CNSC-46. EXTRACELLULAR ATP/ADP PROMOTE GLIOBLASTOMA TUMOR GROWTH THROUGH ACTIVATION OF THE P2Y1 PURINERGIC RECEPTOR AND INDUCTION OF TUMOR CELL-AUTONOMOUS CALCIUM OSCILLATIONS

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain malignancy in adults. Recent observations suggest that GBM cells generate cell-autonomous Ca2+ oscillations that promote tumor growth. Here, we investigated extracellular drivers of these oscillations by imaging patient-derived GBM cells (PDGCs) transduced with GCaMP6s, a genetically encoded Ca2+ indicator. Treatment with extracellular ATP and ADP induced robust potentiation of Ca2+ oscillations, suggesting that activation of cell surface purinergic receptors influences the generation of Ca2+ waves. One of the most highly expressed purinergic receptors in GBM, P2Y1, is a Gaq protein-coupled receptor activated by ADP and ATP that promotes Ca2+ release from intracellular stores. We found that both pharmacologic inhibition and shRNA knockdown of P2Y1 in PDGCs reduced spontaneous and ATP/ADP-induced Ca2+ oscillations. Knockdown of P2Y1 also reduced tumor sphere formation and invasion in vitro, indicating an important role for P2Y1 in generating Ca2+ oscillations and driving tumor growth. Furthermore, to determine whether PDGCs actively secrete ATP to contribute to purinergic signaling, we performed an shRNA knockdown of PANX1, a membrane hemichannel that mediates ATP release, and measured extracellular ATP via bioluminescence. The amount of extracellular ATP was reduced from PANX1 knockdown cells compared to a non-targeting control. Together, these experiments demonstrate the importance of purinergic signaling via P2Y1 in generating tumor cell-autonomous Ca2+ waves that drive GBM growth and suggest a role for PANX1 in actively secreting ATP to contribute to this signaling.