Targeting VEGFR2 with Ramucirumab strongly impacts effector/ activated regulatory T cells and CD8⁺ T cells in the tumor microenvironment

Background: Several studies have established a correlation between the VEGF-VEGFR2 axis and an immunosuppressive microenvironment; this immunosuppression can be overcome by anti-angiogenic reagents, such as ramucirumab (RAM). However, little is known about the immunological impact of anti-angiogenic reagents within the tumor microenvironment in human clinical samples. This study aimed at investigating the effects of RAM on the tumor microenvironmental immune status in human cancers. Methods: We prospectively enrolled 20 patients with advanced gastric cancer (GC) who received RAM-containing chemotherapy. We obtained paired samples from peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs) in primary tumors both pre- and post-RAM therapy to assess immune profiles by immunohistochemistry and flow cytometry. Results: Within the tumor microenvironment, both PD-L1 expression and CD8⁺ T-cell infiltration increased after RAM-containing therapies. In addition, CD45RA^-FOXP3^highCD4⁺ cells (effector regulatory T cells [eTreg cells]) and PD-1 expression by CD8⁺ T cells were significantly reduced in TILs compared with PBMCs after RAM-containing therapies. Patients with partial response and longer progression-free survival had significantly higher pre-treatment eTreg frequencies in TILs than those with progressive disease. In in vitro analysis, VEGFR2 was highly expressed by eTreg cells. Further, VEGFA promoted VEGFR2⁺ eTreg cell proliferation, and this effect could be inhibited by RAM. Conclusions: This study suggests that the frequency of eTreg cells in TILs could be a biomarker for stratifying clinical responses to RAM-containing therapies. Further, we propose that RAM may be employed as an immuno-modulator in combination with immune checkpoint blockade.