PROJECT SUMMARY Colorectal cancer (CRC) is the second leading cause of cancer death in the United States. Conventional cytotoxic chemotherapy remains a key treatment modality for advanced-stage disease prolonging overall survival, yet clinical responses vary substantially among patients. The most widely used chemotherapy regimens for CRC are formulated with 5-fluorouracil (5-FU) as the backbone. The effects of 5-FU have been extensively studied in vitro and in immunodeficient mice. However, what underlies 5-FU’s anti-cancer efficacy in vivo in immunocompetent settings remains poorly understood, the understanding of which will be critical for devising more effective and/or less toxic strategies against CRC. It is well recognized that cancer-cell-intrinsic sensitivity to 5-FU in vitro can partially explain responsiveness to 5-FU in vivo, with the underlying model that for intrinsically sensitive cancers, 5-FU reduces tumor burden primarily through direct cytotoxicity on cancer cells. However, whether this conventional model is accurate in immunocompetent hosts is not clear. Based on preliminary data, we propose to test a revised model for how 5-FU is effective in immunocompetent settings: 5-FU-induced reduction of tumor-burden requires two components, cancer cells’ intrinsic sensitivity to the drug to prime the response and a distinct requirement for immune modulation to play a major role in reducing tumor burden. Lacking either component will reduce 5-FU efficacy in vivo. We will test this revised model through three complementary aims. In the first aim, we will test the dual requirements of cancer- cell-intrinsic chemosensitivity and immune modulation for effective response to 5-FU in vivo. In the second aim, we will determine the molecular and cellular basis of the immune modulation that reduces tumor burden. In the third aim, we will investigate the relationship between the cGAS-STING pathway and chemotherapy response of human colon tumors. Findings from these studies have the potential to transform our understanding of how 5-FU works in vivo and build a bridge between 5-FU effectiveness and anti-tumor immunity. This project also has the potential to unveil new prognostic and therapeutic strategies for CRC patients. Given that 5-FU is also used in other malignancies (e.g., pancreas, gastric, biliary, small intestinal, breast, head and neck), our studies have treatment implications well beyond CRC.