Single-cell RNA-sequencing of murine breast tumor infiltrating immune cells isolated from the site of metastasis in wild-type and short-form Ron knockout mice

Metastatic breast cancer is the overwhelming cause of breast cancer mortality and is still incurable. Development of immunotherapy is an exciting new area of research in metastatic breast cancer; however, the extreme immunosuppressive tumor environment poses a major challenge. Here we provide evidence that a particular isoform of Ron kinase, short-form Ron (SF-Ron), strongly suppresses anti-tumor immune responses and promotes metastatic outgrowth of mammary tumors. We used single-cell RNA-sequencing to identify the CD45+ immune cell populations within the common site of breast cancer metastasis, lung, from either wild-type (WT) or SF-Ron knockout (Ron SF-/-) in the MMTV-PyMT experimental metastasis model. SF-Ron was required for breast cancer metastasis develoment and the skewing of the lung immune microenvironment toward the immune-suppressive pro-tumor microenvironment. Lack of SF-Ron not only enhanced tumor-specific immune response, but also led to accumulation of less differentiated TCF1+ CD4+ T cells in the metastatic lungs that were endowed with type I helper T cell-like (Th1-like) differentiation potential. Mice treated with a small molecule Ron kinase inhibitor produced significantly more tumor-specific CD8+ T cells and reduced metastatic outgrowth. Our study indicates that blocking Ron, specifically SF-Ron, remodels the metastatic lung microenvironment to enhance anti-tumor immunity and clear metastatic lesions, providing strong pre-clinical evidence for Ron kinase inhibitors to augment immunotherapy for treatment of metastatic breast cancer. MMTV-PyMT tumor cells were injected via the lateral tail vein into wild-type and short-form Ron knockout mice (n>8). Lung metastasis were allowed to develop for 28 days. At the end point, lungs occupied by metastases were harvested and pooled in each group, and processing into single cell suspension. The DAPI(-)CD45(+) live tumor metastases infiltrating immune cells were sorted via flow cytometry and processed for single-cell RNA-sequencing via 10x platform.