Identification of Extracellular Vesicle Subsets with nanoFACS

Authors

• JC Jones
• A Morales-Kastresana
• JA Berzofsky

Abstract

Different cells, under different conditions, release distinctive types Extracellular Vesicles (EVs), including 40-120 nm exosomes, that have specific effects that are presumed to be due to the specific protein, lipid, or RNA repertoires of those different types of EVs. We proposed that these different EV “types” could be studied as “subsets,” in the same manner that immune cell subsets are studied with flow cytometry. We developed nanoFACS, a high-resolution, high-speed flow cytometric approach, to identify and sort tumor and immune subsets of EVs released in the context of hypoxia and/or radiation. We used high-sensitivity mass spectrometric protein identification and RNAseq to identify surface markers and exRNA repertoires associated with conventional and radiosurgical doses of radiation. We further compared exRNA repertoires of size-fractionated EVs, and surface-marker-fractionated EV subsets from irradiated and unirradiated conditions, and tested the hypothesis that EV subset enrichment and sorting with high resolution, single particle EV flow cytometry, results in higher sensitivity and specificity for radiation-specific exRNA profiles. Re-analysis of EV subsets sorted by nanoFACS with our system demonstrated >95% purity, with increased sensitivity of the tumor- and state (hypoxia or radiation)- associated miRNA profiles. In vitro assays confirmed biological activity of nanoFACS-sorted particles. Functional single particle subset sorting has not been achieved with any other EV/exosome preparation method. Use of method to sort and study EVs from human plasma offers significant potential for basic biological studies, and can be used as a “liquid biopsy” that provides a non-invasive means to assess tumor status and response to treatment.

Scientific Focus Area: Cancer Biology

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