Project Summary/Abstract The impact of cancer associated fibroblasts (CAFs) and the extracellular microenvironment (ECM) in cancer metastasis has become more and more appreciated. The tumor microenvironment consists of multiple compartments: cellular, physical, and chemical. Each compartment in cancerous tissues differs greatly from normal tissue, yet the role that each plays in metastasis and how they affect each other is poorly understood. CAFs have been shown to promote tumor cell metastasis in part by altering the local collagen microenvironment. CAFs can enhance cancer metastasis in 3 ways; (1) direct association between CAFs and leader cells within the tumor, (2) CAF mechanical alterations of the local microenvironment, and (3) CAF secretions that can signal for tumor metastasis and collagen remodeling. How the chemo-mechanical feedback between CAFs, cancer cells, and the ECM remains poorly understood. Here, our goal is to elucidate the chemo-mechanical feedback function of CAFs on leader cells and the microenvironment that facilitates directed collective cell migration. (1) Quantify the impact of CAFs on collective cell migration and ECM remodeling. (2) To examine the effect of the CAF secretome on local collagen structure and function. We will utilize microfluidic cell-based invasion assays in conjunction with primary breast tumor organoids or reconstituted breast tumor organoids (non-migratory mammary cells mixed with isolated leader cells or CAFs) to probe the effect of CAFs on collective cell migration. We will isolate the role of mechanical forces generated by CAFs within the tumor on leader cell function and the effect of CAF generated forces on collagen deformation and remodeling (1). We will then compare the mechanical effect of CAFs on collective migration to that of the chemical CAF secretions on collagen remodeling (2). We aim to quantify the relative role of both mechanical and chemical models to generate a predictive model of CAF function on collective cell migration. We will verify this model by using engineering techniques to manipulate the microenvironment and observe the effect on collective migration. We will combine this with biological techniques to genetically manipulate CAFs and leader cells to identify how CAFs are capable of effecting collective cell migration.