Glioblastoma Organoid(GBO) invasion of Prolonged Ex Vivo Human Brain Slices: Spatial Transcriptomics Reveals GBO Modulation of Ionotropic Glutamate Receptors, Inducing Neuronal Excitotoxicity

Introduction: Glioblastomas (GBM) are highly aggressive tumors characterized by invasion via destruction of brain parenchyma. Targeting glutamate production signaling through ionotropic glutamate receptors can slow down glioma progression in vivo, yet the detailed mechanisms are not fully understood. We employ a novel model using a long-term organotypic human brain slices and patient-derived GBM organoids(GBOs) to investigate glutamate production by invading tumor cells and the resulting excitotoxicity on neighboring neurons.

Methods: Human brain specimens were obtained from patients undergoing epileptogenic disconnection surgery. Slices were acutely oxygenated in artificial CSF(aCSF) enriched with sucrose, pyruvate, vibratome sectioned (250microns), and cultured with neuroprotective media using ROCK-inhibitor, and transduced with GFP-fluorescent EF1-A. Patient-derived-GBOs were transduced with RFP-fluorescent EF1-A and implanted on slices. Spatial transcriptomics was conducted by comparing GBO core, brain slice edge, and FACS sorting the brain-tumor interface for RFP+(Invasive GBO) vs GFP(invaded parenchyma) cells. Immunofluorescent staining(I.F.) was conducted on whole slices.

Results: I.F. co-staining with MAP2/cleaved-caspase3 showed increased neuronal apoptosis near invading GBO cells compared to edge(P < 0.05). To understand this phenomenon, we analyzed transcriptional changes of invading GBO cells, which upregulated SLC7A11 by 1.8-fold(P < 0.001) once implanted on brain slices, leading to increased glutamate secretion within the brain-tumor interface, a culprit for excitotoxicity on the unwary parenchymal cells. Furthermore, invasive GBOs substantially downregulated their NMDA/AMPA receptors compared to normal brain to prevent auto-excitotoxicity log2FC -9.1&-2.3 respectively(P < 0.001). Additionally, NMDA receptor subunits of Invading GBO cells were reduced by 6.7-fold(P < 0.001) when compared to adjacent brain parenchyma. The invaded brain parenchyma then upregulated SLC1A3 expression to increase glutamate reuptake compared to their uninvaded counterparts at edge of brain slices as a last failed effort for adaptation(P < 0.001).

Conclusion : Our novel brain slice-GBO model underscores the importance of ionotropic glutamate receptors modulation by invasive tumor cells, further elucidating the mechanistic pathway of glutamate production in invasion, which can serve as potential therapeutic targets.