CNSC-60. STIMULUS-EVOKED INCREASES IN INTRACELLULAR CALCIUM PROMOTE SUSCEPTIBILITY TO FERROPTOSIS IN GLIOMA CELLS

Abstract Gliomas integrate within neural networks and receive synaptic input from neurons. We recently reported that sensory stimulus-evoked neuronal activity can elicit cytosolic calcium transients in glioma cells. It is known that perturbing calcium homeostasis promotes reactive oxygen species (ROS) production in the mitochondria of cancer cells and affects their metabolism. We previously identified the mitochondrion as a major source of ROS and lipid peroxidation mediating ferroptotic cell death in glioma. While studies have implicated neuron-dependent calcium influx in glioma progression, we explored whether calcium increases in glioma cells can be leveraged therapeutically to trigger ferroptosis. We utilized repetitive delivery of ATP or glutamate to stimulate calcium bursts in murine p53−/−PDGFA+ glioma cells to mimic neuronal stimulation in vitro. We performed live cell calcium imaging of glioma cells expressing either cytosolic GCaMP6s or mitochondrial matrix-targeted (mito4x) GCaMP6f reporters in the presence or absence of the mitochondrial calcium uniporter negative modulator, MCUi4. We measured total and mitochondrial-specific lipid peroxidation by flow cytometry with BODIPY-C11 and mitoCLox, respectively, with and without MCUi4. Lastly, we performed viability screens to assess the effects of repetitive calcium bursts on susceptibility to the ferroptosis-inducing drug, RSL3. ATP and glutamate stimulated both cytosolic and mitochondrial calcium bursts in glioma cells. Repetitive stimulation led to an increase in both total cellular and mitochondrial-specific lipid peroxidation. MCUi4 attenuated mitochondrial but not cytosolic calcium bursts and decreased lipid peroxidation. Viability screening with RSL3 showed that repetitive stimulations increased glioma cell susceptibility to ferroptosis, which was reversed with co-treatment with the ferroptosis inhibitor Ferrostatin-1. These findings reveal a mechanistic cascade from stimulus-evoked calcium increases to lipid peroxidation and ferroptosis-susceptibility in glioma. Current experiments in our lab are studying these effects in vivo, using physiologic stimulus-evoked neuronal activity to drive glioma calcium bursts and assess therapeutic efficacy when combined with pro-ferroptotic treatments.