Tumor cell heterogeneity drives spatial organization of the intratumoral immune response in squamous cell skin carcinoma

Abstract

Intratumoral heterogeneity (ITH)—defined as genetic and cellular diversity within a tumor—is linked to a failure of immunotherapy in multiple cancer types and to an inferior anti-tumor immune response even in the absence of therapy. To determine how tumor heterogeneity shapes the immune microenvironment and impacts responses to therapy, we modeled heterogeneous tumors comprised of a pro-inflammatory ("hot") and an immunosuppressive ("cold") tumor population. This was done by mixing two squamous skin carcinoma cell lines that alone give rise to tumors with a reproducible immunologically "hot" or "cold" immune phenotype respectively, and labeling these with YFP or RFP fluorescent tags to enable precise spatial tracking. The resulting mixed-population tumors were made up of a patchwork of distinct regions that could be classified as being comprised of YFP+ ("hot") cells, RFP+ ("cold") cells, or a mixture of YFP+ and RFP+ cells. We find the spatial organization of tumor cells creates a blueprint for the spatial organization and functional activity of infiltrating immune cells, defining the architecture of both tumor-infiltrating T cell and myeloid cell compartments. Using a combination of microdissection techniques and single-cell spatial transcriptomics, we show that YFP regions harbor a higher frequency of Th1 cells and IFNγ+ CD8 T cells compared to RFP regions, whereas immunosuppressive macrophages preferentially accumulate in RFP regions. Total CD4 T cells were also enriched in YFP regions, but by contrast, total CD8 T cells—despite their superior function in YFP regions—were of low abundance throughout the heterogeneous tumors. We identify the chemokine Cx3cl1, produced at higher levels by our "cold" tumors, as a mediator of intratumoral macrophage abundance, and show that overexpression of Cx3cl1 in pro-inflammatory ("hot") tumor cells leads to an increase in immunosuppressive CD206+ macrophages. We further interrogated the impact of this spatial patterning of immune cells on the responses of heterogeneous tumors to checkpoint blockade. We find that combination of PD-1 blockade and CD40 agonist is able to increase the Th1 response in "cold" RFP regions, however the T cell response in "cold" regions remains inferior to "hot" regions and treatment achieves only a modest reduction in tumor growth. Collectively, our results demonstrate that the spatial organization of heterogeneous tumor cells has a profound impact on directing the spatial organization and function of tumor-infiltrating immune cells as well as on responses to immunotherapy.

Immunologic tumor microenvironment modulators for turning cold tumors hot

“…For example, studies using PDAC models on mice showed that a mix of two different PDAC cell lines – one that makes immune hot tumors and the other that makes immune cold tumors – created an overall immune cold tumor environment. This finding suggests that the immune cold phenotype may dominate in high‐heterogeneity scenarios [ 219 ]. Additionally, a study in a murine melanoma model demonstrated that high‐heterogeneity tumors displayed a notably diminished CD8 + T cell response when compared to low‐heterogeneity tumors [ 219 ].…”

“…This finding suggests that the immune cold phenotype may dominate in high‐heterogeneity scenarios [ 219 ]. Additionally, a study in a murine melanoma model demonstrated that high‐heterogeneity tumors displayed a notably diminished CD8 + T cell response when compared to low‐heterogeneity tumors [ 219 ].…”