P01.38.B PROTEOMIC ANALYSIS OF EXTRACELLULAR VESICLES DERIVED FROM GLIOBLASTOMA STEM-LIKE CELLS TO CHARACTERIZE CELL HETEROGENEITY AND DIFFERENTIATION POTENTIAL

Abstract BACKGROUND Glioblastoma stem-like cells (GSCs) represent a distinct cellular population within the glioblastoma microenvironment, contributing significantly to intratumoral heterogeneity, therapy resistance, and disease recurrence. GSCs possess the capacity for self-renewal and can differentiate into more specialized cell types, including those expressing neuronal and/or astrocytic markers. Extracellular vesicles (EVs) are membrane enveloped particles that carry molecular cargo, such as proteins, RNAs and other molecules, which can be transferred to recipient cells and modify their phenotype. In glioblastoma, EVs are predominantly associated with pro-oncogenic signaling and angiogenesis. Given that GSCs comprise heterogeneous populations with distinct differentiation potentials, we aim to characterize the proteome of EVs secreted by several patient-derived GSC lines (n>10) to better understand the GSCs heterogeneity and differentiation capacity. MATERIAL AND METHODS We have established a biobank of 24 patient-derived GSC cultures, characterized for tumorigenicity in immunodeficient mice and evaluated for differentiation potential by immunocytochemistry. Initial experiments were conducted using the NCH644 GSCs, which were grown in serum-free medium. EVs were isolated from conditioned medium via ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting for exosomal markers. EV proteins were extracted using RIPA buffer and processed with a modified filter-aided sample preparation (FASP) protocol. Proteomic analysis was performed using a timsTOF HT mass spectrometer coupled with a NanoElute2 HPLC system, followed by data analysis in Spectronaut software. RESULTS We successfully isolated EVs with diameters ranging from 30 to 200 nm, consistent with the size range of exosomes. Western blot analysis confirmed the enrichment of exosomal markers CD63, CD9, Alix, and TSG101 in EV preparations compared to parental GSCs. Proteomic profiling quantified 7,665 proteins in EV samples. Notably, all proteins from the Top 100 most commonly reported EV-associated proteins in the Vesiclepedia database were detected. CONCLUSION We have established a robust workflow for proteomic profiling of EVs released by patient-derived GSCs. This platform will be used to analyze EVs secreted during both proliferative and differentiated states of GSCs, with the goal of identifying protein signatures indicative of astrocytic, neuronal, or mixed differentiation potentials. These findings will advance our understanding of GSC heterogeneity and differentiation dynamics in glioblastoma. SUPPORT/DISCLOSURE This study was supported by National institute for cancer research (Project no.: LX22NPO5102) financed by the European Union - Next Generation EU. The authors declare no conflicts of interests.