Project Summary/Abstract Intrahepatic cholangiocarcinoma (ICC) is the second most common form of liver cancer and is often diagnosed at an advanced or metastatic stage. The only available treatment for unresectable, late-stage ICC is systemic chemotherapy which typically extends patient survival by less than two years. A hallmark of ICC is its dense tumor reactive stroma (TRS), consisting of recruited endothelial cells, macrophages, and myofibroblasts (MFs) from the surrounding liver. The TRS has been implicated in ICC tumorigenesis, and in particular a TRS with more activated MFs is associated with a worse prognosis. Another hallmark of ICC is its heterogeneity, with no oncogenic mutations common to the majority of patients. However, Yes Associated Protein (YAP), the major downstream effector of the Hippo signaling pathway, has been shown to be activated in the majority of human ICC tissue samples. Our lab has developed a novel syngenic mouse model of transplanted ICC, where mouse biliary cells were isolated, cultured as liver organoids, and mutations found in human ICC patients were introduced using CRISPR technology to generate ICC organoids. These ICC organoids can be transplanted subcutaneously or intrahepatically to give rise to ICC tumors which recapitulate key features of the human disease, including the recruitment of a dense TRS. Interestingly, we have observed the highest YAP activity in the MFs of the TRS as opposed to ICC tumor cells in our model. Thus, we hypothesize that YAP activation in the TRS is crucial for ICC tumorigenesis. We propose to test this hypothesis through deletion of YAP in the TRS, as well as deletion of YAP specifically in TRS MFs, which we hypothesize will reduce ICC tumor burden in our in vivo mouse model. To elucidate the mechanism of YAP-driven TRS MF and ICC cell crosstalk, we will develop a novel ICC organoid and TRS MF in vitro co-culture system, where we hypothesize that TRS MFs will promote ICC cell growth and invasion in a YAP-dependent manner. Finally, we will perform single-cell RNA sequencing on control and YAP-deleted TRS MFs to identify YAP-driven secreted ligands in these cells. We will then perform in our ICC organoids a CRISPR-Cas9 screen targeting the receptors corresponding to our top secreted ligand hits in the TRS MFs. This CRISPR-Cas9 loss-of-function screen will be performed in our in vitro co-culture system, and we will thus be able to identify the signaling pathways that mediate crosstalk between TRS MFs and ICC cells. The pathways identified in our screen can be validated in our in vivo ICC mouse model, increasing the validity of our findings. This work will not only identify molecular mechanisms of crosstalk between ICC tumor cells and its associated liver stromal cells, but will identify new therapeutic targets which could be investigated for novel ICC treatment strategies.