Volumetric microscopy of CD9 and CD63 reveals distinct subpopulations and novel structures of extracellular vesicle in situ in triple negative breast cancer cells

Secreted extracellular vesicles (EVs) are now known to play multifaceted roles in biological processes such as immune responses and cancer. The two primary classes of EVs are defined in terms of their origins: exosomes are derived from the endosomal pathway while microvesicles (ectosomes) bud from the cell membrane. However, it remains unclear whether the contents, sizes, and localizations of subpopulations of EVs can be used to associate them with the two primary classes. Here, we use confocal microscopy and high-resolution volumetric imaging to study intracellular localization of the EV markers CD9 and CD63 prior to EV export from cells. We find significantly different spatial expression of CD9 and CD63. CD9 is primarily localized in microvesicles, while CD63 is detected exclusively in exosomes. We also observe structures in which CD63 forms a shell that encapsulates CD9 and interpret them to be multi-vesicular bodies. The morphology and location within the endoplasmic reticulum of these shell-like structures are consistent with a role in differential sorting and export of exosomes and microvesicles. Our in situ imaging allows unambiguous identification and tracking of EVs from their points of origin to cell export, and suggest that CD9 and CD63 can be used as biomarkers to differentiate subpopulations of EVs.