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Cellular Uptake Heterogeneity of Small Extracellular Vesicles

Exosomes, also known as small extracellular vesicles (sEV), represent a subpopulation of EVs of 30‐200nm in diameter that are contained into multivesicular bodies and are released by cells via exocytosis. There has been increased interest to use sEVs for therapeutic purposes due to their immune‐tolerance, potential for targeted delivery and increased half‐life in circulation, compare to other nanoparticles. However, due to the heterogeneity of sEVs as well as the different phenotypic features of recipient cells, the uptake mechanisms can be highly variable and selective. Studies from our lab and others show that, in vitro, not all the cells in a given cell population take up vesicles with similar kinetics and avidity. Therefore, understanding the uptake of sEVs by cells in different physiologic states (eg, quiescent vs. proliferative) as well as the intracellular fate of sEVs is key for their use as therapeutic tools. As proof of concept approaches, we used the interaction of sEVs from various endothelial cells with PC3‐ML prostate cancer cells, a highly metastatic human cancer cell line. Using imaging flow cytometry and super‐resolution microscopy, we determined the uptake of sEVs by a heterogeneous population of prostate cancer cells. sEVs were isolated by sequential ultracentrifugation, labeled with lipophilic dyes (DiD or DiO) and following incubation with recipient cells, nuclei were stained using DAPI or Hoechst dyes. Cells incubated with sEVs overnight showed a highly significant negative correlation between nuclear stain fluorescence and the internalization of labeled sEVs. Using two different nuclear dyes, we found concordant data (DAPI r = ‐.067 p < 0.0001 and Hoechst r = ‐.119, p < 0.0001).. This result was independently confirmed for DAPI nuclear staining using fluorescence microscopy (r = ‐.1352 p = 0.0004). We hypothesized that the negative correlation between nuclear staining and internalization may be cell cycle‐dependent. Indeed, a binary switch was described from endocytosis being “on” in interphase to “off” in mitosis as cells traverse the G2/M checkpoint. Using clathrin‐dependent endocytosis inhibitor dynasore, we found that >80% of the EVs uptake is via endocytosis. Internalization of sEVs was time‐dependent and showed the same negative correlation with nuclear staining at each of the experimental time points (0,5, 1, 2, 4 and 24 hours). We also found that following incubation of cells with sEVs for up to 4hrs, co‐localization with the cellular lysosomal compartment was <5%, suggesting that the majority of the internalized EVs escape lysosomal degradation following internalization. Further understanding of the uptake mechanisms of sEVs are key to set the premises for their use as therapeutic delivery systems in various diseases.