Tmic-78. endothelial regulation of the immunosuppressive phenotype of tumor-associated microglia in glioblastoma

Abstract Glioblastoma (GBM) is considered a non-immunogenic “cold tumor” with an immunosuppressive microenvironment that blocks T-cell infiltration and function, limiting the efficacy of targeted immunotherapy. Tumor-associated microglia and macrophages (TAM) constitute approximately 30-50% of the non-neoplastic immune cells, and they are closely associated with blood vessels in GBM. However, the precise mechanism by which TAM acquire the immunosuppressive phenotype remains poorly understood. In this study, we show that tumor endothelial cells (TEC) from primary and recurrent GBM express inflammatory cytokines and chemokines that influence the function and polarization of microglia. We demonstrate that conditioned media (CM) of TEC promotes the immunosuppressive phenotype of normal microglia and patient-derived TAM. RNA-sequencing of TAM treated with TEC-CM shows upregulation of immunosuppressive cytokines, as well as T-cell co-inhibitory molecules such as PD-L1. Culturing CD8 T-cells in CM of TAM-polarized by TEC inhibits their cytotoxic function and cytokine production, and induces expression of PD-1 and CTLA-4. Furthermore, gene set enrichment analysis uncovers alterations in calcium signaling-related pathways in TAM polarized by TEC-CM. Blocking calcium signaling with small molecule inhibitors in microglia reverses the immunosuppressive phenotype induced by TEC. To determine whether the interactions between GBM, endothelial cells and TAM promotes immunosuppression in vivo, we utilize the humanized immune system mouse model (huNOG-EXL) that harbor human lymphocytes and monocyte populations. In preliminary studies, we found that co-transplantation of GBM tumor cells with TAM and TEC significantly promotes tumor growth compared with GBM cells alone. Single-cell mass cytometry analysis of peripheral blood mononuclear cells (PBMC) pre- and post-transplant, and scRNA-sequencing and spatial-transcriptomic studies of these co-transplanted mice will potentially reveal key insights into local- and peripheral immune responses regulated by tumor endothelial-microglia interactions. Together, our findings highlight a key role for endothelial-mediated calcium signaling in regulation of the immunosuppressive phenotype of microglia in GBM.