Abstract
Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults and is characterized by profound local and systemic immune suppression, which undermines the efficacy of immunotherapies. Cell-to-cell communication within the tumor microenvironment and systemically plays a critical role in cancer progression. Extracellular vesicles (EVs) are lipid bilayer-bound structures secreted by all cell types, serving as key mediators of intercellular communication. In cancer, EVs promote tumor progression by enhancing proliferation, pre-metastatic niche formation, angiogenesis, immune evasion, and therapy resistance. Consequently, targeting GBM-specific EV biogenesis pathways represents a promising strategy to counteract tumor-mediated immunosuppression. To identify GBM-specific mechanisms of EV biogenesis, we sought to determine the subcellular compartment most closely associated with GBM-derived EVs. We performed compartment-based proteomic studies on fractionated cells divided into membrane-associated, cytosolic, nuclear soluble, chromatin-associated and cytoskeletal fractions along with their derived EVs using liquid chromatography mass spectrometry. We found that the compartment most closely associated with GBM-EV proteomes was the membrane compartment (r=0.529, p<0.001), suggesting a membrane-associated origin of GBM-EVs. We further performed Gene Ontology (GO) analysis comparing GBM-EV enriched proteins to those from astrocyte-EVs, which revealed enrichment in several exosome-related biological process pathways (FDR<2.62e-24). Notably, we identified a subset of proteins with chaperone-binding functions, including CDC37 and AHSA1, which are co-chaperone proteins that interact with different isoforms of HSP90. Both CDC37 and AHSA1 were significantly enriched in GBM-EVs (log2FC=3.24 and 3.95, p<0.03), suggesting a potential role in cargo sorting or EV release. These findings support that GBM-EVs are predominantly released as exosomes of membrane origin and suggest that chaperone-associated proteins such as CDC37 and AHSA1 may regulate GBM-specific EV biogenesis. Targeting these mechanisms may offer new therapeutic strategies for disrupting tumor-promoting EV signaling in GBM.