PROJECT SUMMARY/ABSTRACT - MECHANOSURVEILLANCE IN BREAST CANCER METASTASIS Metastatic breast cancer is a devastating disease that impacts the lives of tens of thousands of women each year. Unfortunately, the majority of treatment options for patients diagnosed with metastatic breast cancer are palliative. Current treatment strategies often target cancer cells’ genetic and biochemical abnormalities, but cancer cells also undergo profound changes in their biophysical properties such as cellular stiffness. Targeting biophysical properties offers exciting new therapeutic avenues, but the basic knowledge on the relationship between cancer cell stiffness and regulation of metastasis is incomplete. Emerging evidence suggests that cancer cell stiffness acts as a mechanical input for activation of cytotoxic lymphocytes that destroy cancer cells. Thereby, stiffness becomes an immune vulnerability of cancer cells during the metastatic cascade. This process, where mechanical inputs activate the immune surveillance, is termed mechanosurveillance and it is strongly regulated by the expression of Myocardin related transcription factors A and B (MRTFA/B) in cancer cells. Based on the published and the preliminary data, the overarching hypothesis of this project is that MRTFA driven calcium influx increases cancer cell stiffness and thereby activates mechanosurveillance during breast cancer metastasis. The long-term goal of this project is to determine the molecular mediators of MRTFA associated mechanosurveillance and thereby inform pharmacological methods to exploit this cancer cell vulnerability for better treatment of patients with metastatic breast cancer. The research design includes a set of interdisciplinary approaches, such as transcriptomic and genomic analyses of patient samples and cancer models, atomic force microscopy on patient derived xenograft organoids (PDXos) and on immunocompetent models of metastatic colonization, and histological analyses of human breast cancer metastases. The specific aims of this project are: 1) to determine the contribution of MRTFA driven calcium influx on cancer cell stiffness in PDXos, in vitro, and in situ mouse models by using atomic force microscopy and confocal microscopy. 2) to determine the degree of cytotoxic lymphocyte activation by MRTFA and the associated stiffness in vivo by using multiplexed imaging and machine learning on metastatic tissue samples from patients and by using flow cytometry in metastases in syngeneic mouse models. 3) to determine how to exploit MRTFA associated mechanosurveillance to target metastases by testing the impact of clinically relevant immune checkpoint blockade treatments on metastasis bearing mice. This work will uncover mediators of mechanosurveillance and demonstrate its prevalence in human cancer. Thus, results of this work will impact the immediate use of immune checkpoint blockade and the future strategies to enhance mechanosurveillance in patients with metastatic breast...