PROJECT SUMMARY Despite innovations in diagnosis and treatment, colorectal cancer (CRC) remains one the most common causes of cancer-related deaths in the United States. During tumorigenesis, the extracellular matrix (ECM) drastically stiffens, resulting in a self-amplifying feedback loop, furthering cancer progression; in CRC, increased matrix elasticity is correlated with more advanced stages and worse treatment outcomes. Cells sense tissue stiffness via integrins, which relay biochemical signals down many pathways, including the Rho/ROCK pathway. The Rho/ROCK pathway has been shown to be highly dysregulated in many cancers, but its role in CRC is still highly debated: some studies have shown elevated levels in metastatic lesions, whereas others have correlated inactivation of this pathway with metastasis. Herein, we propose to study the mechanical contributions to RhoA activation and how this may lead to increased proliferation and signaling, with a particular emphasis on comparing the mechanical environments of the colon and the liver—the most common metastatic site, which has been shown to be significantly stiffer than the primary tumor tissue. We will develop a novel hydrogel biomaterial that can stiffen around cells from 1kPa (primary tumor) to 6kPa (liver metastatic tumor) upon exposure to 365nm light over the course of the week, by employing photomediated oxime ligation, a chemistry we have pioneered in our previous work for making photostiffening hydrogels as well as immobilizing full-length proteins in natural and synthetic materials. To encode for subsequent cell release for downstream assays, we will develop a suite of enzymatically sensitive peptide crosslinkers to enable bioorthogonal, user-defined hydrogel degradation. In Aim 2, we will examine RhoA activation in primary patient-derived CRC tumor organoids as a function of progressive stiffening of the matrix, and compare to levels of RhoA activation in metastatic cancer organoid lines in stiff gels, via pull-down assays, Western blotting, and a lentivirally transduced biosensor construct. In Aim 3, we will investigate the mechanical memory of RhoA activation in metastatic tumor organoid lines and whether is reversable upon systematic softening of the matrix back to baseline, primary colon stiffnesses. We expect results from these studies will result in novel insights into the role of matrix mechanics in CRC progression and potentially open the door for biophysical routes of tumor resolution and treatment.