ABSTRACT: Purpose: The aim of this study is to investigate mechanisms driving tumor-promoting mechanisms in cholangiocarcinoma while focusing on transitions from normal cholangiocytes to precancer lesions and from precancer lesion to invasive carcinoma. An original mouse model of intrahepatic cholangiocarcinoma was developed, based on induction of a KrasG12D mutation in cholangiocytes combined with chronic inflammation. RNA-Seq analyses compare the transcriptome of ductular proliferations, intraductal papillary neoplasm of the bile duct and intrahepatic cholangiocarcinoma. A gene cascade involving EGF, KrasG12D, Sox17 and Tns4 was identified to promote tumor progression.
Project description:Here we analyzed mouse and human samples to characterize origin, subtypes, functions and cell-cell interactions of cancer-associated fibroblasts in cholangiocarcinoma, a highly desmoplastic tumor of the liver. Hepatic stellate cell-derived cancer-associated fibroblasts were isolated from two different models of murine intrahepatic cholangiocarcinoma, induced by overexpression of YAP+AKT or KRASG12D in combination with sg-p19, and compared by bulk RNA-sequencing to hepatic stellate cells from two models of liver fibrosis, induced by bile duct ligation or DDC diet. CAF-enriched fractions of from YAP+AKT or KRAS/sg-p19-induced intrahepatic cholangiocarcinoma, were analyzed by single-cell RNA sequencing. A cell suspension from human cholangiocarcinoma, containing all cell populations, was analyzed by single cell RNA-sequencing.
Project description:We report the genertion of a gRNA library that induces mutations often found in intrahepatic cholangiocarcinoma. We use this to screen in vivo mutations that when lost act synergistically with mutant Ras. Specifically KrasG12D and NrasG12V. We have included exome sequencing of these tumours in this data set.
Project description:We report the genertion of a gRNA library that induces mutations often found in intrahepatic cholangiocarcinoma. We use this to screen in vivo mutations that when lost act synergistically with mutant Ras. Specifically KrasG12D and NrasG12V. We have included RNA sequencing of these tumours in this data set.
Project description:Validation of preclinical models of intrahepatic cholangiocarcinoma progression that reliably recapitulate altered molecular features of the human disease would provide an important resource for suggesting and testing of novel target-based therapies against this devastating cancer. In this study, comprehensive gene expression profiling in a novel orthotopic rat model of intrahepatic cholangiocarcinoma progression was carried out in an effort to identify potential therapeutic targets relevant to the progressive human cancer. Microarray analysis was performed on intrahepatic cholangiocarcinomas formed at 10, 15, and 25 days after bile duct inoculation of neu-transformed rat cholangiocytes (BDEneu cells) into rat liver and on peritoneal metastases at the 25 day time period, compared with non-cancerous right liver lobe from the same animals.
Project description:Validation of preclinical models of intrahepatic cholangiocarcinoma progression that reliably recapitulate altered molecular features of the human disease would provide an important resource for suggesting and testing of novel target-based therapies against this devastating cancer. In this study, comprehensive gene expression profiling in a novel orthotopic rat model of intrahepatic cholangiocarcinoma progression was carried out in an effort to identify potential therapeutic targets relevant to the progressive human cancer. Microarray analysis was performed on intrahepatic cholangiocarcinomas formed at 10, 15, and 25 days after bile duct inoculation of neu-transformed rat cholangiocytes (BDEneu cells) into rat liver and on peritoneal metastases at the 25 day time period, compared with non-cancerous right liver lobe from the same animals. Experiment Overall Design: Tumors were collected at 10, 15, 25 days post inoculation from biological triplicates (different rats innoculated simultaneously). Also, biological triplicates of metastatic tumors (Mets) at day 25 were analyzed. Biological triplicates of paired normal right liver lobe (RLL) from the same animals from which tumors/mets were obtained were also analyzed in this study. Thus, a total of 9 rats were used for this study.
