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Abstract

Glioblastoma is the most frequent and lethal brain tumor, showing a high degree of intra- and intertumoral heterogeneity. Despite the multimodal treatments (surgical resection, irradiation, and chemotherapy), overall survival of patients is still less than 15 months. The inevitable relapse of glioblastoma is mainly attributed to the subpopulation of tumor initiating cells, namely glioblastoma stem cells, which are shown to be refractory to standard treatments. The exceedingly heterogeneous nature of glioblastoma creates hurdles in developing effective therapies and makes it extremely difficult to eradicate. This project investigated the role of GSCs-derived sEVs in glioblastoma aggressiveness with the particular focus on the complexity of tumor-derived sEVs in terms of proteins, metabolites, fatty acids, and small RNAs. In this direction, protein, metabolite, fatty acid, and small-RNA contents of sEVs and their parent cells (NCH421k, NCH644, NCH705, and NCH711d) were profiled by utilizing mass spectrometry and highthroughput sequencing. Protein profiling of sEVs and their respective cell lines revealed the enrichment of proteins playing a role in amino acids, carboxylic acids, and organic acids transmembrane transport, together with the ones functioning in growth factor binding (including insulin-like growth factor I and transforming growth factor beta). In line with the proteomic analysis, metabolite screening of GSCderived sEVs also displayed the existence of metabolites associated with alanine, aspartate, glutamate metabolism, arginine biosynthesis, and butanoate metabolism, suggesting the dual role of GSCs-derived sEVs, which is transferring proteins responsible for the transport of amino acids/carboxylic acids and providing metabolites that will be used in amino acid and carboxylic acid metabolism. In conjunction with proteomic and metabolic analysis, profiling of fatty acids (carboxylic acids with long aliphatic chains) has revealed the presence of different fatty acid species, especially saturated fatty acids, in GSCs-derived sEVs, further implying that loading of biological cargos into sEVs is a highly regulated process, and that GSCs-derived sEVs are important sources for tumor cells to maintain their cellular metabolism. Finally, small RNA sequencing of GSCs-derived sEVs and their parent cells was carried out to shed some light on the contribution of sEVs-derived small RNAs to heterogeneity of glioblastoma; however, due to technical problems in sequencing (very low reads counts, poor complexity of sequencing library) the data is unfortunately not reliable for comparative analysis. In summary, this project revealed the complexity of GSCs-derived sEVs in terms of proteins, metabolites, fatty acids, and smRNAs (with limited degree), and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma.