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) has remained unclear. Therefore, the objectives of our study were to evaluate the anti-tumor effects of PD-L1 inhibitors combined with radiotherapy in a mouse model of ESCC and to depict the immune landscape within the tumor microenvironment (TME).

Methods: Murine ESCC cells (mEC25) were injected subcutaneously into the right flanks of C57BL/6 mice. Tumor-bearing mice were exposed to different treatments: IgG antibody (control), anti-PD-L1 antibody, radiation, or radiation + anti-PD-L1 antibody. Tumor growth and survival time of mice were monitored. Tumour immune microenvironment was assessed by flow cytometry, including CD4⁺T cells, CD8⁺T cells, regulatory T cells (Tregs), tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and the activation and exhaustion of CD8⁺T cell. In addition, transcriptomic analysis was used to examine the changes in immune gene expression in the TME.

Results: Radiotherapy combined with anti-PD-L1 inhibitors (radioimmunotherapy) synergistically enhanced anti-tumor immune response, leading to decreased tumor growth and prolonged survival of tumor-bearing mice. The radioimmunotherapy increased the infiltration of CD8⁺ T cells, the ratio of CD8⁺ T cells to Tregs, the population of central memory CD8⁺ T cells (T_CM), interferon-gamma (IFN-γ) secretion of tumor-infiltrating CD8⁺ T cells, and reduced the accumulation of M2-type TAMs and Tregs in the TME in mouse model. In addition, the radioimmunotherapy induced anti-tumor immune response in the spleen and tumor-draining lymph node (TDLN). Moreover, transcriptomic analysis suggested that the radioimmunotherapy promoted the activation of immune regulatory pathways and increased the expression of cytokines such as CXCL9 and CXCL10, thus creating an immunoinflammatory tumor microenvironment.

Conclusions: Our research revealed that anti-PD-L1 inhibitors combined with radiotherapy caused systemic anti-tumor immunity by reshaping the immune microenvironment in a mouse model of ESCC.