Exosomes and Extracellular Vesicles (EV): Research, Diagnostics and Therapeutics Opportunities Agenda

Conference Registration, Conference Materials Pick-Up, Morning Coffee and Breakfast Pastries

Coffee Break and Networking

Networking Lunch in the Exhibit Hall: Visit Exhibitors and Poster Viewing

Technology Spotlight:
Luncheon Technology Spotlight: Enabling Biofluid Biomarker Discovery
Shannon Pendergrast, Chief Scientific Officer, Ymir Genomics

Ymir Genomics is developing a variety of methods and devices that facilitate Biomarker Discovery from Biofluids such as blood, urine, and saliva.

CSF Exosome Biomarkers of Inflammation, Oxidative Stress, and Neuronal Injury in Aging and HIV
Dana Gabuzda, Professor of Neurology, Dana-Farber Cancer Institute and Harvard Medical School, United States of America

This presentation will focus on analysis of human cerebrospinal fluid exosome proteins in aging and HIV, and identification of exosome biomarkers correlating with immune activation, oxidative stress, and neuronal injury.

Technology Spotlight:
Analysis and Insight from Liquid Biopsy Biomarkers
Isaac Houston, Life Science Specialist, QIAGEN

QIAGEN provides a broad range of tools for sample preparation and assay technologies for biomarker isolation, discovery, and verification.  The presentation will provide researchers with effective and efficient workflow tools to overcome challenges in cancer and non-invasive genetic research.  Join QIAGEN to learn how these powerful tools have enabled our customers to achieve valuable insights from various liquid biopsy biomarkers.

Coffee Break

Networking Reception with Beer, Wine and Appetizers. Engage with Fellow Delegates and Enjoy Views of the Charles River and the Boston Skyline

Close of Day 1 of the Conference

Dinner Short Course on Microfluidics for Studying Circulating Biomarkers [Separate Registration Required]

Morning Coffee, Breakfast Pastries and Networking in the Exhibit Hall

Mesenchymal Stem Cell Exosomes: Powerful Agents of Lung Growth and Repair
Stella Kourembanas, Chief, Division of Newborn Medicine, Boston Children’s Hospital; Clement A. Smith Professor of Pediatrics, Harvard Medical School, United States of America

Bronchopulmonary dysplasia (BPD) is the most common complication of prematurity characterized by an arrest of lung growth with reduced alveoli, fewer blood vessels, and abnormal lung function. BPD has significant long-term pulmonary morbidities, including pulmonary hypertension (PH), airway hyperreactivity, abnormal pulmonary function test results, and, in some cases, emphysematous changes that persist into adulthood. Mesenchymal stem cells (MSCs) are recognized as potential cell-based therapy for diseases of the lung. These multipotent cells exhibit beneficial effects through anti-inflammatory, immuno-modulatory, pro-survival and anti-fibrotic mechanisms that are not clearly defined. We showed that bone marrow-derived MSCs or their cell-free conditioned media (CM) prevent and reverse experimental lung injury in the neonatal mouse model of BPD. We isolated exosomes from human MSC-CM and showed that they can prevent lung inflammation and PH in experimental models. Exosomes are small vesicular structures produced by all cells that contain a distinct cargo which not only represents the cell of origin but is differentially-enriched in specific nucleic acid or lipid/protein species. They serve as an important cell-to-cell communication mechanism. We have shown that MSC exosomes restore lung homeostasis through immuno-modulatory pathways, and enable lung-specific progenitor cells to repair lung injury. Current work is focusing on the full characterization of MSC exosomes and their biological function in lung injury, growth, and repair using experimental models of broncho-pulmonary dysplasia. Our goal is to develop the most optimal, well-characterized, and functional MSC exosome preparation for human application.

Development of Therapeutic Approaches Using Extracellular Vesicles to Treat Age-Related Diseases
Paul Robbins, Professor, Department of Biochemistry, Molecular Biology, and Biophysics, and the Institute on the Biology of Aging and Metabolism, University of Minnesota Medical School, United States of America

With aging, there is an inevitable and progressive loss of the ability of tissues to recover from stress, in part through loss of stem cell function. More than 90% of people over 65 years of age have at least one chronic disease, while 75% have at least two. Thus, it is imperative to find a way to target therapeutically the process of aging to compress the period of functional decline in old age. We have demonstrated that injection of adult stem cells isolated from young wild-type mice into a mouse model of accelerated aging conferred significant lifespan and healthspan extension through a paracrine/endocrine mechanism. Moreover, treatment of mice with EVs from young stem cells is able to extend health-span. Progress towards developing clinically relevant approaches using stem cell-derived extracellular vesicles to treat autoimmune and age-related pathologies will be presented.

