| Popis |
Introduction Various populations of small extracellular vesicles (sEVs) are widely heterogeneous, even within a single cell type. These populations are marked by different surface molecules, such as EpCAM, CD63, or phosphatidylserine (PS). This implies that EV subpopulations may carry diverse proteomes, which could be essential for their wide range of biological functions. These variations allow sEVs to be involved in processes, such as the immune response, disease progression, and fibroblast activation into CAFs. This work aimed to explore, how distinct surface markers shape the functional roles of sEVs in the context of head and neck cancer (HNSCC). Methods EVs were isolated from conditioned media (obtained from HNSCC cell line FaDu) that were subjected to differential centrifugation, filtration, and concentration and EVs were isolated using affinity-based methods targeting EpCAM, CD63, and PS on EV surface. Then, isolated EVs were characterised using NTA or DLS, cryo-EM, TEM, WB for EV surface markers, and LC-MS for their proteomic composition. Furthermore, the effect of various groups of EVs on recipient cells was assessed using WB for CAF activation, LC-MS, and measurement of mitochondrial energetics in recipient cells. Results So far, we have been mainly focused on the first subgroup, PS-positive sEVs, as PS is of high importance in the tumour microenvironment. By inhibition of autophagy, specifically lysosomal degradation, we observed the accumulation of proteins in PS-EVs as an alternative pathway for autophagic degradation. This led us to the assumption that isolated EVs are of endosomal origin rather than microvesicles. Accumulation of lysosomal membrane marker, LAMP1, and decrease in plasma membrane marker, basigin, in PS-EVs after bafilomycin treatment supported our previous finding. Summary/Conclusion Our results indicate that the clinical use of autophagy modulators in cancer must be taken cautiously, since they also affect the PS-EVs content. We also observed that PS-EVs derived from cancer cells were uptaken by normal fibroblasts activating them into CAFs. Thus, molecules carried in PS-EVs can be critical for cancer development, progression, and potential treatment resistance. Further research will be focused on other aforementioned subgroups of EVs.
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