Project Summary/Abstract Breast cancer is the most frequent malignancy in women and aging is one of the largest risk factors for the development of breast cancer. The accumulation of oncogenic mutations within incipient tumor cells and age- related changes in the stromal compartment may together impact tumor progression. One change that occurs in the tumor microenvironment is the accumulation of p16INKA4 (p16) positive senescent cells which express various cytokines, chemokines, and enzymes that all together recognized as senescence-associated secretory phenotype (SASP) factors. While SASPs from different cell origins can exert different effects on tumorigenesis, SASP factors secreted by senescent stromal cells can not only directly promote the proliferation of tumor cells, but also create an immunosuppressive microenvironment that ultimately leads to more robust tumor growth. The less ideal response to immunotherapy among breast cancer patients raises the possibilities that senescent stromal cells could be one of the barriers protecting breast tumor cells from current immune checkpoint blockade therapies and that the efficacy of immunotherapy can be boosted when combined with senescent cell elimination strategies. Since immunosuppressive SASP factors are detectable in human breast cancer stroma, I hypothesize that senescent stromal cells contribute to mammary gland tumorigenesis by modulating regulatory T cells (Tregs). Preliminarily, I have shown that depletion of senescent stromal cells can lead to significantly delayed mammary tumor onset in a transgenic mouse model. In addition, I have found that orthotopic mammary tumor transplantation with senescent fibroblasts results in higher tumor burden and more Tregs infiltration. Finally, my single cell RNA-sequencing (scRNA-seq) analysis on murine and human mammary tumor/breast cancer dataset reveals that senescence signature is highly restricted to a specific cancer associated fibroblasts (CAFs) subpopulation and this population expresses elevated levels of several Treg-inducing factors. I thus propose to perform further scRNA-seq analysis to identify gene expression signatures of senescent CAFs across breast cancer subtypes and understand these CAFs’ impact on tumor microenvironment. Furthermore, I will conduct in depth tumor immunity analysis on both transgenic and orthotopic injection mouse models to discuss how tumor immune environment is shaped by the senescent CAFs and whether depletion of senescent stromal cells, using both genetic and pharmacological tools, can exert synergistic effect when combined with immune checkpoint blockade therapies. These aims will not only provide a comprehensive picture of the relationship between senescent stroma and breast cancer development, but also help me pursue my interests in immunology and translational medicine. The training received through this proposal will also strengthen my research skills in immunology and cancer biology. These skills will allow ...