Intrahepatic cholangiocarcinoma (ICC) is an aggressive liver tumor with limited therapeutic options and 5-year survival rates of less than 10%. ICC is characterized by its highly desmoplastic nature, with abundance of cancer- associated fibroblasts (CAF) and extracellular matrix (ECM). The role of CAF and ECM in ICC remain controversial due to the paucity of functional in vivo studies. While the majority of ICC in vitro studies support a cancer-promoting role of CAF, recent studies in endogenously arising pancreatic cancer, a highly desmoplastic tumor with many similarities to ICC, have shown that CAF restrain cancer growth. Here, we seek to answer the question whether CAF promote or restrain ICC, using a number of novel and cell-specific tools to manipulate CAF and tumor cells and their crosstalk in endogenously arising ICC in vivo. We hypothesize that CAF and ECM provide a niche that promotes ICC growth and survival, and that detailed characterization of ICC-CAF crosstalk will identify novel therapeutic targets within the tumor microenvironment. In Aim 1, we will determine the role CAF using novel tools to determine how genetic CAF inhibition or early and late CAF ablation affect ICC growth, proliferative and anti-apoptotic signaling pathways, and mouse survival. In Aim 2, we will investigate pathways that mediate the recruitment, proliferation and activation of CAF in ICC focusing on the hypothesis that tumor cells hijack normal fibrogenic mechanism in the liver via tumor-derived TGFb and PDGF isoforms and TGFb- activating integrins, resulting in accumulation and activation of CAF. In addition to detailed mechanistic studies in knockout mice and in vitro co-cultures, we will determine whether pharmacologic inhibition of CAF activation by FDA-approved drug Nintedanib or integrin-blocking antibodies inhibit ICC growth and prolong mouse survival. In Aim 3, we will determine pathways through which CAF modulate ICC growth, focusing on the hypothesis that CAF-derived ECM activates tumor-promoting signals in the tumor cell compartment. To test this hypothesis, we will investigate ICC development in mice with CAF-specific knockout of Col1a1, or tumor-selective knockout of collagen-sensing receptor discoidin domain receptor 1 (DDR1). In addition, we will determine whether ECM- mediated stiffness and subsequent activation of mechanosensitive signaling in tumor cells promote ICC development. We will measure tumor stiffness, activation of mechanosensitive signaling pathways and DDR1 expression in human CCA samples and correlate these to clinical outcomes. The role of stiffness and DDR1 in ICC growth and CAF-ICC crosstalk will be investigated in more detail in vitro through modulation of stiffness and by co-culturing CAF and ICC cells that lack Col1a1 or DDR1, respectively. In summary, the proposed studies will employ novel tools to answer a long-standing question ...