Temporal Genomic Analysis of Homogeneous Tumor Models Reveals Key Regulators of Immune Evasion in Melanoma

Low intratumor heterogeneity correlates with increased patient survival and immunotherapy response. However, even highly homogeneous tumors are vari-ably aggressive, and the immunologic factors impacting aggressiveness remain understudied. In this study, we analyzed the mechanisms underlying immune escape in murine tumors with low intratumor heterogeneity. We used immunophenotyping and single-cell RNA sequencing to compare the temporal growth of in vivo transplanted, genetically similar, rejected and nonrejected single-cell clones. Nonrejected clones showed high infiltration of tumor-associated macrophages, lower T cell infiltra-tion, and increased T cell exhaustion when compared with rejected clones. Comparative analysis of rejection-associated gene expression programs, combined with in vivo CRISPR knockout screens of candidate regulators, identified macrophage migration inhibitory factor (Mif) as a major con-tributor to preventing immune rejection. Mif knockout resulted in smaller tumors and reduced tumor-associated macrophage infiltration. These results were validated in patients with melanoma. Overall, our homogeneous tumor system can uncover factors regulating growth variability and identifies Mif as critical in aggressive melanoma. Significance: In this study, we find that Mif expression is associated with tumor growth and aggressiveness, specifically in tumors with low heterogeneity. These findings could facilitate the development of new strategies to treat patients with homogeneous, high MIF–expressing tumors that are unresponsive to immune checkpoint therapy.