Tumor cell heterogeneity drives spatial organization of the intratumoral immune response

Intratumoral heterogeneity (ITH)—defined as genetic and cellular diversity within a tumor—is linked to failure of immunotherapy and an inferior anti-tumor immune response. We modeled heterogeneous tumors comprised of “hot” and “cold” tumor populations (giving rise to T cell–rich and T cell–poor tumors, respectively) and introduced fluorescent labels to enable precise spatial tracking. We found the cold tumor cell population exerted a “dominant cold” effect in mixed tumors. Strikingly, spatial analysis revealed that the tumor cells themselves created distinct local microenvironments within heterogeneous tumors: regions occupied by cold tumor cells showed pronounced immunosuppression, harboring increased CD206^Hi macrophages and diminished local T cell function. This inferior T cell activity in cold regions persisted even after immunotherapy and mechanistically was mediated by CX3CL1 produced by the cold tumor cells. An immune cold tumor population within a heterogeneous tumor thus impairs tumor immunity on both a tumor-wide and a highly localized spatial scale.