UNRAVELING GLIAL REMODELING IN SPACE AND TIME DURING GLIOBLASTOMA EVOLUTION

Riccio L¹, De Luca C¹, Rega C¹, Papa M¹ and Virtuoso A¹ | ¹Laboratory of Neuronal Networks Morphology and System Biology, Department of Mental and Physical Health and Preventive Medicine, University of Campania Luigi Vanvitelli, Naples, Italy

Glioblastoma (GBM) is the most aggressive primary malignant tumor of the central nervous system (CNS), characterized by marked molecular heterogeneity, rapid proliferation, extensive invasiveness, and strong resistance to conventional therapies. Despite current standard treatments–surgery, radiotherapy, and temozolomide–median patient survival remains below 15 months. Key contributors to GBM progression include activated glial cells and remodeling of the extracellular matrix. In this study, we investigated the cellular and molecular dynamics of the tumor microenvironment, focusing on astrocytic and microglial responses, and on the modulation of glial markers such as GFAP, Iba1, Connexin 43 (Cx43), and the purinergic receptor P2X4R. Acute and organotypic brain slice cultures were prepared from C57BL/6J mice, into which GL261 glioblastoma cells were injected into the cortex. Tissues were collected 3 and 7 days post-injection to analyze tumor progression. GBM growth was monitored through the proliferation marker Ki67. Histological analysis, confocal immunofluorescence, and western blotting were performed to assess marker expression and localization. GL261 cells engaged with the surrounding neuroglial network, migrating in fascicle-like patterns that extended even into the contralateral hemisphere. This dynamic infiltration was associated with pronounced peritumoral astrocyte activation (increased GFAP) and a robust microglial response, evidenced by morphological changes in Iba1-positive cells. Cx43 expression was altered by tumor invasion, suggesting disrupted intercellular communication, while P2X4R expression dynamics indicated modulation of the local immune response. Consistently, elevated Ki67 levels confirmed the highly proliferative and aggressive behavior of the tumor. Organotypic slice cultures represent a fast and reliable ex vivo platform to investigate GBM invasiveness, migration, and neuroglial interactions, revealing spatiotemporal remodeling of the tumor microenvironment during disease progression. Region-specific glial activation and time-dependent modulation of Cx43 and P2X4R highlight the complex and evolving nature of glial–tumor crosstalk. These findings may help identify potential therapeutic windows and support the development of molecularly targeted treatment strategies.