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.
BackgroundIntratumoral heterogeneity (ITH) is cellular and molecular diversity within a tumor. ITH is linked to failure of immunotherapy in multiple cancer types. One of the suggested mechanisms of this failure is the absence of a productive immune response, which can be driven by the dominance of a tumor population that creates immunosuppressive microenvironment. However, the molecular mechanisms that mediate such dominant immunosuppressive effects and how a dominant immunosuppressive tumor population affects the efficacy of immune checkpoint blockade (ICB) therapy are poorly understood.MethodsWe generated a library of squamous skin cell carcinoma cell lines derived from DMBA+TPA carcinogen-treated mice. Upon transplantation into immunocompetent mice, two cell lines (CF6 and CF9) gave rise to highly and poorly immune-infiltrated tumors, respectively. These two cell lines were tagged with YFP and RFP, respectively, mixed at a 1:1 ratio and injected subcutaneously. The resulting mixed tumors contained a patchwork of distinct regions that are predominantly occupied with YFP (CF6) cells, occupied with RFP (CF9) cells, or are a mixture of YFP and RFP cells. These mixed tumors were microdissected into YFP, RFP, and mixed regions, and the immune infiltrate in each region was analyzed by flow cytometry.ResultsWe found mixed tumors were ”cold” on a whole-tumor level. However, regional analysis showed a higher frequency of total and CD4 T cells in YFP regions compared to RFP regions. In contrast, macrophages exhibited a preferential localization to RFP regions. This suggests that local factors unique to each region drive the immune infiltration patterns of CD4 T cells and macrophages. Interestingly, although CD8 T cells showed a low frequency in all regions, a majority of CD8 T cells from YFP regions produced IFNgamma in response to PMA/Ionomycin stimulation while IFNgamma+ CD8 T cells from RFP regions made up a small fraction. This suggests that the presence of CF9-RFP regions inhibits infiltration of CD8 T cells throughout the tumor, but may not affect the anti-tumoral potential of CD8 T cells that succeed in infiltrating CF6-YFP regions.ConclusionsAn immunosuppressive tumor population rendered our model heterogeneous tumors ”cold” on the whole. Nonetheless, we observed regional patterns in the quantity and quality of immune infiltrates, with local immune infiltration being shaped by the tumor cells present in that region. Understanding how these immune infiltration patterns impact the response of each region to ICB therapy will provide insights into the mechanism by which ITH poses barriers to immunotherapy.
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