Work

Tetraspanin Positivity as a Function of Extracellular Vesicle Size Measured by a Modified Immuno-TEM Protocol

Year: 2025

Type: article

Abstract: 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, are 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 only analyzes EVs that were previously captured based on the presence of tetraspanins, which introduces a bias in their size distribution.

Source: ACS Applied Bio Materials

DOI: 10.1021/acsabm.5c00591