Exosomes and Microvesicles: Research, Biomarker Cargo and Therapeutic Potential Agenda

Conference Registration, Materials Pick- and Continental Breakfast in the Exhibit Hall

Coffee Break and Networking in the Exhibit Hall

Networking Lunch, Discussions with Exhibitors and View Posters

The Role of Extracellular Vesicles and their Content in Glioblastoma Microenvironment
Agnieszka Bronisz, Instructor in Neurosurgery, Brigham and Women's Hospital/Harvard Medical School, United States of America

Provide the overview on the role of extracellular vesicles (EVs) communication in glioblastoma microenvironment. Analysis of proteome and microRNA content of glioblastoma stem cells derived EVs. Present evidence for protein/microRNA functional transfer by EVs. Discuss the prospects of EV protein/microRNA-targeted strategy for brain tumor therapy.

Technology Spotlight:
Rapid isolation, Purification and Detailed Characterization of Extracellular Vesicles from Biological Fluids
Anoop Pal, Chief Scientist - Americas Region, iZON Science

Isolation and purification of extracellular vesicles (EVs) from complex biological fluids without changing their biophysical properties is critical. Moreover, accurate characterization of EV properties (size, size distribution, charge, mobility etc.) and quantification of EVs is paramount to link the impact of biophysical properties of EVs to biological outcomes.  This work presents a simple, repeatable, reliable and cost-effective approach that can be used for purification, isolation and detailed particle-by-particle characterization of EVs.

Technology Spotlight:
New Tools for Exosome Isolation and Analysis: ExoCap Kit for Serum, Plasma & Cell Culture
Hiroaki Sagawa, Senior Manager, JSR Micro Inc

Research in circulating tumor cells, circulating cell-free DNA, extracellular RNA, and Extracellular Vesicles (EVs) have been gaining popularity as a promising non-invasive diagnostic approach. With this backdrop, JSR has focused on the development of EV capture methodologies, keeping in mind future potential uses for EVs in non-invasive diagnostics at the point of care. In this session, a description of a selective capture method of EVs as a potential platform for non-invasive diagnostics will be provided by introducing ExoCap^TM, a magnetic beads based isolation and enrichment method of exosomes from various body fluids and cell culture supernatants. ExoCap consists of Magnosphere magnetic beads particles coupled with antibodies that recognize antigens on the exosome surface, a washing buffer, and a reagent that releases the captured exosomes for analysis. The antibodies against CD9, CD63, CD81, and EpCAM were specifically selected for this kit. ExoCap can easily separate exosomes, without ultracentrifuge or any special equipment. A sample amount of 0.1mL is sufficient. In addition, it is an animal free system which is superior to other methods for Mass Spec analysis. Moreover, this method enables non-destructive purification of exosomes. ExoCap can also be applied to beads based Flow Cytometry immediately after the capture process with 50 µL of samples. To confirm exosomal isolation from diverse body fluids (such as human serum, plasma) and cell culture media, exosomes were examined by western blot, RT-qPCR, particle size distribution measurement, Flow Cytometry, and scanning transmission electron microscopy (TEM). Exosomes isolated by ExoCap had a lipid bilayer membrane, showed a particle size distribution around 100nm, and expressed tetraspanin molecules.

Coffee Break and Networking in the Exhibit Hall

Urinary Exosomes and Microvesicles as Carriers of Diagnostic Markers
Leileata Russo, Research Professor, Mass General Hospital (MGH)/Harvard Medical School, United States of America

Exosomes and microvesicles have been widely examined for their biological role in health and disease. This presentation will cover their potential use as a non-invasive ‘biopsy’ of the genitourinary tract and beyond. We have demonstrated that exosome and microvesicle RNA (collectively referred to as exoRNA) is extremely stable enabling reproducibility and enhancing sensitivity and specificity of analysis. An overview of the current use and reports of exosomes and microvesicles as a diagnostic platform will be given.

