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.

哺乳动物细胞可释放出一系列异质性的细胞外囊泡（extracellular vesicles, EVs），这类囊泡可通过其所携带的货物分子介导细胞间通讯。为解析EV的异质性并探究货物分子是否会被分配至特定的EV亚群中，我们开发了一种名为“EV指纹图谱（EV Fingerprinting）”的方法：该方法通过对定量单细胞EV流式细胞术采集的多参数数据进行降维分析，来区分不同的囊泡亚群。研究发现，EV亚群可通过膜有序度与EV粒径的组合特征加以区分，这两项特征均可通过对掺入脂质双层的亲脂性染料Di-8-ANEPPS的荧光特征进行多参数分析获得。通过分别敲除小GTP酶Rab27a以干扰EV分泌，以及过表达EV相关四跨膜蛋白CD63以干扰EV生物发生，实验结果显示EV亚群与货物分子分布均出现了特异性的显著改变。其中，Rab27a会显著影响所有膜有序度较高的小型EV亚群；而CD63过表达则仅特异性地增加了一类膜有序度处于中等水平的小型EV亚群的生成量。后续的多重实验结果表明，EV的货物分子呈现出明确且非随机的分布特征：CD63与CD81会分别选择性地分配至较小粒径与较大粒径的EV亚群中。本研究不仅提供了一种探究EV生物发生机制的方法，同时也揭示了货物分子的选择性分配如何促成EV的异质性。