e23001 Background: In angiogenesis, VEGF-A and its receptor VEGFR-2, and in lymphangiogenesis, VEGF-C and VEGFR-3, play central roles. An anti-tumor immunotherapy using an agonistic antibody (Ab) to death receptor (DR)-5, the apoptosis inducing receptor, exhibits antitumor effects. VEGF-A or -C inhibition augmented the effects of immunotherapy. However, little is known about the effects of combining anti-angiogenesis, anti-lymphangiogenesis, and immunotherapy. Here, we combined sunitinib, which mainly inhibits VEGFR1-3 and PDGFRs, as anti-angiogenic and anti-lymphangiogenic therapies, with anti-DR-5 Ab as an immunotherapy in mice. Methods: Mice were injected with breast carcinoma 4T1 cells into the footpad, and treated with sunitinib and/or anti-DR-5 Ab. On day 14, mice were anesthetized, and the popliteal lymph nodes and tumors were isolated for immunohistochemistry, flow cytometry, hypoxia, and lymphatic flow analysis. To evaluate the tumor growth rate, mice were injected with 4T1 cells into the hindflank, treated with the combination or monotherapy and sacrificed on day 21. CD4+ and/or CD8+T cells were depleted by anti-CD4 and/or anti-CD8 Ab, respectively. Results: Sunitinib decreased endothelial cells, pericytes, and hypoxic area densities in the tumors suggesting blood vessel normalization. Sunitinib also decreased the amount of evans blue injected into tumors and hyaluronic acid in tumors, suggesting improved lymphatic flow. In draining lymph nodes, sunitinib and anti-DR-5 Ab combination increased the numbers of activated dendritic cells and CD8+ T cells than controls. In tumors, the combination increased the numbers of infiltrating effector CD4+FOXP3- and CD8+ T cells compared to controls. Sunitinib and anti-DR-5 Ab combination augmented the decrease in the tumor growth rate compared to each mono-therapy. CD4+ and/or CD8+ T cell depletion showed anti-tumor effects of the combination were partially CD8+T cell-dependent. Conclusions: Blood vessel normalization and improved lymphatic flow in tumors may improve circulation between the tumors and draining lymph nodes, which has a potential to augment the effects of immunotherapy.
Prolyl Hydroxylase inhibitor repaired chemo-sensitivity through tumor blood vessel normalization in tumor mouse model