Transfert intercellulaire des vésicules extracellulaires mammaires : analyse spatiale tridimensionnelle et perturbation induite par l'exposition à des nanoparticules de dioxyde de titane

Le résumé fourni par la source

Extracellular vesicles (EVs) are nano- to micrometer-sized lipid bilayer-delimited vesicles secreted by all cell types and involved in intercellular communication. EVs originate either from the plasma membrane, typically enriched in the tetraspanin CD9, or from intracellular multivesicular bodies (MVBs), typically enriched in CD63. Although EVs are widely described as long-range messengers and explored as therapeutic delivery vehicles, the spatial dynamics of their transfer between cells remain poorly understood. In my thesis project, I have developed a coculture approach using human mammary MCF-7 cell line to study the direct transfer of endogenous or fluorescent fusions of EV-markers CD9, CD81, and CD63 from donor cell to acceptor cells labelled with non-transferrable fluorescent dyes. High-resolution 3D confocal imaging and automated fluorescence detection and quantification revealed the presence of EV-marker positive punctate structures overlapping with cytosolic dyes, suggestive of EVs secretion and internalization into acceptor cells, i.e., EV transfer. This transfer appeared to accumulate in the vicinity of donor cells compared to distant regions, even under flow conditions, indicating that EV transfer may be more efficient at short distances. Computation of the 3-D coordinates of EV-marker positive spots outside donor cells revealed common, yet unexpected, features of transfer of the three tetraspanins. The transfer of EV markers occurred along a gradient originating from the donor cells, and decreased rapidly within a distance of 20 µm. Furthermore, the transfer was polarized in two dimensions: laterally (x, y), it was “oriented” toward one side of the donor cells, and vertically (z), it accumulated in basal compared to upper planes. Simultaneous monitoring of CD9 and CD81 revealed that CD81 transfer was more efficient than CD9, while CD9 was preferentially enriched in basal regions. Notably, basal CD81-positive structures frequently colocalized with CD9. Live-cell imaging of GFP-CD63 showed colocalization of CD9 in basal planes, suggesting that EV markers detected at basal levels could originate from the plasma membrane. Finally, depletion of syntenin-1 in donor cells, a known regulator of EV biogenesis, decreased the overall rate of EV transfer as expected, but also attenuated both (x,y) and (z) polarization. In the second part of this work, I investigated how EV biogenesis pathways may be impacted by exposure of mammary cells to titanium dioxide (TiO₂) nanoparticles (NPs), which are emerging environmental pollutants widely used in all industrial sectors. In a recent study, we have detected their presence in human and animal milk, without addressing the mechanisms underlying their secretion by the mammary gland. After determining the subtoxic doses in MCF-7 cells, I found that TiO₂-NPs localized in CD63-positive intracellular compartments whose sizes were significantly enlarged compared to those of unexposed cells. CD9 was less detected at the cell surface but localized instead in CD63 internal compartments. Analysis of the TiO₂-NPs exposed MCF-7 secretome, combined with Raman spectroscopy and transmission electron microscopy, provided evidence indicating that mammary EVs may carry TiO₂-NPs. Altogether, this work revealed unexpected spatial features of EV marker transfer between mammary cells and suggests that EV biogenesis pathways may be altered upon exposure to TiO₂-NPs, highlighting a potential role for EVs in NPs intercellular transport.

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