The Influence of Matrix Stiffness on Extracellular Matrix Protein Expression in 3D Encapsulated Mammary Fibroblasts

A disease that transcends all races is breast cancer, the second leading cause of death among women with cancer. One factor, which participates in breast tumor progression, is the extracellular matrix (ECM), an acellular, protein-rich entity, which drives several cell, processes shown to promote tumorigenesis. Specifically, abnormal expression patterns and cross-linking of matrix fibers induces a more dense tissue structure, which has been reported to drive breast cancer progression. Alterations in ECM expression and cross-linking are in part due to carcinoma-associated fibroblasts (CAFs), activated fibroblasts which deposit copious ECM in the breast tumor environment. The goal of this study is to understand how ECM expression patterns change following culture of human mammary fibroblasts (HMFs) in mechanically tuned 3 dimensional gelatin hydrogels, a culture setup that better mimics the natural breast environment. Since matrix stiffness has been reported to drive the myofibroblast phenotype, it is anticipated that altered ECM expression and deposition patterns will accompany HMF culture in mechanically stiff gelatin hydrogels. To alter the stiffness of the gel, microbial transglutaminase, which cross-links lysine and glutamine residues in the gelatin matrix, will be employed and rheology will be subsequently utilized to determine the bulk mechanical stiffness of cross-linked gels. Three gels with various stiffnesses including compliant, moderate, and stiff will be utilized for encapsulation of HMFs. Viability assays, such as the Live/Dead assay, will be utilized to monitor cell viability over the 7-day culture period. To assess whether matrix stiffness induces unique patterns of matrix expression, we will evaluate ECM protein expression using western blot for the following ECM proteins: tenascin-C, fibronectin, laminin and collagens I, IV. In addition, immunofluorescence will be utilized to evaluate cellular deposition of ECM proteins in the gelatin hydrogel. Results from these assays will elucidate whether increasing matrix stiffness induces a more pronounced expression level of the aforementioned ECM proteins. Results from these studies may allow for future development of novel therapeutic agents targeted to the myofibroblast within the breast cancer microenvironment