Defining Therapeutically Targetable Regulators of Tumor-Intrinsic Immune Suppression in Glioblastoma - Glioblastoma (GBM) is the most common and aggressive form of brain cancer in adults with a dismal prognosis and limited therapeutic options. Despite significant advancements in the field, cancer immunotherapy is relatively ineffective in many solid tumors, including GBM. Several factors contribute to this therapeutic failure in GBM including spatial and cellular heterogeneity, a predominantly pro-tumor immune microenvironment, and tumor cell-intrinsic immune evasive and suppressive mechanisms. Characterizing spatially distinct immunosuppressive cell types and the mechanisms they employ to modulate the anti-tumor immune response represents a promising avenue for targeted therapeutics. The broad goal of this proposal is to define spatially and phenotypically unique tumor cell mechanisms that suppress the anti-tumor immune response and are amenable to therapeutic inhibition. This goal will be achieved by leveraging state of the art sequencing and imaging technologies, advanced in vivo genetic screening, and robust in vitro and in vivo validation using genetic inactivation techniques. In Aim 1, spatial proteomics and transcriptomics technologies paired with single-cell RNA-sequencing to visualize distinct spatial domains with local immune-suppression in patient GBM specimens. Transcriptional phenotypes of tumor and myeloid cells unique to these domains will be identified and further analyzed to infer genetic regulators and mechanisms specific to these cells that may be involved in immunesuppression. In Aim 1, candidate genes defined in the previous aim will be investigated in murine glioma models. Initially, an in vivo enrichment screen involving CRISPR/Cas9 gene-editing technology will be performed. In this system, T cells, which are genetically altered to target implanted murine glioma cells but with a repressed cytotoxic capacity, will transfer Cre recombinase to interacting tumor cells. Notably, these tumor cells will possess a ere-induced mCherry cassette; therefore, tumor cells that interact with T cells in the tumor microenvironment will be fluorescently labeled. A gRNA enrichment screen performed on tumor cells isolated from these mice will reveal genes that are likely involved in mediating tumor-T cell interfacing. Subsequent in vivo validation studies involving individual inactivation of these genes will determine which are potently immune-suppressive, defining the top candidates for additional studies focused on in-depth in vivo and in vitro mechanistic exploration. Further experiments utilizing small-molecule inhibitors or blocking antibodies against validated targets will be conducted to assess the efficacy of pre-clinical therapeutic options. These proposed methods of multi-omic spatial analysis. cutting-edge in vivo murine modeling of tumor-immune interaction. and extensive in vivo and in vitro validation will inform the development of novel therapeutic strategies to reprogram the GBM immune microenvironment and augment the anti-tumor immune response. potentially enhancing efficacy of current and emerging immunotherapies.
