Zhyvolozhnyi A. The regulatory role of extracellular vesicles under normal and carcinogenic conditions

Extracellular vesicles (EVs) represent a heterogeneous population of nano/micro-sized membrane vesicles that are constantly secreted into the extracellular environment by nearly all studied cell types under both normal physiological and pathological conditions. EVs contain nucleic acids, proteins with various functional potentials, signaling molecules, and metabolites, allowing them to play a leading role in intercellular communication. The goal of this dissertation was to investigate the production characteristics, molecular composition, and functional properties of EVs isolated from human sweat and the conditioned medium of pseudonormal and tumor cells, and to examine the influence of normoxia/hypoxia conditions and the adaptor protein Ruk/CIN85 on these parameters. It was found that human sweat EVs contain human DNA and RNAs (74% of reads corresponded to the human genome, with 70% being tRNA, 18.5% other RNA types, 5% mRNA, and 1.85% miRNA) and microbial origins (bacteria, archaea, and viruses typical of the skin microbiome). The protein composition of human sweat EVs and those collected from alginate patches revealed the presence of 1,209 unique human proteins, about 20% of which were present in every individual sample, along with the exosome marker CD63, 846 proteins shared with total sweat, and 368 proteins with the alginate patch. In addition, 1,594 proteins of bacterial origin were detected. The effect of hypoxia on renal adenocarcinoma cells led to increased EVs secretion and notable changes in their protein cargo compared to normoxia. LS-MS analysis of "hypoxic" EVs revealed an excessive presence of integrins and proteins involved in cell adhesion. Time gated Raman spectroscopy and surface-enhanced time gated Raman spectroscopy showed distinctive changes in the amide regions of EVs proteins under hypoxia, suggesting that these techniques are promising for studying the effects of external stimuli, such as hypoxia, on EVs composition. Characterization of EVs from the conditioned medium of Renca mouse kidney carcinoma cells overexpressing Ruk/CIN85 (Renca-RukUp) and control Renca-Mock cells under normoxia and hypoxia showed that the quantity of particles produced by Renca-RukUp cells was ten times higher under hypoxia compared to normoxia. In normoxia, the levels of Ruk/CIN85, Alix, and CD81 were elevated in EVs from Renca-RukUp cells compared to Renca-Mock cells. However, under hypoxia, the levels of proteins decreased by more than hundred times in EVs from Renca-RukUp cells, while Ruk/CIN85 and CD81 increased in EVs from Renca-Mock cells. Thus, Ruk/CIN85 emerges as a novel EVs component in tumor cells, potentially playing a regulatory role in controlling EV composition under normoxia and hypoxia. Further characterization of EVs produced by HEK293 human embryonic kidney cells stably transfected with the EGFP-Ruk/CIN85 vector demonstrated that EVs containing EGFP-Ruk/CIN85 can modulate cell proliferation and motility in vitro. The proteins identified in EVs from HEK293 cells with EGFP-Ruk/CIN85 overexpression were mainly metabolic enzymes. EVs produced by 4T1 mouse breast adenocarcinoma cells with upregulated (RukUp) or downregulated (RukDown) expression of Ruk/CIN85 were found to contain different sets of its multiple molecular forms and could modulate the proliferative activity, motility, and invasiveness of 4T1 WT cells. This suggests that Ruk/CIN85 is a constitutive component of tumor cell-derived EVs and plays an active role in carcinogenesis depending on its content in EVs.