Targeting Purinergic Signaling to Block the Effects of Seizures on Glioma Progression - PROJECT SUMMARY There is growing evidence that neuronal activity contributes to glioma proliferation and progression, but the molecular mechanisms by which this occurs are not well understood. Moreover, there has been limited study of how endogenous neuronal activity, including spontaneous epileptic activity and sensory stimulus-evoked activity affects glioma cells. A clearer mechanistic understanding of glioma responses to neuronal activity is critical to identify novel therapeutic avenues. A consequence of neuronal hyperactivity and seizures is ATP release. The purinergic receptor P2RX7 is expressed in human glioma and activated by high concentrations of extracellular ATP, resulting in calcium influx. Calcium influx has been broadly implicated in glioma progression and shown to be a functionally relevant consequence of neuron-glioma crosstalk. However, the role of P2RX7 in glioma has not been well elucidated, and its role in tumor-promoting crosstalk between neurons and glioma cells has not been studied. This proposal will investigate purinergic signaling as a means by which neuronal activity leads to glioma progression at the infiltrative cortical margin. Aims 1 and 2 will utilize glioma cells genetically engineered to express fluorescent reporters of calcium influx (GCaMP6s) and extracellular ATP (GRABATP1.0). In Aim 1, in vitro imaging of glioma cells and ex vivo imaging and electrophysiology of acute tumor slices will be performed to determine the function of purinergic receptors in glioma cell calcium responses to neuronal activity. Aim 1 will also investigate the effects of purinergic signaling on glioma cell proliferation and assess P2RX7 expression in human glioma at the infiltrative margin. Aim 2 will employ an orthotopic diffusely infiltrating GCaMP6s glioma model that elicits neuronal pathology, including hyperexcitability and seizures. In vivo two-photon imaging and two-color wide field optical mapping of neurons and glioma cells will be used to assess the effects of neuronal activity, including epileptic events and stimulus-evoked activity, on glioma cell calcium activity and progression. In both aims, purinergic signaling in glioma cells will be perturbed by P2RX7 knockout or the brain-permeable P2RX7 inhibitor Brilliant Blue G to investigate its function in neuron-glioma crosstalk and glioma cell proliferation and progression. As part of a comprehensive training plan, this project will be conducted at Columbia University Irving Medical Center under the mentorship of my sponsor in collaboration with an interdisciplinary team of physician- and basic scientists who will provide all the expertise and resources needed for me to develop a strong foundation in brain tumor biology, ex vivo electrophysiology, and in vivo brain imaging, and an enhanced ability to reason through research challenges. Clinical training will occur at NewYork-Presbyterian Hospital, home to the Herbert Irving Comprehensive Cancer Center and Neurological Institute of New York. Integrated research and clinical training will provide insight into clinical applications of basic brain tumor research and facilitate mentorship by a dedicated group of clinical and research faculty. This fellowship will support a long-term goal for a career as an independent physician-scientist working at the intersection of neuroscience and neuro-oncology.
