Exploring Neural Stem Cell Modulation by Glioblastoma EVs from Cell Lines of Distinct Origins

Background. Tumor-associated microenvironmental signaling plays a critical role in cancer proliferation by affecting adjacent healthy cells. Glioblastomas (GBMs), highly aggressive brain tumors, actively reshape their microenvironment through various mechanisms, notably via extracellular vesicles (EVs), which mediate intercellular communication. These EVs likely influence neural stem cells (NSCs), essential for brain homeostasis, neurogenesis, and repair. GBMs often arise near the subventricular zone (SVZ), rich in NSCs, suggesting potential two-way cellular interactions. Recent findings propose that NSCs may even serve as a cell of origin for GBM, though it remains unclear whether this is due to glioblastoma-induced signaling or intrinsic NSC traits. While SVZNSCs are implicated in GBM progression and recurrence, little is known about their direct response to tumor-derived signaling. This study aimed to explore how GBM cell lines of distinct origin affect NSC behavior, focusing on calcium signaling, a crucial second messenger involved in proliferation, differentiation, and cellular communication. Methods. GBM cell lines A172 and U87-MG were cultured in DMEM high-glucose media with 10% EV-depleted FBS for 72 h. EVs were isolated using 12% PEG precipitation and characterized via nanoparticle tracking analysis and ELISA for CD63, HSP70, and APOA1. NSC H9 cells were cultured in KnockOut DMEM/F-12 with StemPro supplement, bFGF, and EGF (20 ng/ml each), and stimulated with GBM EVs (~1250 EVs/cell) or control EVs from unconditioned media signaling was measured using Oregon Green-488 BAPTA- influx via L-type channels and RyR-mediated release from the endoplasmic reticulum. signaling change in NSC after stimulation with GBM EVs and its dependence on cell line EVs. The A172 EVs significantly increased the number signals in affected NSC by 25% compared to unaffected NSC (p<0.01 Kolmogorov-Smirnov, K-S, test), the difference was absent after application of nifedipine indicating increased levels of L-type Ca2+ channels. In NSC affected by U87 EVs Ca2+ signaling was not changed; however, the amplitude of signals was significantly increased by 29% after nifedipine (p<0.01, K-S test). The following caffeine application increased the amplitude by 16% (p<0.05, K-S test), and the number of signals increased by 126% (p<0.001, K-S test) indicating increased levels of RyR in NSC affected by U87 EVs. signaling is altered by GBM-derived EVs, affected by A172 EVs shows possible facilitation of NSC proliferation and differentiation. On the other hand, strong increase in RyR levels in NSCs due to U87-MG EVs shows potentiation of response of affected cells to possible influence by environmental molecular mediators of proliferation or differentiation, like growth factors, or inflammatory interleukins. Since transient Ca2+ signaling regulates cell differentiation, migration and proliferation, different effects of A172 and U87-MG EVs shows selective shaping of GBM cellular environment.