Nano-flow cytometry of single extracellular vesicles reveals subpopulation differences across cell types and pharmacological perturbations

Extracellular vesicles (EVs) are lipid bilayer-enclosed particles released by most cell types, which can transfer signals and cargoes between cells. EVs released by a single donor cell source are increasingly recognized as extremely heterogeneous, in terms of size, intracellular origin, and cargo composition. Analyzing large numbers of EVs at the single vesicle level is therefore the only way to truly decipher their heterogeneity. Here, we developed a reliable pipeline of single EV analysis using a nanoparticle-dedicated flow cytometer, which detects particles and measures their size down to 55 nm in diameter, without the need for vesicle pre-immobilization or fluorescent label. We show that titrating each antibody, eliminating unbound antibodies and using EVs devoid of the analyzed markers as negative controls are required to reliably quantify the proportion of EVs bearing none or any combination of two markers, as well as to measure their sizes. We thus observed, depending on the cell source (human cell lines MDA-MB-231, HeLa, A549), variable proportions of EVs bearing none of the CD9, CD81 and CD63 tetraspanins often used to define EVs, and of single- and double-positive EVs for each of these markers. We also observed CD29 (ITGB1) as a protein detected as frequently on EVs as CD9, while other transmembrane proteins (CD44, SSEA-4, CD98), were detected in a small proportion of EVs, and mostly of relatively large size. Finally, we used this pipeline to uncover differential effects of small molecule drugs on subtypes of EVs, and showed that Homosalate increased the proportion of CD9+/CD63+ EVs while two other drugs, Dipivefrin hydrochloride and Metaraminol bitartrate, instead increased the proportion of CD9-/CD63+ EVs. Overall, nano-flow cytometry allows to reliably quantify proportions of EV subpopulations suggested by bulk analyzes of EV markers, at single EV resolution.