Mid-Morning Break

The Role of Core Facilities and Emerging Technologies in Maximizing Rigor and Reproducibility of EV Quantification and Characterization and Following MISEV Guidelines
Rachel DeRita, Director, Extracellular Vesicle Core Facility, University of Pennsylvania School of Veterinary medicine, United States of America

It remains very clear in the field of extracellular vesicle (EV) research that the rapid rate of increase in publications and expansion of interdisciplinary clinical EV interest has created the need for increased standardization and access to the appropriate technologies to uphold these standards. As the first core facility in the United States with the sole intention of creating a space where users can both isolate and characterize EVs, we provide a central location for the facilitation of EV research via access to multiple technologies (both established and emerging) such as resistive pulse sensing, nanoparticle tracking analysis, ultracentrifugation, high-performance liquid chromatography, flow cytometric analysis of EVs and additional immune or fluorescence-based EV characterization techniques. We surveyed a group of leading scientific investigators and researchers in varying stages of their scientific careers in the Mid-Atlantic region of the US.  The survey data demonstrate applications of greatest current and future interest to be employed in a shared lab resource. The current demand is highest for isolation services, ultracentrifugation and NTA, with a gradually increasing demand for immunophenotying analyses such as the ExoView chip array, fluorescent NTA and flow cytometry. We additionally present strategies and data-based examples of how shared resource facilities can facilitate multifactorial and rigorous EV characterization in accordance with MISEV guidelines, and encourage collaboration among EV researchers. In order to answer the larger remaining questions in the EV field such as the isolation of specific EV subsets, EV tracking between cells and the use of EVs for biomarker discovery and drug delivery, it is essential that shared resource facilities interact not only with investigators, but with each other to integrate the necessary resources to progress.

Lunch Break

Revealing the Diversity of EVs with Single Vesicle Analysis
John Nolan, CEO, Cellarcus Biosciences, Inc., United States of America

Extracellular vesicles (EVs) appear to be involved in many physiological processes and have attracted great attention as potential diagnostic and therapeutic targets. The realization of this potential has been hampered by the lack of analytical methods with the necessary specificity, sensitivity, and quantitative rigor needed for robust hypothesis testing, biomarker development, and EV engineering. New analytical tools with improved specificity and sensitivity are revealing the diverse constituents of biofluid preparations that had been previously referred to as “exosomes” and “microvesicles,” and are forcing a re-evaluation of both methods and nomenclature. Single vesicle analysis represents the ultimate level of EV analysis, and we have been using single vesicle flow cytometry (vFC) to count, size, and measure the molecular cargo on individual EVs at a large scale. I will review the state of EV analytics, highlight critical elements of single EV analysis, and share insights into EV diversity that have emerged from the application of quantitative analytical methods to the study of EVs.

Accurate Concentration of Biological Nanoparticles – Why and How?
Jean-Luc Fraikin, CEO, Spectradyne

Extracellular vesicles (EVs), virus and non-viral drug delivery vehicles are rapidly moving from the research lab into real-world applications.  Why should we care about measuring the concentration of these biological nanoparticles accurately? In the case of a COVID vaccine for example, we should care because particle concentration is the dose!  In a broad range of applications, particle concentration is a critical parameter that must be carefully controlled in order to develop safe and effective products.  This talk will include a comparison of methods and examples to illustrate how accurate concentration measurements are important for good quality science.

Know What’s In Your Sample: Phenotyping of Extracellular Vesicles (EVs) with Fluorescent Nanoparticle Tracking Analysis (f-NTA)
Sven Rudolf Kreutel, Chief Executive Officer, Particle Metrix GmbH and CEO, Particle Metrix Inc., USA