Signaling Pathways Regulating Extracellular Vesicle RNA
Alain Charest, Associate Professor, Beth Israel Deaconess Medical Center, Cancer Research Institute, Department of Medicine, Harvard Medical School, United States of America

Technology Spotlight:
Surface Charge and Fluorescence: Biochemical Analysis of Vesicles by Nanoparticle Tracking Analysis
David Palmlund, Sales Engineer, Particle Metrix, Inc.

Nanoparticle Tracking Analysis (NTA) combined with surface charge measurement and fluorescence detection capability enables the user to gain bio-chemical information about the particle surface. Biological particles such as virus, virus-like particles (VLPs), vesicles, liposomes and protein aggregates can be specified, quantified and differentiated from inorganic particles such as dust, bubbles or precipitates.

Coffee Break and Networking

Exosome Engineering for Protein Delivery Via Optogenetic Approach
Chulhee Choi, Professor and Chair, BioMedical Imaging Center, Korea Advanced Institute of Science and Technology (KAIST), President, ILIAS Biologics Incorporated, Korea South

In this presentation, an opto-genetically engineered exosome system, named ‘exosomes for protein loading via optically reversible protein–protein interaction” (EXPLOR) that can deliver soluble proteins into the cytosol via controlled, reversible protein–protein interactions (PPI) will be introduced. By integrating a reversible PPI module controlled by blue light with the endogenous process of exosome biogenesis, cargo proteins can be loaded into newly generated exosomes. Treatment with protein-loaded EXPLORs was shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in both a time- and dose-dependent manner. Previously, it has been shown to delivery mCherry, Cre enzyme, Bax, and Super repressor I?B proteins as functional proteins in the target cells and in vivo. In this presentation, the results for follow-up studies will also be discussed.

Influence of Glioblastoma-derived Extracellular Vesicles on the Tumor Microenvironment
Marike Broekman, Neurosurgeon, Dept of Neurosurgery, Universitair Medisch Centrum Utrecht, Netherlands

Extracellular vesicles (EVs) have been shown play a role in glioblastoma biology. Here, we will present data on EV mediated modification of microglia, macrophages and T-cells in vivo.

Stressing Out the Neighbors: Stressed Exosomes (“SexOsomes”?) Passage Stress Phenotypes to Recipient Cells
Michael Graner, Professor, Dept of Neurosurgery, University of Colorado Anschutz School of Medicine, United States of America

Cancer cells undergo numerous stresses, many of them self-inflicted, but often do not appear to suffer the consequences of those stresses. In some cases, the stress responses are actually beneficial to the tumor cells, providing them with potent resilience to their less-than-hospitable environments. One consistent tumor stress is the Unfolded Protein Response (UPR), an endoplasmic reticulum-based stress-management system with sensors, transducers, and effectors that result in a transcriptional/translational landscape rearrangement leading to resolution of the stress, or cellular apoptosis. However, tumors may incorporate the UPR into their stress portfolio to survive or even thrive amidst their environmental insults. We propose that exosomes from stressed cells (stressed exosomes, or “sexosomes”) are able to induce stress response phenotypes in recipient, unstressed cells, thus enabling stress responses without having to experience the actual stress. Our analysis in this report goes to the molecular level, monitoring proteome changes in glioma cells when those cells are exposed to exosomes released from UPR-stressed cells. We find high overlap in the proteomes of stressed cells and unstressed cells that receive “sexosomes”, suggesting that tumors may unify their overall stress responses despite their inherent heterogeneity. The implications for general tumor biology, and in particular, therapeutic resistance, are highlighted.

Extracellular Vesicles and their Fellow Travelers in HIV Infection
Kenneth Witwer, Associate Professor, Johns Hopkins University School of Medicine, United States of America

Retroviruses such as HIV represent hijacked extracellular vesicles (EVs). As we and others have reported, altering the EV exposure of cells in culture has measurable effects on growth, survival, and behavior. We now find that under EV-depleted conditions, retrovirus-susceptible cell lines and primary cells produce more HIV. Baseline production is restored with add-back of EVs from ultracentrifuge pellets. Furthermore, we observed increased infectivity of both X4 and R5 tropic viruses produced under EVD versus EVR conditions. Gene expression, lipid production, and cellular microRNAs were probed as possible contributors to these phenomena, revealing minimal roles for microRNAs but potential involvement of lipid biogenesis pathways. However, standard EV depletion protocols may also deplete other constituents of serum, including lipoprotein particles, which associate with and may mimic EVs in some characterization assays. We submit that the possible functional partnerships of EVs and lipoproteins are a rich topic for further investigation.

