Impacts of Combining PD-L1 inhibitor and Radiotherapy on the Tumour immune microenvironment in a Mouse Model of Esophageal Squamous Cell Carcinoma

Abstract

Background：The combination of radiation with immune checkpoint inhibitors (ICIs) has been demonstrated to display synergistic effects in solid cancers. Nevertheless, the anti-tumor effect of combining radiation with programmed cell death 1 ligand 1 (PD-L1) inhibitor in esophageal squamous cell carcinoma (ESCC) remains unclear. Therefore, the objectives of our study were to evaluate the anti-tumor effects of PD-L1 inhibitors combined with radiotherapy in ESCC mouse model and to depict the immune landscape within the tumor microenvironment (TME). Methods: A syngeneic C57BL/6 subcutaneous xenograft mouse model was applied to evaluate the anti-tumor efficacy of different treatment protocols according to tumor growth curve and survival time. Tumour immune microenvironment was assessed by flow cytometry including CD4⁺T cells, CD8⁺T cells, regulatory T cells (Tregs), tumor-derived macrophage (TAM), myeloid-derived suppressor cell (MDSC), and the expression of CD8⁺T cell activation, exhaustion, and memory state markers. In addition, transcriptomic analysis was used to examine the immune gene expression changes in tumor microenvironment. Results: Radiotherapy combined with anti-PD-L1 inhibitors synergistically enhanced anti-tumor immune response via boosted the infiltration of CD8⁺ T cells, increased the ratio of CD8⁺ T cells to Tregs and population of central memory CD8⁺ T cells (T_CM), enhanced interferon gamma (IFN-γ) secretion by tumor-infiltrating CD8⁺ T cells, and reduced the accumulation of M2-type TAMs and Tregs in the TME in mouse model. In addition, radioimmunotherapy also induced a better immunophenotype in spleen and tumor draining lymph node (TDLN). Consequently, radioimmunotherapy appeared greater benefit in antitumor effects and mice survival. Moreover, our transcriptomic analysis suggested that radioimmunotherapy promoted the expression of immunostimulation-related regulatory pathways and cytokines that shape the immunoinflammatory tumor microenvironment. Conclusions: Our research indicated that anti-PD-L1 inhibitors combined with RT promotes systemic anti-tumor immunity by improving the immune microenvironment in a mouse model of ESCC.