In the last decade, Nanoparticle Tracking Analysis (NTA) has emerged as a vital and fast characterization technology for Extracellular Vesicles, Exosomes and Microvesicles. in the size range from 30 nm to 1 µm. While getting a particle size distribution, the user typically cannot discriminate whether the particle is a vesicle, protein aggregate, cellular trash or an inorganic precipitate from scatter data alone. The fluorescence detection capability of NTA enables the user to gain specific biochemical information about the particle surface and increases the resolution and the discrimination power. However, traditional NTA instruments are equipped with one laser wavelength in operation. This limits the detection of biomarkers to only a couple of fluorescence dyes. For change of excitation wavelength, it was necessary to change the laser source, a time and sample consuming step. To overcome this issue Particle Metrix developed a unique QUATT laser technology and now allows the rapid analysis of biomarker concentrations or ratios with four excitation lasers on the same sample. Here we report the multi-fluorescence detection of four independent biomarkers (CD63, CD81, CD9 and CD41) in one NTA sample with the new Particle Metrix ZetaView® QUATT for the first time.

Analyzing Extracellular Vesicles With The Amnis® ImageStream®X Mk II High Gain Mode
Haley Pugsley, Manager and Senior Scientist, Luminex Corporation

The importance of EVs has become apparent, as they are key mediators of intercellular communication. However, quantifying and characterizing EVs in a reproducible and reliable manner is challenging due to their small size—exosomes range from 30 to 100 nm in diameter. Flow Cytometry is an attractive method for measuring EVs; however, this has been challenging since fl ow cytometers were designed to measure and detect cells. High Gain mode on the Amnis® ImageStream®X Mk II Imaging Flow Cytometer was designed to address the challenges of measuring particles that are much smaller than cells. This presentation demonstrates the improved small particle detection, including EVs, using this new High Gain mode.

Mid-Afternoon Break

Nuclear Transport of Extracellular Vesicles
Aurelio Lorico, Professor of Pathology, Touro University Nevada School of Medicine, United States of America

We have recently identified a novel nuclear path, where Rab7+ late endosomes enter into nuclear envelope invaginations and mediate the nuclear translocation of EV-derived biomaterials, such as proteins and RNA. The close association of three proteins; endoplasmic reticulum (ER)-localized vesicle-associated membrane protein (VAMP)-associated protein VAP-A, oxysterol-binding protein (OSBP)-related protein ORP3 and small GTPase Rab7 is essential for the entrance and/or maintenance of Rab7+ late endosomes into NEI and the nuclear transfer of EV-derived biocomponents (e.g., proteins and nucleic acids) that in turns may regulate the gene expression in host (or EV-targeted) cells (Santos et al., J. Biol. Chem. 2018).

Close of Day 1 of the Conference

Extracellular Vesicles in Cytokine Regulation
Leonid Margolis, Head, Section of Intercellular Interactions, Eunice Kennedy-Shriver National Institute of Child Health and Human Development, NIH, United States of America

Extracellular vesicles play an important role in human immuno-modulation mediated by cytokines both in normal condition and in pathologies. We found that both myocardial infarction and in HIV-1 infection extracellular vesicles are associated with immune activation, an important driver of the disease.

Coordination of Functionally Related mRNAs and Proteins in Granules, GW/P bodies, and Vesicles
Jack Keene, James B. Duke Professor, Duke University Medical Center, United States of America

