RNA sequencing of blood-brain barrier (BBB) microvessels from mice with orthotopic GBM showing the benefit of triple therapy combining anti-immune checkpoint therapy with dual-anti angiogenic therapy.

Glioblastoma multiforme (GBM) is a non T cell-inflamed cancer characterized by an immunosuppressive microenvironment that impedes dendritic cell maturation and T cell cytotoxicity. The alleviation of immunosuppression might be a prerequisite for succesful immune checkpoint therapy in GBM. We here combine anti-angiogenic and immune checkpoint therapy and demonstrate improved therapeutic efficacy in syngeneic, orthotopic GBM models. We observed that blockade of vascular endothelial growth factor (VEGF), Angiopoietin-2 (Ang-2) and programmed cell death protein-1 (PD-1) significantly extended survival compared to vascular targeting alone. In the GBM microenvironment, triple therapy increased the numbers of cytotoxic T-lymphocytes that inversely correlated with myeloid-derived suppressor and regulatory T cells. Furthermore, transcriptomic analysis of GBM microvessels indicates a global vascular normalization that was highest after triple therapy. Our results propose a rationale to overcome limitations of VEGF monotherapy by integrating the synergistic effects of VEGF/Ang-2 and PD-1 blockade to reinforce anti-tumor immunity through a normalized vasculature. Overall design: Brain microvessels were isolated from treated and untreated adult WT BL6/57 mice from tumor bearing brain region at 21 days post transplation followed by RNA sequecning. Three biological replicates were included for each group pooling 3 mice for each set. Contra-lateral brain region within each group and sham operated mice served as controls.