The intercellular transfer of extracellular vesicles markers CD63, CD9 and CD81 is spatially polarized and restricted to cell vicinity

Extracellular vesicles (EVs) are small lipid structures secreted by cells that originate from the cell surface (typically enriched in the tetraspanin (tspan) CD9) or from multivesicular bodies (typically enriched in the tspan CD63). Current methods for studying EVs involve concentrating and purifying EVs, without providing information about the distance or amount of EVs that may transfer from one cell to another. Here, we developed a coculture assay of human mammary MCF-7 cells to study the transfer of mCherry-CD81 or mCherry-CD9 from "donor" cells to a lawn of "acceptor" cells stained with cell tracker blue or green (CTB/CTG), non-transferrable fluorescent dyes. Using confocal fluorescence microscopy, we observed the presence of spots containing mCherry-CD81 or mCherry-CD9 outside donor cells, concentrated at short distance from donor cells and that overlapped with CTB signal, suggestive of their internalization in acceptor cells. Endogenous CD63, CD81 and CD9 also transferred more efficiently at short distances, even in the presence of a flow, as shown by immunostaining cocultures of wild type and KO CD-63, or -9, or -81 cells with antibodies directed against these tspans. Computation of the (x,y,z) coordinates of tspans-containing spots revealed a double polarized transfer: in (x,y), it distributed along a gradient that started from donor cells and decreased with the distance, and in (z), it was stronger in basal compared to upper planes, a (z) polarization that was affected by syntenin-1 depletion in donor cells. Simultaneous monitoring of CD9/CD81 transfer from into double CD81/CD9 KO cells showed that cells transferred more CD81 spots than of CD9. At the basal level, CD63 and CD81 spots were plasma membrane derived as they almost always contained CD9+, and resembled membranous remnants of migration. However, live cell imaging showed migration independent secretion of EVs in the extracellular space, in upper planes. Altogether, not only is our coculture assay suitable for the direct qualitative and quantitative study of EV-transfer, but it highlighted shared three-dimensional features of EV markers transfer between cells.