Gene expression is determined by transcriptional and post-transcriptional mechanisms that coordinate regulatory layers and are localized in many cellular sub-environments. Post-transcriptional regulatory layers consist of trans RNA-binding proteins and multiple copies of cis messenger RNAs (mRNAs) and noncoding RNAs (miRNAs and other ncRNAs) that together form modular RNA regulons (RBPs) that in turn, coordinate the latter state of gene expression on a global level. The multi-targeting of mRNAs in RNA regulons include cis binding sites that are: a) RBP sequence specific, b) miR binding sites, c) modified mRNAs (e.g. methylated), that together produce functionally related proteins that can be found in cellular vesicles, bodies or granules by translation. Thus, RNA regulons are found in nearly all species and coordinate RNA turnover, localization, and/or translation globally in response to biological activation or repression. RNA regulons have myriad ramifications for biological coordination. Over time, dozens of RNA regulons have been reported in many biological systems and in dozens of species. For examples from the field: 1) PUF RNA regulons coordinate fungal metabolism and pathogenesis dating back over 500 million years; 2) trypanosome RNA regulons coordinate parasite differentiation in the blood at a time when transcription is silenced. 3) dozens of mammalian RNA regulons have biological and physiological ramifications in which mRNAs encoding functionally related proteins bound to several of the ~1,500 RBPs now known RBPs. Thus, RNA regulons can efficiently utilize and locate protein building blocks and regulatory factors that function as complex traits. Moreover, these overlapping mRNA subsets are highly dynamic and responsive to intrinsic and extrinsic signals. To understand these dynamic processes our lab developed Digestion Optimized-RIP-seq that is quantitative and applicable to understanding how combinatorial mechanisms regulate dynamic coordination of RNA targets during growth and differentiation, as well as revealing novel RNA regulons in cancer, neurodegeneration and liver development/regeneration. Our goal is to teach these technologies to students and fellows in order to decipher the remodeling of RNA regulons so to reveal underlying mechanisms of disease.

Single Particle Detection of Extracellular Vesicles and Native SARS-CoV-2 Virions by Microfluidic Resistive Pulse Sensing
Zoltan Varga, Biological Nanochemistry Research Group, Research Centre for Natural Sciences (RCNS), Hungary

Microfluidic resistive pulse sensing (MRPS) is the nanoscale implementation of the Coulter principle in a microfluidic cartridge. As such, MRPS is a non-optical, single-particle detection method to measure the size distribution and concentration of nanoparticles. MRPS can be used to characterize extracellular vesicles (EVs). Enveloped viruses such as SARS-CoV-2 are spherical biological nanoparticles in the hundred nanometer size range, therefore MRPS is the method of choice for their characterization. In this study, MRPS was used to measure the size distribution of various EVs and SARS-CoV-2. The analysis of the supernatant of infected Vero cells indicated an envelope diameter of approximately 80 nm for the SARS-CoV-2 virions, which is in good agreement with previous cryo-EM studies. The detection of more than ten thousand virions made it possible to determine the size distribution of SARS-CoV-2 and paves the way for the MRPS-based detection of COVID-19 infection.

Mid-Morning Break

Extracellular Vesicle Glycosylation
Johnathon Anderson, Assistant Professor, University of California-Davis, United States of America

Up to 50% of the proteins encoded in the human genome are predicted to contain glycosylation sites. Aberrant glycosylation is a hallmark of several diseases, including many types of cancer from different origins. Although the functional significance of such patterns of glycan expression and presentation are not yet well characterized, tumor growth, metastases and immunoregulation have all been associated with such abnormal displays of glycosylation within the tumor microenvironment. Our group is interested in understanding glycosylation dynamics in the context of cancer-associated fibroblasts (CAFs), and mesenchymal stromal cells (MSCs), which serve as a source of CAFs in some types of cancer. One of the most remarkable changes in cancer glycosylation is the aberrant expression of sialic acid–bearing glycans called sialoglycans. Cells within the tumor bed are often covered with a dense layer of sialoglycans which have emerged in recent years as potent immune modulators that promote tumor immune evasion. Sialoglycans serve as the cognate ligands for a family of immune checkpoint receptors called sialic acid–binding immunoglobulin-like lectins (Siglecs), which are expressed by various populations of leukocytes. Preclinical studies have shown that tumor associated sialoglycans negatively influence immune cell function by interacting with the immune-inhibitory Siglec family members. Our group is interested in how sialoglycan expression is regulated in MSCs and CAFs, and what functional role they may play in their derived extracellular vesicles.

EVs and Viruses Panel Discussion by Lucia Languino and Michael Graner, Conference Chairpersons

• Status of the Field
• What have we learnt and what are the research trends
  
  
• The Panelists will weigh-in on these topics -- The Panelists Are:

Esther Nolte-‘t Hoen
Fatah Kashanchi
Jack Keene
Jan Lötvall
Jennifer Jones
Leonid Margolis

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