Extracellular Vesicles & Cell-Free RNAs 2023 Agenda

Welcome and Introduction by Conference Co-Chairs: Professor Morgan and Professor Graner -- Ballroom B
Michael Graner, Professor, Dept of Neurosurgery, University of Colorado Anschutz School of Medicine
Terry Morgan, Professor of Pathology and Laboratory Medicine, Oregon Health and Science University, United States of America

Immune-Associated Extracellular Vesicles in Blood
Shannon Stott, Assistant Professor, Massachusetts General Hospital & Harvard Medical School, United States of America

Electrokinetic-based Microchip for Purification and Characterization of Small Extracellular Vesicles
Leyla Esfandiari, Associate Professor of Biomedical Engineering, University of Cincinnati, United States of America

Small extracellular vesicles (sEVs) are lipid-bilayer delimited particles that are naturally secreted by all cells and have emerged as valuable biomarkers for a wide range of diseases. Efficient isolation of sEVs while maintaining yield and purity is crucial to harvest their potential in diagnostic, prognostic, and therapeutic applications. However, because of the complex nature of samples and the heterogeneous physicochemical properties of EVs, their accurate isolation and characterization from body fluids raises significant challenges in clinical settings. We have developed and patented a simple, yet powerful electrokinetic-based microchip capable of rapid and label-free purification of sEVs from body fluids by applying a significantly low electric field. The device also tailored with a sensing module to further characterize sEVs based on their dielectric properties by measuring their impedance. Thus, the microchip has significant potential to serve as a bioanalytical tool for liquid biopsy.

Extracellular Biology and Liquid Biopsies: What is Next?
Jennifer Jones, NIH Stadtman Investigator, Head of Transnational Nanobiology, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, United States of America

Mechanisms of Extracellular Vesicle Biogenesis that Regulate Wound Healing
Brian Eliceiri, Professor, UC San Diego, United States of America

Small extracellular vesicles (EVs) are important mediators of intercellular signaling that carry biologically active protein payloads relevant in wound healing.  However, whether EVs are analyzed from wound fluid or other biological fluids, EVs are heterogenous, reflecting in part the release of EVs from different cell types.  To address the central question of EV heterogeneity in parallel with the unmet need for in vivo sources of EVs with low platelet contamination, we have developed a macroporous scaffold for the subcutaneous implantation and subsequent collection of EVs that is applicable for the study of EV activity in any defined mouse model. Using polyvinylalcohol (PVA) sponges as the scaffold, we first show that the rapid infiltration of immune cells of hematopoietic origin is accompanied a substantial and heterogenous population of EVs. Second, we show how single vesicle flow cytometry (vFC) addresses the heterogeneity challenge by quantifying the expression of surface markers that map to specific subpopulations of EVs relevant to cellular origin.  Third, we show how in vitro two-dimensional cell culture, although common, introduces significant bias in EV release that is addressed with the PVA scaffold in vivo model. Together these studies, define a novel model establishing the biochemical basis and biological activity of EVs in the biology of wound healing.

Placental Extracellular Vesicles; Regulators of Maternal Physiology
Larry Chamley, Professor and Head of Department of Obstetrics and Gynaecology, University of Auckland, New Zealand

The human placenta produces vast quantities of extracellular vesicles into the maternal blood continuously during pregnancy. Biodistribution studies indicate that the majority of these EVs are taken up from the maternal blood rapidly in the lungs, liver kidneys and spleen. Functional studies demonstrate that normotensive placental EVs can protect against the development of hypertension long-term while EVs from pregnancies complicated by preeclampsia a hypertensive disease of pregnancy activate the maternal endothelium and induce a pro-constrictive phenotype in resistance arteries.  These functional studies suggest that the protein and/or regulatory RNA cargos of placental EVs have a long-lasting regulatory effect on  the maternal cardiovascular system.

