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.
Intratumoral heterogeneity (ITH) – cellular and molecular diversity within a tumor – is linked to failure of immunotherapy in multiple cancer types. A high degree of ITH is associated with poor infiltration of T cells into the tumor and resistance to immune checkpoint blockade (ICB) therapy. To determine how distinct tumor populations within heterogeneous tumors shape the immune microenvironment and how this impacts therapy response, we modeled heterogeneous tumors composed of an RFP-tagged immunosuppressive tumor population and a YFP-tagged pro-inflammatory tumor population. The resulting tumors contained a patchwork of distinct regions with YFP +cells, RFP +cells, or a mixture of YFP +and RFP +cells. Analysis of the immune infiltrates in each region revealed a higher frequency of total CD4 T cells, Th1 cells and IFNg +CD8 T cells in YFP regions compared to RFP regions, whereas macrophages exhibited the opposite pattern. PD-1 blockade and CD40 agonist combinatorial antibody therapy induced an increase in Th1 abundance in RFP regions, but the treatment did not clear the tumors. Together, these results reveal that distinct regional immune infiltration pattens within the tumor are driven by the local tumor cells present in each region, and that the treatment-induced global improvement of Th1 infiltration alone is not sufficient to induce tumor clearance. Moreover, we identified Cx3cl1 as a driver of a dominant immunosuppressive tumor microenvironment, marked by an increase in immunosuppressive CD206 +macrophages and decrease in anti-tumoral neutrophils and monocytes. These results suggest that an immunosuppressive tumor population drives immunotherapy resistance of heterogeneous tumors and Cx3cl1 as a new therapeutic target. Supported by grants from Parker Institute for Cancer Immunotherapy (Project Grant)
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