Knowledge of immune cell phenotypes in the tumor microenvironment is essential for understanding mechanisms of cancer progression and immunotherapy response. We profiled 45,000 immune cells from eight breast carcinomas, as well as matched normal breast tissue, blood, and lymph nodes, using single-cell RNA-seq. We developed a preprocessing pipeline, SEQC, and a Bayesian clustering and normalization method, Biscuit, to address computational challenges inherent to single-cell data. Despite significant similarity between normal and tumor tissue-resident immune cells, we observed continuous phenotypic expansions specific to the tumor microenvironment. Analysis of paired single-cell RNA and T cell receptor (TCR) sequencing data from 27,000 additional T cells revealed the combinatorial impact of TCR utilization on phenotypic diversity. Our results support a model of continuous activation in T cells and do not comport with the macrophage polarization model in cancer. Our results have important implications for characterizing tumor-infiltrating immune cells.

Summary Regulatory T (Treg) cells reside in lymphoid organs and barrier tissues where they control different types of inflammatory responses. Treg cells are also found in human cancers, and studies in animal models suggest that they contribute to cancer progression. However, properties of human intratumoral Treg cells and those present in corresponding normal tissue remain largely unknown. Here, we analyzed features of Treg cells in untreated human breast carcinomas, normal mammary gland, and peripheral blood. Tumor-resident Treg cells were potently suppressive and their gene expression pattern resembled that of normal breast tissue, but not of activated peripheral blood Treg cells. Nevertheless, a number of cytokine and chemokine receptor genes, most notably CCR8, were upregulated in tumor-resident Treg cells in comparison to normal tissue resident ones. Our studies suggest that targeting CCR8 for the depletion of tumor-resident Treg cells may represent a promising immunotherapeutic approach for the treatment of breast cancer.

SUMMARYKnowledge of the phenotypic states of immune cells in the tumor microenvironment is essential for understanding immunological mechanisms of cancer progression and immunotherapy responses, as well as the development of novel treatments. By combining single-cell RNA-seq data from over 45,000 immune cells collected from eight primary breast carcinomas, as well as matched normal breast tissue, peripheral blood, and lymph node, we created an immune map of breast cancer. We developed a preprocessing pipeline, SEQC, and a Bayesian clustering and normalization method, Biscuit, to address the computational challenges inherent to single-cell RNA-seq data, enabling integration of data across patients. This atlas revealed significant similarity between normal and tumor tissue resident immune cells. However, we observed continuous tumor-specific phenotypic expansions driven by environmental cues. Our results argue against discrete activation states in T cells and the polarization model of macrophage activation in cancer, with important implications for characterizing tumorinfiltrating immune cells.