CNSC-96. TARGETING NEURON-GLIOMA CHLORIDE CROSS-TALK IN GLIOBLASTOMA

Abstract Neuronal activity and synchrony in both normal and pathological states are regulated by excitatory and inhibitory synaptic inputs, determined by ion channel permeability across the cell membrane. This study investigates neuron-glioma cross-talk in chloride flux and the role of chloride, the most abundant neuronal anion, in influencing neuronal excitability and glioblastoma (GBM) pathophysiology. We hypothesized that GBM’s aggressive proliferative and invasive phenotype is linked to chloride dysregulation and that inhibiting glioma chloride flux could reduce tumor proliferation and glioma-induced neuronal hyperexcitability. The direct effects of chloride flux on glioma growth and invasion were tested using optogenetic stimulation of chloride opsin-expressing glioma cells both in vitro and implanted in vivo in patient-derived xenograft (PDX) mice. To explore chloride cross-talk mechanisms between neurons and glioma cells, we performed single-nucleus RNA sequencing (sNuc-seq) of GBM-neuron co-cultures, analyzing 54,000 cells, and revealed the upregulation of genes involved in chloride homeostasis, including the voltage-gated ClC-3 chloride channel and the sodium-potassium-chloride cotransporter 1 (NKCC1). Mass spectrometry-based proteomic analysis of culture supernatant was performed to identify mechanistic targets and paracrine factors mediating chloride transfer. Light-activated chloride pumping into glioma cells significantly reduced tumor proliferation in vitro and in vivo, underscoring the link between intracellular chloride concentration and tumor growth. sNuc-seq also showed upregulation of NKCC1 and the ligand-gated chloride channel GABAA receptor in tumor-associated neurons co-cultured with NKCC1-overexpressing glioma cells, suggesting shared chloride signaling mechanisms in the tumor microenvironment. Proteomic profiling of conditioned media identified glioma-derived MK2 (MAPKAPK2) as a driver of neuronal NKCC1 upregulation and chloride dysregulation. Pretreatment of GBM cells with the MK2 inhibitor ralimetinib restored chloride balance and reduced neuronal hyperexcitability. Collectively, these findings reveal a paracrine mechanism of chloride transfer between glioma cells and neurons, highlighting chloride signaling as a novel therapeutic target to inhibit neuronal hyperexcitability and tumor proliferation in glioblastoma multiforme (GBM).