Everything We Think We Know About Extracellular Vesicle Cargo is Wrong
Terry Morgan, Professor of Pathology and Laboratory Medicine, Oregon Health and Science University, United States of America

Extracellular vesicles (EVs) comprise a range of submicron particles, including small EVs (exosomes ~75-150 nm) derived from endosomal biogenesis, larger microvesicles (< 1um) that bud directly from the cell membrane, and fragments of cells undergoing necrosis and/or apoptosis. These lipid encapsulated EVs contain surface cell-specific protein markers and a variety of RNAs that provide insights into their source and potential function (e.g. regulating angiogenesis and immune microenvironments).  Methods like density gradient ultracentrifugation followed by size-exclusion chromatography (SEC) provide a heterogeneous mixture of EVs from a variety of cell sources, and in a variety of sizes. Affinity capture lacks size-specificity, which is important because larger microvesicles are thought to have entirely different contents and biological functions than small EVs (exosomes). These methodological shortcomings have limited adequate EV profiling and characterization.  To address this need, the Morgan laboratory uses multiplex nanoscale fluorescent activated cytometric sorting (nanoFACS) with 40nm resolution to image, count, and isolate cell- and size-specific EVs. Importantly, this next generation approach provides highly efficient flow sorting of multiplex-labeled EVs at the 0.3 nanoliter droplet scale (5x better than commercially available FACS machines using our custom made sorting nozzle and EV-specific protocol).  We have completed validation studies characterizing flow sorted placental EVs according to guidelines put forth by the International Society of Extracellular Vesicles (ISEV), including nanoparticle tracking analysis, CryoEM, ELISA, and microRNA content within small EVs compared with flow sorted 100nm liposomes spiked into the same plasma samples. Spiked in liposomes are an important negative control for background contamination inherent in any EV isolation method and novel to our EV isolation approach.

Digital Flow Cytometry for the High-Throughput Multiplexed Analysis of Single Extracellular Vesicles and Particles
Daniel Chiu, A. Bruce Montgomery Professor of Chemistry, University of Washington, United States of America

Extracellular vesicles and particles (EVPs) play a central role in liquid biopsy, intercellular communication, and disease transmission and progression, and are emerging therapeutic tools. To better understand the biology of EVPs and fully unlock their diagnostic and therapeutic potential, it is critical to access quantitative information regarding their concentration, size, and biological heterogeneities. To meet this need, we have developed a single-molecule sensitive flow platform, which uses a high-throughput 12-channel flow analyzer that detects each and every fluorescent molecule flowing through a microfluidic channel, and enables multiparameter characterization of EVPs, including single-EVP phenotyping, sizing, and the absolute quantitation of EVP concentrations and biomarker copy numbers.  This new flow technology should have a broad range of applications, from analysis of single EVPs such as exosomes or RNA-binding proteins to characterization of therapeutic lipid nanoparticles, viruses, and proteins; it also provides absolute quantitation of non-EVP samples such as dyes, beads, and Ab-dye conjugates.

Screening Tests using Micro- and Nanofluidics for Early Detection of Ovarian Cancer with Extracellular Vesicles Serving as the Input
Steve Soper, Foundation Distinguished Professor, Director, Center of BioModular Multi-Scale System for Precision Medicine, The University of Kansas, United States of America

We are developing screening tests consisting of novel hardware, biomarkers, and assays to service a number of diseases, including the early detection of cancer and viral infections. The commonality in these tests is that they consist of microfluidic devices made from plastics via injection molding. Thus, our tests can be mass produced at low-cost to facilitate bench-to-bedside transition and point-of-care testing (PoCT) for large scale population screening. The assays are based on the use of liquid biopsy markers as the input, which can be secured in a non- to minimally-invasive manner appropriate for screening. Recently we have focused on developing plastic nanofluidic devices, which provides unique opportunities for single-entity analyses – these devices can also be injection molded. In this presentation, we will discuss the evolution of our fabrication efforts of plastic-based microfluidic and nanofluidic devices as well as their surface modification to make devices appropriate for screening using extracellular vesicles (EVs) as the input. Then, we will discuss the application of the device and the associated assay for selection of rare liquid biopsy targets (EVs) from clinical samples to serve as screening tests. The specific application we will discuss is the analysis of EVs for the early detection of ovarian cancer. Ovarian cancer is the 5th most deadly cancer for women in the US and has a 46.2% 5-y survival rate. Unfortunately, ~85% of cases are diagnosed at a late stage of disease providing pore outcomes for these patients. Therefore, new strategies for early detection are required. The screening test we are developing consists of a microfluidic chip for EV affinity selection, which consists of a high density array of pillars surface-decorated with antibodies to efficiently select EVs followed by the label-free enumeration to determine elevated levels of EVs in the plasma of patients suspected of having ovarian cancer. Unique EV-associated surface proteins were discovered for selection of ovarian cancer EV specifically for the early detection of disease. The selected EVs were counted using a nano-Coulter Counter chip (nCC), which consisted of in-plane nanopores. Both steps of the screening test described here were carried out using a microfluidic and nanofluidic chips integrated to a control board for automating sample processing with results produced within 20 min.

