Tetraspanin positivity as a function of extracellular vesicle size measured by a modified immuno-TEM protocol
The identification of surface markers that correlate with specific subpopulations of extracellular vesicles (EVs) is important for EV identification, classification, purification, sorting, and functional analysis. Tetraspanins such as CD9, CD63 and CD81 were once considered to be universal markers of exosomes: small EVs released into the extracellular space when late endosomes / multivesicular bodies fuse with the plasma membrane. In contrast, plasma membrane-derived ectosomes (also called microvesicles) have a different biogenesis, were often regarded as being larger than exosomes, and display a different surface proteome. However, recent studies have shown that tetraspanins such as CD9 and CD81 are highly enriched on ectosomes derived from various sources. Thus, it is currently unclear how tetraspanin content correlates with specific EV subpopulations. Here, we present a modified immuno-TEM protocol that can be easily applied to heterogeneous EV populations comprising both small and large EVs (and presumably also a collection of exosomes and ectosomes). In EVs purified from U-2 OS cells by size-exclusion chromatography, we show that the percentage of particles positive for CD9 and CD81 is significantly higher in the subpopulation of EVs [≤] 100 nm (i.e., small EVs). These results also explain discrepancies in the size distribution profiles that we obtained using the same EV preparations by alternative single-vesicle characterization platforms such as nano flow cytometry and SP-IRIS / ExoView. The latter, when used to capture tetraspanin-positive particles, returns a population that is relatively small in size.