Abstract

Deciphering the Vesicle Code: Making Sense of EV Heterogeneity

John Nolan, CEO, Cellarcus Biosciences, Inc.

Cells release extracellular vesicles (EVs) that can carry molecular cargo, including lipids, proteins, and nucleic acids, to nearby or distant cells and affect their function. Understanding the mechanisms of EV biogenesis, uptake and transport has the potential to lead to new biomarkers, diagnostics, and therapeutics. A limit to realizing this potential is the ability to measure individual EVs and their cargo quantitatively and reproducibly. Conventional bulk biochemical analyses, which report only the total amount of cargo in an EV preparation, cannot effectively assess compositional heterogeneity. Single EV analysis methods are required, but conventional tools are challenged to accurately measure small, dim particles. We have developed a vesicle flow cytometry (vFC) method that can detect and size individual EVs to 70 nm, and measure surface cargo to ~25 molecules/EV. vFC allows the identification and characterization of vesicle sub-types within heterogeneous populations in culture supernatants of biofluids. Single EV analysis using vFC reveals striking differences in the expression of canonical vesicle cargo such as tetraspanins, as well as other surface markers including integrins, tumor markers, and carbohydrates on EVs in biofluids and even EVs collected from cultured cell lines. vFC-based single EV immunophenotyping will enable us to understand the origins and functions of EVs in much the same way cell-based immunophenotyping has enabled a detailed understanding of the immune system, and lead to a new generation of EV-based biomarkers, diagnostics, and therapeutics.