203 NKCC1 Dependent GABAergic Signaling is Necessary for GBM Migration and Neural Network Integration

INTRODUCTION: A hallmark of the incurable nature of GBM is its highly migratory phenotype, making surgical resection only a method that prolonged the inevitable death of the patient. Neuron-glioma interactions have been shown to play a vital role in tumor cell migration, the level of functional connectivity in neuron-glioma networks and glioma-induced neuronal hyperexcitability. All of which have been shown to lead to worse prognosis. Preliminary from our lab shows that in tumor regions with high functional connectivity show excitatory GABAergic signaling mediated through NKCC1. We hypothesize that knocking down NKCC1 will lead to downregulated glioma cell migration, proliferation and network connectivity. METHODS: In neuron-glioma coculture, NKCC1 was knocked down through shRNA or the pharmacological inhibitor Bumetanide, and the effect on network activity and synchrony of neurons was captured using microelectrode array (MEA) and real-time calcium imaging. GBM cells with NKCC1 intact or NKCC1 knockdown were transplanted onto organotypic human and mouse cortical slices and performed live cell imaging to assess for tumor cell migration, depth of invasion, and presence of tumor microtubes. We performed immunostaining on both organotypic brain slices transplanted with GBM cells as well as neurons in co-culture with GBM cells to quantify proliferation and neuron-glioma synapses. RESULTS: GBM with NKCC1 knocked down demonstrated a decrease in network excitability and synchrony, tumor cell migration, depth of invasion, and network integration, but showed an upregulation in tumor microtube number and length. NKCC1 knockdown cells also showed decreased proliferation and number of neuron-glioma synapses. CONCLUSIONS: NKCC1-mediated excitatory GABAergic signaling is necessary for GBM network integration, migration, glioma proliferation, and Knocking down of NKCC1 has uncovered a possible therapeutic vulnerability in tumor cell migration.