Microenvironment T-Type calcium channels regulate neuronal and glial processes to promote glioblastoma growth

Abstract

Background: Glioblastoma (GBM) is the most common primary malignant brain tumor. The aim of this study was to elucidate the role of microenvironment and intrinsic T-type calcium channels (Cav3) in regulating tumor growth and progression.

Methods: We grafted syngeneic GBM cells into Cav3.2 knockout mice to assess the role of microenvironment T-Type calcium channels on GBM tumor growth. We performed single-cell RNA-seq (scRNA-seq) of tumors from WT and Cav3.2 KO mice to elucidate the regulation of tumors by the microenvironment. We used neurons from WT and Cav3.2 KO mice in co-culture with GBM stem cells (GSC) to assess the effects of Cav3.2 on neuron/GSC synaptic connections and tumor cell growth.

Results: Cav3.2 KO in the microenvironment led to significant reduction of GBM growth and prolongation of animal survival. scRNA-seq showed that microenvironment Cav3.2 regulates neuronal and glial biological processes. Microenvironment Cav3.2 downregulated numerous genes associated with regulating the OPC cell state in GBM tumors such as SOX10 and Olig2. Neuronal Cav3.2 promoted neuron/GSC synaptic connections and GSC growth. Treatment of GSCs with the Cav3 blocker mibefradil downregulated genes associated with neuronal processes. The Cav3 blocker drug mibefradil synergized with temozolomide (TMZ) and radiation to reduce in vivo tumor growth and prolong animal survival.

Conclusions: Together these data reveal a role for microenvironment Cav3 in promoting GBM tumor progression through regulating neuronal and glial processes particularly associated with the OPC-cell state. Targeting both intrinsic and microenvironment Cav3 with the inhibitor mibefradil significantly enhanced the anti-GBM effects of TMZ and radiation.

Figure 1:. Microenvironment Cav3.2 promotes tumor progression and reduces survival.

A) Schematic of experimental workflow for WT and Cav3.2 KO experiments. B) Representative MRIs and quantification showing that microenvironment Cav3.2 KO leads to significant reduction in GBM tumor volume. Red arrows indicate the tumor location. C & D) Kaplan Meir survival curves for WT and Cav3.2 KO mice in GL261 and CT2A xenograft tumors respectively. E) Representative immunofluorescence staining of Ki67 in WT and Cav3.2 KO with quantification showing a significant reduction in proliferation index. ***<0.001

Figure 2:. Microenvironmental Cav3.2 regulates neuronal and glial processes.

A) Schematic showing the experimental workflow for single-cell RNA-seq tumor isolation. B) UMAP of the distinct cell types isolated from WT and KO mice. C) Gene ontology Biological Pathways of downregulated genes in Cav3.2 KO tumors. D) Cnetplot of GO BP pathways and associated genes. E) Gene ontology Cellular Compartment of downregulated genes in KO tumors. F) Cnetplot of GO CC pathways and corresponding genes.

Figure 3:. Cav3.2 KO downregulates OPC cell state genes.

A) Network graph of the top 25 co-expressed genes in the Tumor 4 module of the hdWGCNA analysis. B) Gene Ontology Biological Pathways for the Tumor module 4. C) Enrichment score of the OPC cell state in WT and Cav3.2 KO tumors. D) Expression of Sox10 in WT and Cav3.2 KO tumors showing decreased expression. E) Representative immunofluorescent staining of SOX10 (Red) in WT and Cav3.2 KO tumors (GFP) with quantification of %SOX10 positive cells on the right. F) Spatial transcriptomic data of 266T showing the different zones of tumor infiltration (Left), OPC-like cells (Right) and Sox10 expression (Bottom) demonstrating enrichment of Sox10 in the OPC-like cells in the transition zone.

Figure 4:. Neuronal Cav3.2 promotes GSCs growth and neuronal connections.

A) Representative immunofluorescent images of Glioma Stem cells G34 (Red) co-cultured with WT and Cav3.2 KO neurons. Proliferative cells are labeled with EDU (Green) with quantification of proliferation index to the right. B) Representative immunofluorescent images of G34 cells subjected to WT and Cav3.2 KO Conditioned medium with quantification of proliferation index to the right. C) Representative immunofluorescent images of Glioma Stem cells G34-GFP (Green) co-cultured with WT and Cav3.2 KO neurons labeled with Neurofilament (Pink) with quantification of neuron projections to the right. D) Representative immunofluorescent staining of VGLUT1 (Red) in WT and Cav3.2 KO tumors (GFP) with quantification of percent VGLUT1-GFP positive cells on the right.

Figure 5:. Mibefradil synergizes with standard of care to reduce tumor volume and downregulate neuronal processes.

A) Representative MRIs of Vehicle, Mibefradil, TMZ + Radiation, Mibefradil + TMZ+ Radiation. B) Quantification of tumor volume. C) Volcano plot of the differentially expressed genes from GSCs treated with mibefradil. D) Dotplot of the Gene ontology Biological Pathway for the deregulated genes upon GSC treatment. ***<0.001