a significant impact as a novel modality for cancer treatment.

OT06.O02. Engineered extracellular vesicles mediate the in situ propagation of antibacterial signaling enabling biofilm eradication: in vitro and in vivo studies

Postdoctoral Scholar Tatiana Cuellar‐Gaviria, Maria Rincon‐Benavides, Hatice Topsakal, Ana Salazar‐Puerta, Mia Kordowski, Pranav Rana, Orlando Combita‐Heredia, Daniel Wozniak, Daniel Gallego‐Perez, Natalia Higuita‐Castro

Introductory and Oral Session: OT06 Diverse Sources of EV Therapies, Eureka, May 9, 2024, 4:00 PM ‐ 5:35 PM

Introduction: The rise of antibiotic‐resistant bacteria is poised to evolve into a significant worldwide health emergency, underscoring the urgency for innovative treatment approaches. Methicillin‐resistant Staphylococcus aureus (MRSA) infections are often acquired after invasive medical procedures and are difficult to treat. In this work, we report a nanotechnology‐based approach to generate engineered extracellular vesicles (eEVs) loaded with an antimicrobial peptide that mediates the disruption of MRSA‐biofilm formation in a murine wound model.

Methods: The antimicrobial peptide LL‐37 was used as model cargo. eEVs loaded with LL‐37 were generated by electroporation of mouse primary alveolar epithelial cells (mPAEC) with a plasmid coding for LL‐37. eEVs were isolated from the transfected cells by size exclusion chromatography and concentrated using VivaSpin concentrator. eEV were characterized via Western blot, cryo‐EM and Nanosight and loading efficiency via qRT‐PCR and ELISA. Naïve mPAEC were exposed to 3x103 EVs/cell to evaluate the dynamics of eEV uptake and ability to transfer molecular cargo. In vivo evaluations involved the use of tissue‐nanotransfection to transfect a LL‐37‐plasmid into the wound of a murine wound‐model infected with MRSA. The impact on bacterial load was assessed after 4‐days of daily treatment.

Results: In vitro evaluation showed successful transfection of the LL‐37‐plasmid into mPAECs. The isolated eEVs had an average size of 144 nm and showed positive expression of EV markers CD63, TSG101 and β‐tubulin, and a reduced expression of calnexin. eEVs were effectively packed with LL‐37, as wells as transcripts coding for this antimicrobial peptide when compared to the Sham controls EVs. Also, we found that eEVs were readily taken‐up by naïve cells and were able to induce upregulation of the LL‐37 gene in the recipient cells. In vivo studies showed a significant decrease in the bacterial load in mice tissue‐nanotransfected with the LL‐37‐plasmid. Current studies include a more comprehensive evaluation to determine the role of the eEVs released by the transfected tissue in mediating a paracrine transport of the peptide.

Conclusion: Antimicrobial peptides and their transcripts can be packed within eEVs and are efficiently delivered into recipient cells. This type of antimicrobial eEVs could potentially be harnessed for future therapeutic applications.