Experimental Microglial Secretome Screening in Rat Organotypic Brain Slice Cultures.

Abstract

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults and is characterized by extensive interactions between tumor cells and glioblastoma-associated microglia and macrophages (GAMM). In the peritumoral region, where residual glioma cells contribute to recurrence, resident microglia represent the predominant immune population within the GAMM and, in general, may shape early tumor-supportive responses. The objective of this study was to establish an ex vivo model to investigate microglia-specific responses to glioma cells in intact brain tissue. Three-dimensional organotypic brain slice cultures (OBSCs) retaining endogenous microglia were generated and compared with microglia-depleted slices produced by pharmacological CSF1R inhibition with PLX5622. OBSCs were co-cultured for seven days with fluorescently labeled rat glioma spheroids or non-tumorous astrocyte spheroids under defined treatment conditions. Conditioned media were collected for multiplex chemokine analysis, while fluorescence microscopy was used to quantify spheroid invasion and outgrowth. Following culture, OBSCs were cryosectioned and immunostained to characterize cellular and molecular changes in the peri-spheroidal tissue. The model preserved the three-dimensional brain parenchymal environment and enabled comparison of glioma-associated responses in the presence and absence of resident microglia without contributions from peripheral macrophages. Microglial depletion enabled the identification of microglia-dependent changes in chemokine profiles and spheroid behavior during the defined culture period. This experimentally tractable platform models early glioma-microglia interactions and supports mechanistic studies and evaluation of microglia-directed therapeutic strategies in a physiologically relevant tissue context.