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Extracell Vesicles Circ Nucleic Acids 2020;1:20-56 I http://dx.doi.org/10.20517/evcna.2020.10 Page 29

10. Advancing extracellular vesicle characterization with quantitative single molecule
localization microscopy

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Authors: Kathleen M Lennon , Adam L Maddox , Andras Saftics , Matthew S Brehove , Devin L
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Wakefield , Saumya Das , Kendall Van Keuren-Jensen , Gagandeep Singh , Tijana Jovanovic-Talisman 1
E-mail: klennon@coh.org
Affiliations:
1 Beckman Research Institute, City of Hope, Duarte, CA, USA.
2 Cardiology Division, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.
3 Neurogenomics Division, Translational Genomics Research Institute, Phoenix, AZ, USA.
4 City of Hope, Duarte, CA, USA.
Abstracts: Quantitative Single Molecule Localization Microscopy (qSMLM) can quantify individual
biomolecules in cells with nanoscale precision. However, because of several technical hurdles, a similarly
robust quantification is often difficult to achieve when the methodology is applied to extracellular vesicles
(EVs). One significant challenge has been to rigorously isolate and characterize disease- specific and
tissue-specific subpopulations of EVs. Our approach to probe EV subpopulations entails three main
advancements: 1) more effective methods to label the EVs for SMLM imaging; 2) an optimized protocol
for affinity isolation and imaging of EVs; and 3) advanced algorithms to robustly detect size and count
the number of biomolecules within individual EVs. Briefly, size exclusion chromatography was first used
to isolate EVs from either cultured cell media or patient plasma. Then, EV membranes were labeled with
fluorescent reagents: dyes, lectins, or antibodies. The excess fluorescent molecules were removed by size
separation using concentrating filters, core beads, or size exclusion columns. Finally, EVs enriched in
specific receptor(s) were affinity isolated onto coverslips, imaged, and analyzed. Using this comprehensive
approach, we have quantified EVs isolated from the plasma of patients who have pancreatic cancer.
Specifically, we have determined the number of isolated EVs, their size, and the abundance of several
biomarkers. Compared to healthy controls, patients with pancreatic cancer exhibited a distinct population
of larger EVs enriched in epidermal growth factor receptor and carbohydrate antigen 19-9. Ultimately,
when this approach is paired with traditional methods, it may shift the paradigm for comprehensively
prehensively characterizing cancer-specific and organ-specific EVs.

11. Hypocalcemic-induced exosome secretion drives differential metastatic progression of
epithelial ovarian cancer

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Authors: Amy H. Lee , Deepraj Ghosh , Nhat Quach , Michelle R. Dawson 1,2
E-mail: amy_lee1@brown.edu
Affiliations:
1 School of Engineering, Center for Biomedical Engineering, Brown University, Providence, RI, USA.
2 Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI, USA.
Abstracts:
Introduction: Serous epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy and
peritoneal fluid (ascites) build-up is common in late stage EOC. This ascites is an abundant source of
cell-secreted exosomes. Gynecological conditions are often associated to decreased blood serum calcium
concentrations and elevated exosome production. Exosomes are continuously secreted through the
endosomal pathway; however, their release can be triggered by various physiologic stimuli, leading to
exosome heterogeneity. We have demonstrated that mimicking hypocalcemia conditions by chelating
extracellular calcium releases a unique population of exosomes from an EOC cancer cell line. We