Multiparametric Single-Vesicle Flow Cytometry Resolves Extracellular Vesicle Heterogeneity and Reveals Selective Regulation of Biogenesis and Cargo Distribution

Mammalian cells release a heterogeneous array of extracellular
vesicles (EVs) that contribute to intercellular communication by means
of the cargo that they carry. To resolve EV heterogeneity and determine
if cargo is partitioned into select EV populations, we developed a
method named “EV Fingerprinting” that discerns distinct
vesicle populations using dimensional reduction of multiparametric
data collected by quantitative single-EV flow cytometry. EV populations
were found to be discernible by a combination of membrane order and
EV size, both of which were obtained through multiparametric analysis
of fluorescent features from the lipophilic dye Di-8-ANEPPS incorporated
into the lipid bilayer. Molecular perturbation of EV secretion and
biogenesis through respective ablation of the small GTPase Rab27a
and overexpression of the EV-associated tetraspanin CD63 revealed
distinct and selective alterations in EV populations, as well as cargo
distribution. While Rab27a disproportionately affects all small EV
populations with high membrane order, the overexpression of CD63 selectively
increased the production of one small EV population of intermediate
membrane order. Multiplexing experiments subsequently revealed that
EV cargos have a distinct, nonrandom distribution with CD63 and CD81
selectively partitioning into smaller vs larger EVs, respectively.
These studies not only present a method to probe EV biogenesis but
also reveal how the selective partitioning of cargo contributes to
EV heterogeneity.