Astrocyte-Glioblastoma Stem Cell Interactions via Extracellular Vesicles Contribute to Distinct Vascular Structures.

Glioblastoma (GBM) is a highly malignant astrocytic tumor characterized by marked heterogeneity and therapeutic resistance. Cancer stem-like cells (CSCs) drive recurrence within specialized microenvironments, such as perivascular niches. Glioblastoma stem cells have been considered to interact with surrounding stromal cells, including astrocytes. To investigate these cell communications, we used a co-culture system of glioblastoma KMG4 cells and immortalized human astrocytes (NHA-TS) on hydrogels. Co-culture on hydrogel induced stemness- and epithelial-mesenchymal transition-related genes. Glioblastoma- and astrocyte-derived extracellular vesicles (EVs) were incorporated into reciprocal cells. NHA-TS-derived EVs regulated stemness of KMG4 cells, whereas KMG4-derived EVs increased expression of vascular development-related genes, such as THBS1 and ANGPT1 in astrocytes. Proteomic analysis identified COL1A1 and THBS1 in KMG4 and NHA-TS co-culture EVs. Spatial transcriptomic analysis of human GBM tissue demonstrated THBS1 and COL1A1 expression in perivascular regions. Culturing KMG4 cells on PNaSS gels upregulated pericyte-associated genes such as ACTA2, PDGFRB, HIGD1B. Within the perivascular microenvironment, EV-mediated interactions between glioblastoma cells and astrocytes support the induction of stemness and the differentiation of GBM cells toward a pericyte-like phenotype, promoting perivascular niche formation and microvascular proliferation. The hydrogel-based co-culture model thus provides a simple and effective platform for dissecting tumor-stroma communication in the glioblastoma microenvironment.