Immiscible Aqueous Droplets for Biosensing, Wound Healing Models, and Exosome Manipulation
Shuichi Takayama, Professor, Georgia Research Alliance Eminent Scholar, and Price Gilbert, Jr. Chair in Regenerative Engineering and Medicine Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology & Emory University School of Medicine, United States of America

Liquid-liquid phase separation (LLPS) occurs physiologically inside each of our cells. LLPS can also be observed with various polymer systems such as poly(ethylene glycol) PEG, dextran (DEX) where immiscible aqueous two-phase systems (ATPS) are formed. This talk will describe ATPS fundamentals as well as application of ATPSs in cellular biopatterning including for wound healing studies, exosome isolation, and molecular biosensing.

NCATS Program on Exosome Therapeutics for Regenerative Medicine (ExTReMe)
Danilo Tagle, Director, Office of Special Initiatives, National Center for Advancing Translational Sciences at the NIH (NCATS), United States of America

Extracellular vesicles (EVs), membrane-bound particles containing a variety of RNA types, DNA, proteins and other macromolecules, are now appreciated as an important means of communication between cells and tissues, both in normal cellular physiology and as a potential indicator of cellular stress and other environmental exposures and early disease pathogenesis. EVs have pleiotropic actions in physiological and pathological conditions.  EVs are commonly heterogeneous in size, ranging from 20 to 1,000 nm in diameter depending on their origin and mechanism of release, direct shedding or budding from the plasma membrane. Exosomes are vesicles with a diameter of 20–100 nm formed by the inward budding of endosomal membranes to form large multivesicular bodies (MVBs) and released extracellularly when MVBs fuse with the plasma membrane.  Exosomes have recently been studied for their potential use in therapy as a 1) targeted and non-immunogenic delivery system for drugs or biological molecules, and 2) in the maintenance of tissue homeostasis and their contribution to tissue repair and regeneration. This presentation will summarize NIH-funded activities in exploring the therapeutic applications of exosomes along with application of new experimental models, including organ-on-chip (OOC) systems and in vitro approaches to extend findings.

Opiates and HIV Alter Exosomal EV microRNA Cargo
Andrea Raymond, Associate Professor, Herbert Wertheim College of Medicine, Florida International University, United States of America

Opiate abuse increases the risk of HIV transmission and exacerbates Human Immunodeficiency Virus (HIV) neuropathology by increasing inflammation and modulating immune cell function. However, the exact mechanism(S) for these observations is unclear Exosomal EVs(xEV) contain miRNAs that are differentially expressed due to HIV infection or opiate abuse. Here we developed a preliminary exosomal-miRNA biomarker profile of HIV-infected PBMCs in the context of opiate use.  Functional studies show that xEVs derived from peripheral blood mononuclear cells(PBMCs) exposed to opiates reduced viability and function of neurons. Using Nanostring microRNA arrays we show that morphine and HIV induced differential miRNA expression in PBMC-derived exosomes, potentially identifying a biomarker of opiate use disorder(OUD) and  mechanisms of action or novel therapeutic targets involved in OUD, neuropathology, TNF-a signaling pathway, NF-kB signaling pathway, autophagy, and apoptosis in context of HIV infection.

Extracellular RNAs Associated with Neurodegenerative Disease and Injury
Kendall Van Keuren-Jensen, Professor and Deputy Director, Translational Genomics Research Institute, United States of America