Project Summary/Abstract Colorectal cancer (CRC) is the third most frequent cancer worldwide with increasing incidences every year. It is the leading cause of cancer related mortality, predominantly due to metastatic disease. CRC majorly metastasizes to the liver, and almost 30% of CRC patients develop hepatic metastasis resulting in severely poor outcomes and limited treatment options outside of surgery. Our overarching objective is to examine how CRC cells metastasize and adapt to the liver microenvironment in physiologically relevant, organoid transplantation based CRC models that harbor genetic abnormalities commonly found in CRC patients. Our understanding of how the liver microenvironment shapes the responses of the tumor cells is significantly impeded by a lack of in-vivo CRC models that recapitulate metastatic disease. To overcome the limitations of the current CRC models, we propose the use of a murine model utilizing colonoscopy guided orthotopic transplantation of genetically engineered organoids. We have identified that these models faithfully reproduce the characteristics of human CRC, including liver metastasis commonly observed in patients. Using CRISPER/Cas9 based editing, we will genetically engineer human and murine CRC organoids bearing mutations associated with poor prognosis in patients. We will use our in-vivo model to generate pro and non-metastatic lines from these organoids, followed by characterization of the differential molecular changes in the tumor cells and microenvironment that influence disease progression. Our preliminary data shows the differential regulation of epithelial to mesenchymal (EMT) transcription factors such as Twist1 in pro-metastatic organoids as compared to non-metastatic organoids. We will characterize the role of these candidate EMT transcription factors in CRC liver metastasis (Aim 1), and identify molecular mechanisms that differentiate metastatic from non-metastatic tumor cells. Aim 2 of our proposal will study the effect of high fat diet (HFD)-mediated obesity on liver metastasis in CRC. HFD and obesity have been increasingly shown to influence intestinal stem cell behavior and tumorigenesis, however, its influence on disease progression and metastasis remains unknown. Using our CRC in-vivo models, transcriptomic sequencing and functional validation studies, we will dissect the molecular effects of a high fat diet on tumor and liver microenvironment, liver metastasis and overall outcome. Public/Health/Relevance: Successful completion of this study will identify mechanisms integral to initiate and maintain metastasis as well as the role of HFD-induced obesity in this process, revealing therapeutically targetable vulnerabilities in physiologically relevant models of CRC.