Brain Microenvironment Calcium Channels Modulate Glioblastoma

About This Grant

ABSTRACT The research described in this application has a strong potential to benefit American health because it will lead to a better understanding and to new desperately needed therapies for Glioblastoma (GBM), the deadliest and most incurable primary brain tumor that kills more than thirteen thousand Americans each year. We previously showed that T-Type calcium channels (Cav3) are upregulated in GBM, where they promote tumor growth by regulating tumor cell signaling and gene transcription. The previous work focused on tumor cell-intrinsic Cav3. More recently, we discovered that Cav3.2 are also expressed in key GBM microenvironment (GME) cells including neurons and oligodendrocyte precursors (OPC). Importantly, we found that knockout (KO) of Cav3.2 in the GME significantly inhibits the growth of GBM tumors, suggesting an important role of GME Cav3.2 in regulating GBM growth. We also found that GME Cav3.2 regulate GME/GBM interactions, decreasing the OPClike cell state and inhibiting glutamate signaling pathways from neurons to tumors. GME Cav3.2 KO also led to decreased excitatory postsynaptic currents in GBM tumors. Based on the above, we hypothesize that GME Cav3.2 play an essential role in regulating the GME and influencing GBM malignancy. We will test this hypothesis, uncover its mechanistic basis, and its translational implications. We will first determine the roles and mechanisms of action of neuronal Cav3.2 and OPC Cav3.2 in GBM (Aim 1). We will use co-cultures of WT and Cav3.2 KO neurons and glioblastoma stem cells (GSC), as well as pharmacological blockage of Cav3 to assess the role of the channels on functional synapses between glutamatergic neurons and GBM cells and on GBM malignancy parameters. We will also collect conditioned media from WT and Cav3.2KO neurons and OPCs and use Mass Spectrometry to identify secreted factors from these GME cells that regulate GBM growth. We will then determine the cell specific contributions of GME Cav3.2 on GBM progression in vivo (Aim 2). We will utilize neuron cell specific and OPC cell specific Cav3.2 KO mice to determine the relative involvement of neuronal and OPC Cav3.2 in GBM malignancy and tumor growth. We will assess tumor growth, survival, neuron/GBM synapse function and OPC/GBM function. To gain insight into the mechanism of cell type specific Cav3.2KO on GBM we will perform single cell RNA-seq and spatial transcriptomics and determine the effects of Cav3.2 on GBM cell states, and GBM/GME interactions. We will also test new therapeutic strategies for the combined targeting of Cav3 and associated factors in the GME (Aim 3). We will test the therapeutic effects of combining the FDA approved repurposed Cav3 blocker mibefradil with glutamate inhibitors and OPC activity inhibitors in mouse models of GBM. The use of animal experiments is essential for this project because there exists no other model that recapitulates the complexity of the GME and that is adequate for testing the drug combination therapies. Altogether, the proposed work will improve our understanding of GBM/GME and lead to the development and testing of new therapies against one of the deadliest human cancers.