Networking Lunch: Visit Exhibitors and Engage with Fellow Delegates

Technology Spotlight:
Luncheon Technology Spotlight: Next Generation Affinity-based Extracellular Vesicle Isolation Technologies that Rely on a Synthetic Peptide or Hyaluronic Acid
Anirban Ghosh, Research Scientist, Atlantic Cancer Research Institute (ACRI)

We have developed and validated two robust affinity-based EV-capture technologies that use (i) a synthetic peptide and (ii) hyaluronic acid for improved scalability, quality, platform versatility, cost-effectiveness, clinical compatibility and liquid biopsy assays (biomarker and mutation detection).

Technology Spotlight:
Automated Proteomic Characterization of Single Exosomes
George Daaboul, Chief Scientific Officer, nanoView Biosciences, Inc.

Exosome research requires validation of prepared samples that has traditionally been performed using nanoparticle tracking analysis (NTA) combined with proteomic analysis for exosomal proteins. Recently, Flow Cytometry (FC) has been used to combine the sizing and proteomic information about samples under analysis. While a mature tool for large cellular-sized analyses, FC has limitations resulting from the small-size of exosomes, specifically in terms of the number of available surface epitopes and low-signal relative to background levels, especially in complex samples. nanoView Diagnostics has recently developed a label-free visible-light microarray imaging technique that allows multiplexed enumeration and sizing of individual nanovesicles captured on the sensor in a one-step assay direct-from-sample and can work with samples volumes as small as 5 µl. The exosome characterization technology is a high-throughput technique that can improve standardization of exosome preparations and facilitate translation of exosome based liquid biopsies.

Cell Communication and Therapeutic Delivery via ARMMs (ARRDC1-mediated Microvesicles)
Quan Lu, Associate Professor, Harvard School of Public Health, United States of America

My lab discovered a new type of extracellular vesicles known as ARMMs (ARRDC1-mediated microvesicles), which bud directly at the plasma membrane and are distinct from exosomes. I will discuss the molecular mechanism and physiological function of ARMMs. I will also present most recent data on how ARMMs may be harnessed for therapeutic delivery of bioactive protein and RNA molecules.

Companion Animal Studies Advance Understanding of Disease-Relevant Circulating Exosome specific miR Signatures
Andrew Hoffman, Professor, Director -- Regenerative Medicine Laboratory, Tufts University, United States of America

Companion animal disease models strongly resemble human diseases and biofluids from these animals are readily accessible sources of exosomes for longitudinal studies.  We present an analysis of miRNomic data from circulating exosomes derived from dogs with naturally occurring heart diseases also found in humans (mitral valve disease, arrythmogenic right ventricular cardiomyopathy).   Target mRNA and gene networks underpinning miR-target interactions were identified in silico for candidate miR.   The process of functional, biological, and clinical characterization of circulating exosome specific miR in companion animals will be discussed.

Circadian Rhythm Modulates the Ability of Pulmonary-derived Extracellular Vesicles to Alter Target Marrow Cell Phenotype
Laura Goldberg, Assistant Professor of Medicine, Brown University/Rhode Island Hospital, United States of America

We are interested in how circadian rhythm influences extracellular vesicle (EV)-mediated inter-cellular communication.  To begin exploring whether circadian oscillations alter EV function, we employed a well-established in vitro system in which lung-derived EVs, when co-cultured with murine bone marrow cells, induce the bone marrow cells to express pulmonary epithelial cell-specific mRNA and protein. Using this readily manipulated in vitro system, we were able to vary the circadian time-point of both the lung harvest for EVs and the bone marrow cell harvest for target marrow cells prior to co-culture. We found that 1) EVs, when harvested from lung at distinct circadian time-points, differentially altered the expression of pulmonary epithelial specific mRNAs in target bone marrow cells in culture, and 2) altering the circadian time-point of the target whole bone marrow cells, and co-culturing with lung-derived EVs similarly resulted in statistically significant differences in pulmonary epithelial mRNA expression due to circadian oscillations of the recipient marrow cells. These data indicate that circadian rhythm is likely an important component of EV-mediated inter-cellular communication. Our ongoing work is aimed at elucidating the mechanisms by which circadian rhythm influences EV-mediated communication with bone marrow cells. We hope such studies will provide insight into the molecular mechanisms by which EVs alter the mRNA expression profile of target WBM and help optimize EV manipulations for therapeutic interventions in the future.

