Impacts of Combining Pulsed Low Dose Rate Radiotherapy (PLDR) and Anti-PD-1 Antibody on Tumor Growth and Its Mechanism for Lung Cancer in Mice

Abstract

As an emerging anti-tumor strategy, Immunotherapies has been approved for the treatment of a variety of tumors. Recent data suggest that the efficacy of radiotherapy in various cancers can be augmented when combined with immune checkpoint blockade. Then, pulsed low dose rate radiotherapy (PLDR) is a new radiotherapy segmentation method. Therefore, this study investigated the inhibitory effect of PLDR combined with anti-PD-1 antibody on lung cancer in mice and its impact on tumor immune microenvironment. By transplanting murine LLC cells into the right leg of C57BL/6 mice with immune activity, a transplanted subcutaneous tumor model was established. The mice were randomly divided into five groups: control, conventional radiotherapy (RT)±anti-PD-1 antibody (mAb),PLDR±anti-PD-1 antibody (mAb). RT was delivered as a dose of 5 × 2 Gy whereas PLDR involved delivering a dose of 2 Gy as 10 pulses of 0.2 Gy, each 3 minutes apart and lasting for 5 days. Anti-PD-1 antibody and isotype control were administered intraperitoneally once every three days at a dose of 5mg/kg, three times in total. The tumor, blood and tumor-draining lymph nodes (TDLNs) were harvested after treatment, and a single cell suspension was prepared for flow cytometry to analyze the changes in the immune microenvironment of the tumor tissue, the expression of PD-L1. PD-1 and the activated systemic immune response; Finally, this study explored a mechanism able to explain the observed synergy of combined therapy. PLDR combined with anti-PD-1 antibody can better inhibit the growth of tumor than RT,PLDR and RT combined group. Survival analysis demonstrated a statistically significant advantage for PLDR+anti-PD-1 than other groups. Median survival with PLDR+anti-PD-1mAb was 63d compared with 54d with RT+anti-PD-1mAb,41d with RT and 40d with PLDR and 33 with control. Meanwhile both RT and PLDR induced up-regulation of PD-L1 expression on tumor surface and PD-1 expression on lymphocytes. Then, the frequency of CD4+,CD8+T cells were higher in the PLDR combined treatment group in tumor blood and draining lymph nodes, and synergistically reduce the local accumulation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs) than others. Finally, the main reason for the better tumor inhibition effect of PLDR combined group is that it upgrades the number and activity of CD8+T cells in tumor. PLDR combined with anti-PD-1 antibody can result in better tumor growth inhibition and significantly delay the survival time of mice, which was mainly through the cytotoxic T cell-dependent mechanism, meanwhile increasing the infiltration of CD4 + and CD8 +T-cells in tissues.