Proteomic and miRnomic investigations of senescence-derived exosomes to identify circuits modulating cancer chemoresistance

Cellular senescence is a biological process characterized by a state of proliferative arrest. This process can initially inhibit the proliferation of tumor cells; however, it may also promote resistance to therapy. These pathological effects are attributed to the acquisition of an enhanced secretory profile, the senescence-associated secretory phenotype (SASP), which is rich in inflammatory molecules. Extracellular vesicles (EVs) derived from cancer cells undergoing therapy-induced senescence may provide a pathway for these senescent cells to dispose of proteins, thus contributing to cancer cell survival and resistance to chemotherapy. In this study, using a lung cancer cellular model, we aimed to characterize the proteomic and miRNA content of exosomes associated with early and late senescence, along with the SASP, with the aim of investigating the role of these molecules in modulating cancer chemoresistance and tumor microenvironment. Tumor cells, both senescent and proliferative, were treated to enrich the corresponding EVs, which were successfully extracted to recover the corresponding protein and miRNA cargos. We performed a comprehensive proteomic analysis of exosomal vesicles to explore their roles in cellular senescence and intercellular communication. Exosomal proteins were extracted, reduced, alkylated, and trypsin-digested, followed by TMT labeling and fractionation. Mass spectrometry (nanoLC-ESI-Q-Orbitrap-MS/MS) was used to identify and quantify protein expression profiles. A total of 2,788 proteins were detected, with bioinformatics analysis revealing significant enrichment in biological processes related to nucleosome assembly, chromosome organization, and cellular component organization. Key cellular components included extracellular spaces, vesicles, and plasma membranes. These findings underscore the critical functions of exosomal proteins in cellular communication, particularly in the context of senescence, highlighting their potential as biomarkers and therapeutic targets in age-related diseases.