202 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. METHODS: GBM tissues was collected from 30 patients. GBO were established and transduced with RFP-fluorescent EF1-A. Brain specimens were obtained from three epilepsy patients undergoing disconnection surgery and transduced (GFP-fluorescent EF1-A). Co-cultures of GBOs with brain slices were established. Spatial transcriptomics compared 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 utilized. RESULTS: Within GBM patients, 60% had seizures requiring medical management. MAP2/cleaved-caspase3 I.F. showed increased neuronal apoptosis near invading GBOs versus edge(P<0.05). Transcriptional changes of invading GBOs, showed upregulated SLC7A11 by 1.8-fold(P<0.001) leading to increased glutamate secretion within brain-tumor interface, a culprit for excitotoxicity. Invasive GBOs substantially downregulated NMDA/AMPA receptors compared to normal brain, preventing auto-excitotoxicity with log2FC -9.1&-2.3 respectively(P<0.001). The invaded brain parenchyma upregulated SLC1A3 expression to increase glutamate reuptake compared to uninvaded counterparts at edge of slices as a last failed effort(P<0.001). AMPA receptor inhibitor, CNQX, decreased neuronal apoptosis near the tumor invading edge(P<0.001). SLC7A11 expression on overall patient survival was validated via data from REMBRANDT, with a significant correlation between GBM SLC7A11 expression and survival, where patients with low SLC7A11 expression had an increased survival of 9-months (P=0.028). CONCLUSIONS: Our novel brain slice-GBO model enabled for the first time the direct correlation between patient clinical symptomatology, GBM pathologies, and drug testing. This study underscores the importance of ionotropic glutamate receptors modulation by invasive cells, further elucidating the mechanistic pathway of glutamate production, which can serve as potential therapeutic targets.