Enabling Urine Biomarker Discovery
Shannon Pendergrast, Chief Scientific Officer, Ymir Genomics, United States of America

The discovery of urinary extra-cellular biomarkers has been impeded by the lack of efficient methods for the isolation of extra-cellular vesicles (exosomes and microvesicles), protein, and nucleic acid.  Ultracentrifugation, considered the gold standard for vesicle isolation from many biofluids, is efficacious but laborious, and like most commercially available methods, is unable to consistently isolate enough quality material from small volumes of urine for protein or RNA-based biomarker discovery.  Ymir Genomics has invented a novel precipitation reagent that rapidly and efficiently isolates extra-cellular biomolecules and vesicles from urine samples yielding a quantity suitable for biomarker discovery.  Ymirite is more efficient than either ultracentrifugation or commercial precipitation methods for isolating extra-cellular nucleic acids and vesicles from a variety of urine samples as evaluated by immunoblot, Nanosight, Electron Microscopy, miRNA microarray, and qRT-PCR. Moreover, unlike all other methods, our reagent was able to isolate vesicles from very dilute samples. Ymir Genomic’s Precipitation Reagent offers a method that, in less than 30 minutes, isolates intact extra-cellular vesicles, protein and RNA suitable for biomarker discovery from urine samples.

Technology Spotlight:
From Sample to Insight: QIAGEN´s Approach Pioneering the Liquid Biopsy Revolution
Constanze Kindler, Senior Global Product Manager, QIAGEN

Liquid Biopsy has the potential to transform healthcare and biomedical research. The presentation will give an overview on QIAGEN’s approach to provide integrated solutions for isolation of circulating biomarkers and their analysis. The presentation will cover new and existing QIAGEN solutions ranging from sample stabilization, analyte purification, to analysis.

Networking Cocktail Reception for All Delegates, Speakers, Sponsors and Exhibitors

Close of Day 1 of the Conference. Extracellular Vesicles Dinner Short Course Begins.

Morning Coffee, Breakfast Pastries, and Networking

Evaluation of a Hybrid Exosome/Virus Vector System for Gene Transfer and Therapy in Preclinical Models of Human Disease
Casey Maguire, Assistant Professor of Neurology, Harvard Medical School, United States of America

Adeno-associated virus (AAV) vectors have shown remarkable efficiency in a number of preclinical models of disease in several organs including the eye, brain, muscle, heart, and liver. A major limitation to long term transduction, especially via the systemic route, is pre-existing immunity (humoral and cell-mediated) as well as the high vector load required to achieve sufficient levels of gene expression in the desired organ. Natural nanoparticles released from all cells, called exosomes, may have utility in creating better AAV vectors for human gene therapy. We have previously shown that harvesting AAV vectors from the media of 293T producer cells contains AAV vectors associated with exosomes (called exo-AAV). We have gone on to demonstrate that 293T-derived exo-AAV can evade neutralizing anti-AAV antibodies and greatly increase transduction in mice. Here we will report on our findings of the gene transfer capabilities of exo-AAV to several organs of interest in human disease including the brain, liver, muscle, eye, and cochlea.

Exosomes as Therapeutic RNAs Delivery Vesicles
Anastasia Khvorova, Professor, RNA Therapeutics Institute, University of Massachusetts Medical School, United States of America

Applications of RNA interference for neuroscience research have been limited by a lack of simple and efficient methods to deliver oligonucleotides to primary neurons in culture and to the brain. Here, we show that primary neurons rapidly internalize hydrophobic siRNAs (hsiRNAs) added directly to the culture medium without lipid formulation. We identify functional hsiRNAs targeting the mRNA of huntingtin, the mutation of which is responsible for Huntington’s disease, and show that direct uptake in neurons induces potent and specific silencing in vitro. Moreover, a single injection of unformulated hsiRNA into mouse brain silences Htt mRNA with minimal neuronal toxicity. Thus hsiRNAs embody a class of therapeutic oligonucleotides that enable simple and straightforward functional studies of genes involved in neuronal biology and neurodegenerative disorders in a native biological context.

