CNSC-09. H3G34-mutant Diffuse Hemispheric Gliomas exhibit cellular niches of GABAergic interneuron-like tumor cells with autonomous changes in membrane potential.

Abstract Background Diffuse hemispheric high-grade gliomas, H3G34-mutant (DHG-H3G34), are among the deadliest brain tumors of adolescents and young adults. Our recent study showed that DHGH3G34 cells recapitulate a cellular hierarchy from Radial Glia-like progenitor cells to more mature astrocytic-like (AC-like) and GABAergic interneuron-like (eIN-like) cells. How these cell states are arranged in tumor tissue and how they communicate with each other in the microenvironment remains poorly understood. Methods Spatial Transcriptomics – A Custom panel for 10x-Xenium was designed for DHG-H3G34 cell programs for FFPE samples. Calcium Imaging - The genetically encoded calcium indicator GCAMP8s was used for network dynamics characterization. Voltage Imaging - The genetically encoded voltage indicator ASAP3 was used for detecting changes in membrane potential. Results We observed that AC-like cells were among the most abundant cellular programs across all patient samples and were enriched for GJA1 transcripts, (a gap junction protein-coding gene Connexin-43 responsible for cellular communication and network formation). Indeed, in our in vitro patient-derived cell models, we found rhythmic calcium activity propagation in tumor cell networks with bimodal frequency. Connexin-43 knockdown in DHG-H3G34 models resulted in reduced calcium network dynamics, including decreased kinetics and intensity, as well as downregulation of gene signatures associated with astrocytic and mesenchymal cells and reduced cell proliferation. Additionally, we identified GABAergic interneuron-like niches containing rare cells that were enriched for SLC32A1 transcripts, which encode the presynaptic vesicular GABA transporter (vGAT). In DHG-H3G34 in vitro models, we detected protein expression of vGAT, GABA production, as well as voltage-gated sodium channels, which are involved in membrane potential regulation. Using voltage imaging in monoculture DHG-H3G34 models, we observed autonomous alterations in membrane potential. These findings suggest that DHG-H3G34 cells may exhibit endogenous electrical activity with GABAergic transmission properties. Conclusion DHG-H3G34 cells display network dynamics with bimodal rhythmic frequencies, regulated by the gap junction protein Connexin-43, which promotes proliferation. Finally, eIN-like cancer cells display presynaptic features indicative of potential autonomous GABAergic synaptic transmission.