Decoding the Impact of Glioblastoma EVs on Neural Stem Cells via Calcium Dynamics and Transcriptomics

Abstract

Glioblastoma (GBM) is an aggressive brain tumor marked by rapid growth, frequent recurrence, and resistance to treatment. Emerging research shows that GBM cells interact with neural stem cells (NSCs) through extracellular vesicles (EVs). Understanding how GBM-derived EVs influence NSCs is key to revealing tumor–microenvironment dynamics and identifying novel therapeutics. Because calcium signaling governs cell proliferation, migration, and differentiation, changes in intracellular calcium may provide an early indicator of EV-driven NSC reprogramming. This study examined how GBM cell lines from different origins affect NSC behavior, focusing on calcium-signaling changes and validating findings with next-generation sequencing (NGS). Human neural stem cells (NSC H9 line) were treated with EVs isolated from GBM cell lines A172 and U87-MG conditioned media using 12% polyethylene glycol (PEG) precipitation. Cells were exposed to GBM-derived EVs (~1250 EVs per cell) for 17 h for Ca2+ signalling analysis and for 24 h for NGS. For Ca²⁺ imaging, NSCs were loaded with 2 µM Oregon Green-488 BAPTA-1 dye and imaged at 2 Hz for 200 s using a 40× objective. Nifedipine (2 µM) and caffeine (5 mM) were applied sequentially to assess L-type Ca²⁺ channel activity and ryanodine receptor (RyR)-mediated Ca²⁺ release, respectively. The pilot study revealed that GBM EVs influence Ca²⁺ signaling in NSCs, with effects dependent on the cell line. A172 EVs increased Ca²⁺ signal frequency in affected NSCs, which was blocked by nifedipine, suggesting elevated L-type Ca²⁺ channel activity. In U87 EV– treated NSCs, signal amplitude increased with nifedipine and further rose with caffeine, indicating enhanced RyR activity. NGS analysis revealed changes in RyR, SLC8B1 and LETM1 gene expression, suggesting increased Ca²⁺ concentration in mitochondria leading to augmented metabolic activity in mitochondria. Changes in Ca²⁺ signaling induced by EVs point to potential cross-talk between cytosolic and mitochondrial Ca²⁺ pathways. The study shows that A172 EVs enhance Ca²⁺ signaling, potentially promoting NSC proliferation and differentiation, whereas U87-MG EVs strongly elevate RyR levels, increasing NSC sensitivity to environmental cues such as growth factors or inflammatory interleukins. These functional effects are further supported by NGS analysis, which confirms the underlying molecular changes driving these EV-mediated responses.