Project description:The increased M-NM-1-smooth muscle-actin positive cancer-associated fibroblastic cells (CAF) in the desmoplastic stroma may relate to a more aggressive cancer and worse survival outcomes for intrahepatic cholangiocarcinoma (ICC) patients We developed a novel 3-D organotypic culture model by co-culturing M-NM-1-SMA positive CAF and cholangiocarcinoma cells in a collagen matrix. Cholangiocarcinoma cell lines were established by isolating M-NM-1-SMA positive cancer-associated fibroblastic cells (CAF) (BDEsp-TDFE4) and cholangiocarcinoma cells (BDEsp-TDEH10) from tumors arising from bile duct inoculation of spontaneously-transformed low grade malignant rat BDE1 cholangiocytes (BDEsp cells). These tumor-derived cells lines were then grown in a rat tail type I collagen gel matrix, alone or in co-culture, and their gene expression profile were compared.
Project description:Intrahepatic cholangiocarcinoma (ICC) is a lethal cancer arising from intrahepatic bile ducts in the liver. In the intraductal growth (IDG) type, also referred to as intraductal papillary neoplasm of the bile duct (IPNB), a rare subtype of ICC, cancer cells originating within the bile duct usually invade and infiltrate the nearby liver tissues, resulting in local invasion. IPNB can serve as a good disease model to study the mechanism of local invasion, which is the earliest step of metastasis. Here, we aimed to identify the genes associated with local invasion by comparing the somatic mutations of the matched IPNB tissues with and without local invasion from a set of 11 patients using the Ion AmpliSeq Comprehensive Cancer Panel. Somatic mutation profiling analysis identified RNF213 as a potential candidate gene associated with local invasion in ICC patients. We established stable RNF213-knockdown and shControl cholangiocarcinoma (CCA) cell lines (HuCCT1, YSCCC, and KKU-100). Next, the expression profiling of the RNF213-knockdown and shContol CCA cell lines was analyzed by RNA sequencing.
Project description:Characterization of preclinical models of intrahepatic cholangiocarcinoma progression that reliably recapitulate altered molecular features of the human disease. Here, we performed comprehensive gene expression profiling of cholangiocarcinoma tumors arising from bile duct inoculation of different grade malignant rat cholangiocytes. Tumors arising from bile duct inoculation of spontaneously-transformed low grade malignant rat BDE1 cholangiocytes (BDEsp cells) compared to tumors arising from the inoculation of high grade malignant erbB-2/neu- transformed BDE1 cholangiocytes (BDEneu cells) into the livers of syngeneic rats.
Project description:Characterization of preclinical models of intrahepatic cholangiocarcinoma progression that reliably recapitulate altered molecular features of the human disease. Here, we performed comprehensive gene expression profiling of cholangiocarcinoma tumors arising from bile duct inoculation of different grade malignant rat cholangiocytes.
Project description:Background and Aims: The SWI/SNF ATP-dependent chromatin remodeling complex regulates transcrip-tional machinery access and is critical in normal physiology and cancer development. Pbrm1, a key subunit of this complex, is frequently mutated in intrahepatic cholan-giocarcinoma (ICC), though how its loss contributes to tumorigenesis remains poor-ly understood. This study aims to explore the role of Pbrm1 in liver physiology and its involvement in ICC development. Methods: Liver-specific Pbrm1 knockout (Pbrm1 KO) mice were generated to assess the ef-fects of Pbrm1 loss under various conditions. These mice were exposed to a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet to induce cholestatic injury and were also subjected to a high-fat diet to evaluate susceptibility to liver steatosis. Chromatin accessibility and gene expression under both normal and injury condi-tions were examined. Additionally, the impact of Pbrm1 loss was analyzed in com-bination with an activating KrasG12D mutation to study cancer development. Results: Pbrm1 KO mice exhibited increased susceptibility to cholestatic injury, with an en-hanced ductular reaction. Loss of Pbrm1 reduced chromatin accessibility at hepato-cyte-specific and metabolically important genes, though RNA expression remained unaffected during homeostasis. Following cholestatic injury, hepatocyte-specific gene expression was significantly reduced compared to wild-type controls. Pbrm1 KO mice also showed heightened vulnerability to high-fat diet-induced liver steato-sis. When combined with KrasG12D mutation, Pbrm1 KO/KrasG12D mice had shorter survival and were more likely to develop cholangiocarcinomas, whereas Pbrm1 wild type/KrasG12D mice predominantly developed hepatocellular neoplasms. Conclusion: Pbrm1 maintains chromatin accessibility for hepatocyte differentiation-related genes. Its loss promotes differentiation toward cholangiocytes during injury or tu-morigenesis, driving ICC development.