Co-inhibition of TGF-β and PD-L1 pathways in a metastatic colorectal cancer mouse model triggers interferon responses, innate cells and T cells, alongside metabolic changes and tumor resistance.

Colorectal cancer (CRC) is the third most prevalent cancer worldwide with a high mortality rate (20-30%), especially due to metastasis to adjacent organs. Clinical responses to chemotherapy, radiation, targeted and immunotherapies are limited to a subset of patients making metastatic CRC (mCRC) difficult to treat. To understand the therapeutic modulation of immune response in mCRC, we have used a genetically engineered mouse model (GEMM), "KPN", which resembles the human 'CMS4'-like subtype. We show here that transforming growth factor (TGF-β1), secreted by KPN organoids, increases cancer cell proliferation, and inhibits splenocyte activation in vitro. TGF-β1 also inhibits activation of naive but not pre-activated T cells, suggesting differential effects on specific immune cells. In vivo, the inhibition of TGF-β inflames the KPN tumors, causing infiltration of T cells, monocytes and monocytic intermediates, while reducing neutrophils and epithelial cells. Co-inhibition of TGF-β and PD-L1 signaling further enhances cytotoxic CD8⁺T cells and upregulates innate immune response and interferon gene signatures. However, simultaneous upregulation of cancer-related metabolic genes correlated with limited control of tumor burden and/or progression despite combination treatment. Our study illustrates the importance of using GEMMs to predict better immunotherapies for mCRC.