CNSC-24. A CHOLINERGIC NEURON-TO-TUMOR CIRCUIT ENHANCES HUMAN GLIOBLASTOMA PROGRESSION VIA SYNAPTIC AND PARACRINE MECHANISMS

Abstract Neuronal influences on malignant brain tumor pathogenesis and progression have been increasingly recognized in the central nervous system. In glioblastoma (GBM), mainly glutamatergic inputs have been identified, and so far, they have largely been studied in the context of interspecies xenotransplantation models. We and others have recently leveraged viral tools, such as rabies virus-based retrograde tracing, to define brain-wide connectivity of GBM, which suggested the potential for GBM to receive projections from diverse neuronal subtypes, including modulatory neurons. Here, we explored the cholinergic neuron-to-glioma circuit in in vivo and all-human models to elucidate the impact of cholinergic neuronal activity on GBM biology. In mice, we leveraged electron microscopy to provide structural evidence for a basal forebrain cholinergic neuron (BFCN)-to-GBM synapse. Stimulation of BFCNs via chemogenetic approaches increased tumor cell proliferation. We further found that CHRM3 mediates the effects of BFCNs on GBM proliferation by either overexpressing CHRM3, whose specific activation increased proliferation, or knocking down CHRM3 in tumor cells, which extended mouse survival. We also developed a co-culture system employing patient-derived glioblastoma organoids and human induced pluripotent stem cell (hiPSC)-derived cholinergic neurons. In this system, we provided evidence of structural human cholinergic synaptic inputs onto GBM cells via trans-monosynaptic tracing and electron microscopy and functional synaptic interactions through the CHRM3 receptor via calcium imaging. Deep single-cell RNA sequencing of co-cultures compared to GBM monocultures further revealed shifts in tumor transcriptional profiles toward a more proliferative state, with contributions from both diffusible factors and direct contacts. Finally, pharmacological blockade of cholesterol biosynthetic pathways attenuated these effects, suggesting that lipid metabolism at least partially mediates the pro-proliferative effects of cholinergic neurons on GBM. Collectively our findings uncover a cholinergic circuit that drives GBM progression via contact-dependent and independent programs, highlighting a previously underappreciated modulatory pathway for GBM.