Protein Composition Reflects Extracellular Vesicle Heterogeneity

Abstract

Extracellular vesicles (EVs) are membrane-enclosed particles that are released by virtually all cells from all living organisms. EVs shuttle biologically active cargo including protein, RNA, and DNA between cells. When shed by cancer cells, they function as potent intercellular messangers with important functional consequences. Cells produce a diverse spectrum of EVs, spanning from small vesicles of 40-150 nm in diameter, to large vesicles up to 10 μm in diameter. While this diversity was initially considered to be purely based on size, it is becoming evident that different classes of EVs, and different populations within one EV class may harbor distinct molecular cargo and play specific functions. Furthermore, there are considerable cell type-dependent differences in the cargo and function of shed EVs. This review focuses on the most recent proteomic studies that have attempted to capture the EV heterogeneity by directly comparing the protein composition of different EV classes and EV populations derived from the same cell source. Recent studies comparing protein composition of the same EV class(es) derived from different cell types are also summarized. Emerging approaches to study EV heterogeneity and their important implications for future studies are also discussed.

Keywords: biomarkers; cancer; extracellular vesicles; heterogeneity; protein profiling.

Figure 1.

Heterogeneity of EV classes released by cells. Cells release EVs that can be classified into either s-EVs (40–150 nm) or l-EVs (200 nm–10 μm) according to their size. s-EVs encompass endosome-derived exosomes, which can be further subdivided into small (Exo-S) and large (Exo-L) exosomes, as well as nonendosomal small ectosomes. l-EVs include large ectosomes, microvesicles, migrasomes, exophers, apoptotic bodies, and large oncosomes.

Figure 2.

Heterogeneity of EVs recovered from prostate cancer cell media by ultracentrifugation at 10 000 × g. Transmission electron microscopy image showing particles of different sizes and densities in the EV pellet stained with osmium tetroxide. Prior to pelleting EVs at 10 000 × g for 30 min, conditioned cell media was cleared by two low-speed centrifugation steps: 300 × g for 10 min, followed by 2800 × g for 10 min. Scale bar: 600 nm

Figure 3.

Complexity of exosomes secreted by polarized epithelia. A) Polarized epithelial cells secrete s-EVs with different protein cargo from the apical (green) versus basolateral (yellow) sides of the cell, potentially exposing them to different recipient cells. These s-EVs can be further subdivided into small (Exo-S, 60–80 nm) and large (Exo-L, 90–120 nm). B) Differences in tropism as well as available recipient cells may result in different EV populations signaling with different recipient cells. For instance, the basolateral secreted EVs have a greater chance to come into contact with endothelial cells and fibroblasts than apical secreted EVs.