The therapeutics efficacy of an anticancer treatment is often restricted by tumor fibrous tissue and tumor microenvironment. Stromal matrix barriers create a sanctuary for breast cancer, while the prolonged pro-fibrotic stimuli facilitate cancer cell growth and survival by establishing an immunosuppressive environment. Relaxin, a small peptide hormone, demonstrated dual anti-fibrotic and pro-immunogenic effects in various disease models including several solid cancers. Treatment with relaxin significantly reduced the expression of major tumor extracellular matrix components such as collagens, fibronectin, and elastin. The degradation of fibrotic tumor matrix following relaxin treatment led to reduced cancer cell drug resistance. Additionally, it was shown that relaxin can change tumor macrophage population from pro-inflammatory to pro-resolution enabling T cell- mediated cancer cell killing and macrophage phagocytosis. However, due to a short half-life in vivo, delivery of recombinant relaxin requires continuous infusion. Relaxin signals through its cognate G protein-coupled receptor RXFP1, which is expressed in tumor associated fibroblasts and infiltrating immune cells. We propose targeting the integrity of the tumor microenvironment with the first-in-class small molecule agonist of RXFP1 developed in our laboratories. The lead compound, ML290, shows high activity, oral bioavailability, in vivo stability, and excellent pharmacological properties. It is well tolerated by animals, shows no toxicity in vivo, and does not increase cancer cell proliferation and invasiveness nor does it affect extracellular matrix remodeling in healthy tissues. Our preliminary data indicate that the ML290 treatment of mice with HER2-positive breast cancer reduces tumor size and tumor fibrotic content. The overall goal of this project is to demonstrate anticancer activity of relaxin receptor agonist, ML290, in preclinical models of breast cancer, regardless of cancer subtype. We will test the anti-cancer efficacy of ML290 in primary and metastatic breast cancer models, analyze changes in tumor ECM composition, recruitment of immune cells, and genomic/proteomic response in stromal and cancer cells to treatment. We will analyze the effect of combination treatment of ML290 with immune checkpoint blockade and anti-HER2 immunotherapeutics. The pharmacological re-programing of stromal cancer microenvironment by ML290 will provide a new therapeutic approach for breast cancer suppression.