AbstractMacrophage migration inhibitory factor (MIF) is an upstream regulator of innate immunity, but its expression is increased in some cancers via stabilization with HSP90-associated chaperones. Here, we show that MIF stabilization is tumor-specific in an acute colitis-associated colorectal cancer (CRC) mouse model, leading to tumor-specific functions and selective therapeutic vulnerabilities. Therefore, we demonstrate that a Mif deletion reduced CRC tumor growth. Further, we define a dual role for MIF in CRC tumor progression. Mif deletion protects mice from inflammation-associated tumor initiation, confirming the action of MIF on host inflammatory pathways; however, macrophage recruitment, neoangiogenesis, and proliferative responses are reduced in Mif-deficient tumors once the tumors are established. Thus, during neoplastic transformation, the function of MIF switches from a proinflammatory cytokine to an angiogenesis promoting factor within our experimental model. Mechanistically, Mif-containing tumor cells regulate angiogenic gene expression via a MIF/CD74/MAPK axis in vitro. Clinical correlation studies of CRC patients show the shortest overall survival for patients with high MIF levels in combination with CD74 expression. Pharmacological inhibition of HSP90 to reduce MIF levels decreased tumor growth in vivo, and selectively reduced the growth of organoids derived from murine and human tumors without affecting organoids derived from healthy epithelial cells. Therefore, novel, clinically relevant Hsp90 inhibitors provide therapeutic selectivity by interfering with tumorigenic MIF in tumor epithelial cells but not in normal cells. Furthermore, Mif-depleted colonic tumor organoids showed growth defects compared to wild-type organoids and were less susceptible toward HSP90 inhibitor treatment. Our data support that tumor-specific stabilization of MIF promotes CRC progression and allows MIF to become a potential and selective therapeutic target in CRC.