Comparative marker analysis of extracellular vesicles in different human cancer types

Several cell types, including tumour cells, secrete extracellular vesicles (EVs), and tumour-derived EVs play a role in cancer initiation and progression. These vesicles include both a common set of membrane and cytosolic proteins and origin-specific subsets of proteins that likely correlated to cell type-associated functions. To confirm the presence of EVs in the preparations, researchers have identified so-called EV marker proteins, including the tetraspanin family proteins and such cytosolic proteins as heat shock 70 kDa protein 4 (HSP70) and tumour susceptibility gene 101 (TSG101). However, studies have shown that some EV markers are not always present in all EVs, which not only complicates the identification of EVs but also precludes the quantitative evaluation of EV proteins. Thus, it is strongly required to explore well-conserved EV marker proteins that are present at similar levels, regardless of their tissue or cellular origin. In this study, we compared the presence of 11 well-known EV marker proteins by immunoblotting using EVs isolated from 4 human prostate cell lines and 5 human breast cell lines, including cancer cells with different phenotypes. We found that all the tested EVs were positive for CD9 and CD81, with similar abundance that was irrespective of the EV origin. In contrast, other EV marker proteins, such as TSG101, Rab-5b and CD63, were detected in an inconsistent manner, depending on the origin of the EVs. Thus, we propose that the detection of CD9 and/or CD81 should ensure the presence of EVs.