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Page 6 Graner. Extracell Vesicles Circ Nucleic Acids 2020;1:3-19 I http://dx.doi.org/10.20517/evcna.2020.08

Laura showed that transfer of EVs from astrocytes, generated from patients with amyloid ALS, to healthy
neurons induced neuronal cell death. Scores of small RNA levels were significantly altered in those EVs,
including downregulation of miR494, which affects pathways controlling axonal growth cone collapse.
Supplementation of miR494 into astrocyte EVs and application of the EVs onto neurons rescue neuron
survival, neuronal length, and complexity (increases in nodes and intersections). These studies reveal the
potential of therapeutic cargo for use in EV transfer, based on knowledge of the EV-driven pathology.

Crislyn D’Souza-Schorey (University of Notre Dame, US) spoke of microvesicles (MVs), which are
extracellular vesicles that bud from cell surfaces, and the loading of pre-miRNA cargo into tumor EVs. She
noted the extraordinary heterogeneity of shed vesicles, particularly those released by tumor cells. Her group
has identified ARF6, which is involved in endosomal/endocytic recycling; ARF6 activation correlates with
increased miRs in MVs. They also found that XPO5 is a binding partner of ARF6; XPO5 plays roles in pre-
miR transport from the cell nucleus to the cytoplasm, and is found in tumor MVs, which contains a pre-miR
processing machinery. Tumor MVs contain complexes of ARF6/XPO5 and pre-miRs; translocation of XPO5
to the MVs involve a complicated interaction involving CK2, RAN and cytohesion family guanine exchange
factors. These routes suggest that the intricate mechanisms for the delivery of cellular cargo into different EV
types further display the complexity of these biologic and pathologic processes.

Qin Zhang (Vanderbilt University) finished out the session discussing a recently identified form of
extracellular particle, the exomere. Exomeres are non-membranous particles (~35 nm diameter) of unknown
biogenesis and no clear biologic function. Researchers previously used a biophysical separation technique
called asymmetric-flow field-flow fractionation (AF4) to isolate and identify exomeres; Qin performed high
speed ultracentrifugation (UC) on cell culture medium (167K × g; collect supernatant; UC again @ 167K
× g; harvest pellet) to obtain similar extracellular particles, which were proteomically distinct from small
EVs/exosomes. Exomere-enriched proteins included glycolytic enzymes and AGO RNA binding proteins.
Exomeres also contained the EGFR ligand, AREG, which in that format was capable of inducing prolonged
EGFR signaling in recipient cells, leading to enhanced tumor organoid growth. Another exomere-enriched
protein is the sialyltransferase, STGAL1 (membrane and soluble forms), which is highly enzymatically active
in exomere form on recipient cells. Curiously, the SARS-CoV-2 spike protein receptor, ACE2, is displayed
on exomeres from some cell lines and can bind the viral spike protein via the S1 receptor binding domain,
suggesting that exomeres could serve as decoys against SARS-CoV-2 spike protein-initiated infection.

The final session of the first day of ASEMV2020 was devoted to analytical techniques, and was hosted by
Kendal van Keuren-Jensen (Translational Genomics Research Institute, US) and Tijana Jovanovic-Talisman
(City of Hope, US). The first speaker was Lisa Meyer (Exosome Diagnostics, US; Germany) who presented an
exosome/EV separation and characterization workflow using products from several partner companies. This
included a kit-based separation technology, an automated Western blot device in the form of a capillary flow
electrophoresis apparatus, and validated primary antibodies against exosome/EV and theoretical non-EV
proteins to determine potential contaminants. She showed results confirming the detectability of appropriate
target proteins in the correct (EV-containing) fractions from the separation scheme. The system provides
scalability, rapid quantification, and good detection limits. They are expanding the antibody repertoire and
improving the separation modules, going forward.

Ryan McNamara (University of North Carolina, Chapel Hill, US) introduced a super-resolution microscopy
technique called direct stochastic optical reconstruction microscopy (dSTORM). As exosome/EV sizes
are below the limits of resolution of conventional light microscopy, more advanced forms of imaging are
necessary to accurately discern single EVs and their characteristics, thereby avoiding non-physiologic
manipulations that are likely pursued in what might be done for electron microscopy. dSTORM has multi-
channel capability that could detect two different membranes dyes within the lens apochromatic correction