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 IFNψ+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 CD206Himacrophages. 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE237233 | GEO | 2024/12/12

REPOSITORIES: GEO