P01.41.A MASS SPECTROMETRY-BASED PROTEOMIC ASSESSMENT OF GLIOBLASTOMA STEM-LIKE CELL DIFFERENTIATION

Abstract BACKGROUND Glioblastoma stem-like cells (GSCs) represent a tumor-initiating subpopulation that plays a central role in driving glioblastoma heterogeneity, therapeutic resistance, and recurrence. Characterized by their capacity for self-renewal and multipotent differentiation, GSCs can give rise to cell types expressing neuronal and glial markers. To investigate the proteomic landscape underlying these properties, we performed mass spectrometry-based proteomic profiling of seven patient-derived GSC lines. Our analysis focused on identifying proteins associated with stemness and differentiation potential, offering insights into the molecular mechanisms that sustain GSC function and plasticity. MATERIAL AND METHODS Patient-derived GSCs were cultured in the presence of EGF and bFGF to maintain stem-like conditions, followed by induction of differentiation through growth factor withdrawal and exposure to fetal calf serum for ten days. Cells were lysed using RIPA buffer, and protein samples were prepared using our modified filter-aided sample preparation (FASP) protocol. Proteomic analyses were performed using both data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes on a timsTOF HT mass spectrometer coupled to a nanoElute 2 HPLC system. Raw data were processed using Spectronaut software with the directDIA workflow and a hybrid spectral library for protein identification and quantification. RESULTS We analyzed the cellular proteomes of seven patient-derived GSC lines and their differentiated counterparts. By combining DDA for spectral library generation with DIA for quantification, we identified and quantified over 11,500 proteins. These included key markers of stemness (e.g., Nestin and SOX-2), neuronal differentiation (e.g., Microtubule-associated protein 2 and Tubulin beta-III), and glial identity (e.g., Glial fibrillary acidic protein and Oligodendrocyte transcription factors 1 and 2). We observed a decrease of stemness markers and variable changes of neural markers during differentiation. CONCLUSION We performed proteomic analyses to investigate the serum induced differentiation of patient-derived GSCs and found evidence of limited differentiation under these conditions. While stemness markers showed a decreasing trend in differentiating cells, changes in neuronal and glial markers displayed high variability across cell lines. Our proteomic workflow enables the characterization of GSC heterogeneity and differentiation responses, supporting its utility for studying GSC plasticity. SUPPORT/DISCLOSURE This study was supported by National institute for cancer research (Project no.: LX22NPO5102) financed by the European Union - Next Generation EU as part of the Czech Recovery Plan. The authors declare no conflicts of interests.