Nano-Plasmonic Exosome (nPLEX) Analysis
Hyungsoon Im, Associate Professor, Center for Systems Biology, Mass General Hospital (MGH)/Harvard Medical School, United States of America

This presentation will review a recent progress of nPLEX (nano-plasmonic exosome) technology. The sensor is based on transmission surface plasmon resonance (SPR) through periodic nanohole arrays. Target-specific exosome binding to the array causes SPR signal changes, which enables sensitive and fast detection of exosomes. We applied the first generation nPLEX system to detect exosomes collected from ovarian cancer patients.

Microfluidic Isolation of Cancer Cells: Overcoming the Liquid Biopsy Status Quo
Silvina Ribeiro-Samy, Researcher, International Iberian Nanotechnology Laboratory (INL), Portugal

Development of a size-based rare cell capture device for point-of-care liquid biopsy that shall maximize the clinical value of circulating biomarkers.

Role of Extracellular Vesicles in Fetal Lung Morphogenesis Mediated by Mechanical Signals
Juan Sanchez-Esteban, Associate Professor of Pediatrics, Staff Neonatologist, Women & Infants Hospital of Rhode Island, Brown University, United States of America

Incomplete development of the lung secondary to extreme prematurity or pulmonary hypoplasia can cause neonatal death and serious long-term morbidities. Currently, the management of these conditions is primarily supportive.  Lung morphogenesis has significant dependence on mechanical signals. However, the mechanisms by which mechanical forces accelerate lung development are not fully-characterized. Extracellular vesicles (EVs), including exosomes and microvesicles, are increasingly recognized as a novel mode of cell-to-cell communication. Shedding vesicles are released from many cell types and have been identified in a variety of body fluids. Moreover, EVs were found to be important for tissue morphogenesis in drosophila. However, the role of EVs in fetal lung development is unexplored. Our preliminary studies show the presence of EVs in the lumen of the fetal lung. In addition, physiologic levels of mechanical strain stimulate the release of EVs in fetal lung epithelial cells. Moreover, incubation of fetal epithelial cells with EVs mimics the effect of stretch on cell differentiation. These preliminary studies suggest that signaling mediated by EVs could be important for fetal lung development. Currently, we are investigating the role of EVs in fetal lung development using ex vivo and in vivo models.

Inter- and Intra-Tumoral microRNA Heterogeneity
Agnieszka Bronisz, Instructor in Neurosurgery, Brigham and Women's Hospital/Harvard Medical School, United States of America

Despite the importance of molecular subtype classification of glioblastoma multiforme (GBM), the extent of extracellular vesicle (EV)-driven molecular and phenotypic re-programming remains poorly understood. To reveal complex subpopulation dynamics within the heterogeneous intra-tumoral ecosystem, we characterized microRNA expression and secretion in phenotypically diverse subpopulations of patient-derived GBM stem-like cells (GSC). As EVs and microRNAs convey information that drives phenotype and re-arrange the molecular landscape in a cell type-specific manner, we argue that intra-tumoral exchange of microRNA augments the heterogeneity of GSC that is reflected in highly heterogeneous profiles of microRNA expression in GBM subtypes.

Terminal Complement Components are Critical in the Release of Cellular RNA in Circulation
Ionita Ghiran, Assistant Professor of Medicine, Beth Israel Deaconess Medical Center, United States of America

Despite of over 10 years of intense research, the intimate mechanisms responsible for extracellular vesicles (EVs) formation (exosomes and microvesicles), and the release of cellular RNA species (exRNAs) in circulation are currently known. The complement system is comprised of over 20 soluble and membrane bound proteins with critical roles in recognizing, binding, and removal of foreign particles as well as initiating and regulating innate and acquired immune responses. Activation of the complement system occurs during both, normal (circadian variation), and pathological conditions through either classical, alternative, or lectine pathways leading to the formation and transient insertion of C5b-9/Mac pore complex into cellular plasma membrane. We hypothesis that a) MAC-insertion promotes a sudden, significant and transient water and Ca++ influx, leading to: i) endocytosis of the affected area, followed by delivery of C5b-9/MAC-containing plasma membrane into the multi vesicular body (MVB), and its incorporation into exosomes, or ii) exocytosis of the C9 channle/MAC-affected plasma membrane patch followed by microvesicles (MVs) formation. In addition, the size of the MAC/C5b-9 pore, 12 nm, is large enough to: i) allow cytoplasmic RNA species to be transferred into the MVB following endocytosis of C5b-9/MAC-containing plasma membrane, and ii) RNA species located near the plasma membrane to be released in the extracellular space upon C5b-9/MAC insertion. Our results, for the first time implicate MAC/C5b-9 as: i) a possible channel responsible for exosomes and microparticle biogenesis, and ii) loading of cytosolic RNAs into the exosomes, and iii) the direct release of cytoplasmic RNA species into the circulation (exRNAs).

Close of Day 2 of the Conference