P02.10.B REGULATION OF GLIOBLASTOMA CANCER STEM CELLS AND PROLIFERATION THROUGH VOLTAGE GATED SODIUM CHANNEL

Abstract BACKGROUND Glioblastoma Multiforme (GBM) is one of the most devastating cancer known. Despite decades of research, we still lack an efficient treatment. The heterogeneity in the cell-type composition along with the presence of a subpopulation of cells with high tumorigenic capacity named glioblastoma stem cells (GSCs), make GBM extremely hard to treat. An extensive body of works supports the hypothesis that an aberrant functional expression of membrane ion channels mediates the progression of solid cancer tumors. Potassium, calcium, and chloride channels have been largely correlated with carcinogenesis. However, little is known about the voltage-gated-sodium channel (Nav) in GBM. In fact, the role of this membrane ionic permeability in the GBM progression and relapse is yet to be unveiled. MATERIAL AND METHODS Experiments have been performed on human GSCs obtained from surgical specimen at the Neurosurgery Department of IRCCS-AOU San Marino IST (Genova, Italy), from patients who had not received therapies before intervention. The mRNA profile of the cell lines as well as the transcript expression of stemness markers were evaluated both in control condition and in the presence of the Nav channel blocker Tetrodotoxin (TTX, 30μM). The protein content for the stemness markers and their intermediates was quantified using Western-Blot analysis. Nav-mediated inward currents were recorded from single cells and measured in voltage clamp by applying consecutive voltage steps of +10 mV from a holding potential of -70 mV and up to +60 mV. Transient inward current was calculated on the peak subtracting the baseline leak currents. Current density (pA/pF) was calculated as the ratio between the peak current recorded at +20 mV and the capacitance of the cell. Resting membrane potential (RMP) was also assessed in each recorded cells. RESULTS We have identified a subpopulation of GBM cells (in GBM Proneuronal subtype) that functionally expressed TTX-sensitive inward currents. Transcriptomics investigation reveals the significant expression of SCN1A mRNA. We have shown that Nav density positively correlates with the RMP in GBM cells. Additionally, Nav blockade promotes glioma cell proliferation and G1/S accumulation. Pharmacological blockade of Nav-mediated currents has shown a significant impact on some stemness markers both at the mRNA and protein expression level. A regulatory downstream pathway, modulated by Nav has also been investigated. CONCLUSION Our evidenc