CNSC-05. The Gabaergic Interneuronal and Neural Progenitor Lineage of the Diffuse Hemispheric Glioma, H3 G34-Mutant, Show Calcium Events in the Form of Intercellular Waves and Spontaneous Electrical Activity

BACKGROUND High-grade gliomas are one of the deadliest pediatric brain cancers. A diffuse subtype of hemispheric high-grade gliomas in adolescents and young adults is assigned as DHGH3G34-mutant. Notably, DHGH3G34-mutant gliomas distinctively contain cancer cell types resembling progenitors and GABAergic interneurons (eIN-like). Normal GABAergic interneurons influence neural network dynamics in the brain by communicating with other cells through the transmission of calcium and electrical signals. Our goal is to investigate the spatial organization of eIN-like glioma cells and evaluate whether they form networks and exert electrical activity relevant to tumor progression. METHODS Calcium Imaging: We used the Genetically encoded calcium indicator GCAMP7s with hDCX tdTomato promoter reporter construct. Voltage Imaging: We used the voltage indicator ASAP3 for detecting changes in membrane potential. Light microscopy: The whole fixed DHGH3G34-mutant PDX brain was mounted and imaged using the Zeiss Lightsheet 7 Microscope. RESULTS Using single-cell RNA sequencing and immunocytochemistry on DHGH3G34-mutant primary samples, we found that eIN-like cells express DCX (doublecortin) and form structures observed in ganglionic eminences. Calcium imaging revealed that DHGH3G34-mutant cells exhibit intercellular calcium waves. To track eIN-like cells, we developed a tdTomato fluorescent reporter for the DCX gene promoter activity (hDCX). Combining hDCX with GCAMP7s, we conducted calcium imaging and observed rhythmic calcium events in eIN-like cells at a frequency of 62.5mHz. Additionally, voltage imaging demonstrate that DHGH3G34-mutant cells show spontaneous action potential bursts. Furthermore, using light sheet microscopy on DHGH3G34-mutant PDX models, we observed glioma cells spreading into the contralateral hemisphere of the brain parenchyma. CONCLUSION DHGH3G34-mutant tumors represent highly invasive gliomas distinguished by the presence of progenitors and cells of the GABAergic interneuron lineage, exhibiting calcium waves and spontaneous action potential bursts. We are currently delving into the biological implications of these distinctive traits to gain insights into novel therapeutic strategies for the unique biology of DHGH3G34-mutant gliomas.