Non-coding RNAs as Circulating Markers and Exosomal Therapeutic Targets in Neurological Disorders
Chaya Brodie, Professor, Henry Ford Hospital, United States of America

Coffee Break and Networking in the Exhibit Hall

Characterization of plasma Extracellular Vesicles with Human Age
Erez Eitan, Researcher, Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, United States of America

Extracellular vesicles (EVs; also known as exosomes) are released from most cells into the extracellular environment and circulation. Thus, EVs play a potentially important role in intercellular communication between different cell types throughout the body.  Research has primarily focused on characterizing the proteins, lipids, and RNAs contained in EVs from various disease states, including cancer. As many of the pathological conditions where EVs have been implicated are age-related, it is likely that there changes in EVs with age in humans.  However, little is known about whether there are age-dependent changes in EV concentration (size and number), composition or function with human age.  We used a longitudinal cohort of 75 individuals from the Healthy Aging in Neighborhoods of Diversity across the Life Span (HANDLS) study to characterized EV concentration and surface proteins. We found a significant reduction in their concentration with age. Establishing a profile of EVs with human age will further aid in the development of diagnostics and therapies using EV-designed technologies for age-related diseases.

Networking Lunch, Discussions with Exhibitors and View Posters

Technology Spotlight:
Lunch Technology Spotlight: Increasing the Sensitivity of Next-Generation Gene Expression Workflow for microRNAs from Bodily Fluids, Extracellular Vesicles and Small Sample Input - Sample Preservation, RNA Extraction and Sequencing Library Preparation
Bernard Lam, Senior Research Scientist, Norgen Biotek Corporation

Heterogeneity of Cancer-Derived Extracellular Vesicles
Dolores Di Vizio, Professor, Cedars Sinai Medical Center, United States of America

Cancer cells release oncogenic cargo in bioactive extracellular vesicles (EVs) that can alter the tumor microenvironment and elicit behavioral responses in target cells. This discovery points to a role in tumor evolution for a conserved and finely regulated biological process that allows intercellular transfer of bioactive proteins, nucleic acids and lipids in the form of pre-assembled plasma membrane structures. In the last few years, it has emerged that every cell can release different types of EVs, with selective cargo and function. Studies on exosomes, for example, have demonstrated that diverse types of exosomes can be released by tumor cells. Several new methodologies are being interrogated to differentiate between exosomes with different cargo/functions. Different populations of exosomes as well as of larger EV types including the large oncosomes could provide clinically relevant information.

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

Cancer cells undergo a number of stresses, many of them self-inflicted, but often do not appear to suffer the consequences of those stresses. In some cases, the stress responses may actually prove 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 and 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.

Microfluidic Phenotyping of Circulating Exosomes for Cancer Diagnosis
Yong Zeng, Associate Professor, University of Florida, United States of America

EGFR Signaling Regulates Extracellular Vesicle microRNA Contents
Alain Charest, Associate Professor, Dept of Neurosurgery, Tufts University Medical Center, United States of America

The focus of the research here is on leveraging clinically relevant genetically engineered mouse models of primary malignant brain cancer to study central aspects of gliomagenesis and molecular responses to therapeutic interventions. Recent work from the laboratory involves studies on the control exerted by EGFR signaling on extracellular RNA composition in Glioblastoma Multiforme.

Smartphone-based Optofluidic Exosome Diagnostic for Concussion Recovery
Jina Ko, Researcher, University of Pennsylvania, United States of America

We developed a smartphone-based optofluidic platform to measure brain-derived exosomes. Sample-to-answer on our chip is <1 hour and the key innovation is an optofluidic device that can detect enzyme amplified exosome biomarkers, and is read out using a smartphone camera. Using this approach, we profiled GluR²⁺ exosomes to detect and monitor concussion.

Short Coffee Break

Microfluidics for Exosome Analyses
Hakho Lee, Associate Professor, Director of the Biomedical Engineering Program, Massachusetts General Hospital / Harvard Medical School, United States of America

Nanoplasmonic 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 an exosome analysis technology named nPLEX (nano-plasmonic exosome). 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.

Potential Role of Exosomes in the Development of Malignant Mesothelioma
Phillip Munson, Researcher, University of Vermont, United States of America

Preliminary insights on the characterization of exosomes, and their cargo, from normal and asbestos exposed epithelial cells, as well as studies on the effects, and in vivo localization, of these exosomes on possible target mesothelial cells and tissues.

Mechanism and Application of ARRDC1-mediated Microvesicles (ARMMs)
Quan Lu, Associate Professor, Harvard School of Public Health, United States of America

I will discuss the molecular mechanism and physiological functions of ARMMs. I will also present data on how ARMMs may be used for therapeutic delivery of bioactive large molecules.

Close of Day 2 of the Conference