Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by hepatic lipid accumulation, inflammation, and progressive fibrosis. Acetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step of de novo lipogenesis and regulates fatty-acid β-oxidation in hepatocytes. ACC inhibition reduces hepatic fat content and markers of liver injury in NASH patients; however, the effect of ACC inhibition on liver fibrosis has not been reported. A direct role for ACC in fibrosis was evaluated by measuring de novo lipogenesis, procollagen production, gene expression, glycolysis, and mitochondrial respiration in hepatic stellate cells (HSCs) in the absence or presence of small-molecule inhibitors of ACC. ACC inhibitors were evaluated in rodent models of liver fibrosis induced by diet or the hepatotoxin, DEN. Fibrosis and hepatic steatosis were evaluated by histological and biochemical assessments. In TGF-β-stimulated HSCs, ACC inhibition reduced activation as measured by both α-SMA expression and collagen production. ACC inhibition prevented a metabolic switch necessary for induction of glycolysis and oxidative phosphorylation during HSC activation. While the molecular mechanism by which DNL inhibition blocks glycolysis and oxidative phosphorylation is unknown, we definitively show that HSCs require DNL for activation. Consistent with this direct anti-fibrotic mechanism in HSCs, ACC inhibition reduced liver fibrosis in a rat CDHFD model and in response to chronic DEN-induced liver injury that lacked hepatic lipid accumulation. In addition to reducing lipid accumulation in hepatocytes, ACC inhibition also directly impairs the pro-fibrogenic activity of HSCs. Small molecule inhibitors of ACC may reduce liver fibrosis by both reducing lipotoxicity in hepatocytes and directly reducing HSC activation, providing a mechanistic rationale for the treatment of patients with advanced liver fibrosis due to NASH.

Acid ceramidase inhibition promotes YAP/TAZ degradation to inactivate fibroblasts, decrease matrix stiffness, and reduce fibrosis in mice.

Syngeneic, immunocompetent allograft tumor models recapitulate important aspects of the tumor microenvironment and have short tumor latency with predictable growth kinetics, making them useful for trialing novel therapeutics. Here, we describe surgical techniques for orthotopic and heterotopic pancreatic ductal adenocarcinoma (PDAC) tumor implantation and characterize phenotypes based on implantation site. Mice (n=8 per group) were implanted with 104 cells in the pancreas or flank. Hy15549 and Han4.13 cell lines were derived from primary murine PDAC (Ptf1-Cre; LSL-KRAS-G12D; Trp53 Lox/+) on C57BL/6 and FVB strains, respectively. Single-cell suspension and solid tumor implants were compared. Tumors were treated with two intravenous doses of FOLFIRINOX and responses evaluated. All mice developed pancreatic tumors within 7 days. Orthotopic tumors grew faster and larger than heterotopic tumors. By 3 weeks, orthotopic mice began losing weight, and showed declines in body condition requiring euthanasia starting at 4 weeks. Single-cell injection into the pancreas had near 100% engraftment, but solid tumor implant engraftment was ∼50% and was associated with growth restriction. Orthotopic tumors were significantly more responsive to intravenous FOLFIRINOX compared with heterotopic tumors, with greater reductions in size and increased apoptosis. Heterotopic tumors were more desmoplastic and hypovascular. However, drug uptake into tumor tissue was equivalent regardless of tumor location or degree of fibrosis, indicating that microenvironment differences between heterotopic and orthotopic tumors influenced response to therapy. Our results show that orthotopic and heterotopic allograft locations confer unique microenvironments that influence growth kinetics, desmoplastic response and angiogenesis. Tumor location influences chemosensitivity to FOLFIRINOX and should inform future preclinical trials. This article has an associated First Person interview with the first author of the paper.

Liver biopsy, the gold standard for assessing liver fibrosis, suffers from limitations due to sampling error and invasiveness. There is therefore a critical need for methods to non-invasively quantify fibrosis throughout the entire liver. The goal of this study was to use molecular Magnetic Resonance Imaging (MRI) of Type I collagen to non-invasively image liver fibrosis and assess response to rapamycin therapy. Liver fibrosis was induced in rats by bile duct ligation (BDL). MRI was performed 4, 10, or 18 days following BDL. Some BDL rats were treated daily with rapamycin starting on day 4 and imaged on day 18. A three-dimensional (3D) inversion recovery MRI sequence was used to quantify the change in liver longitudinal relaxation rate (ΔR1) induced by the collagen-targeted probe EP-3533. Liver tissue was subjected to pathologic scoring of fibrosis and analyzed for Sirius Red staining and hydroxyproline content. ΔR1 increased significantly with time following BDL compared to controls in agreement with ex vivo measures of increasing fibrosis. Receiver operating characteristic curve analysis demonstrated the ability of ΔR1 to detect liver fibrosis and distinguish intermediate and late stages of fibrosis. EP-3533 MRI correctly characterized the response to rapamycin in 11 out of 12 treated rats compared to the standard of collagen proportional area (CPA). 3D MRI enabled characterization of disease heterogeneity throughout the whole liver. EP-3533 allowed for staging of liver fibrosis, assessment of response to rapamycin therapy, and demonstrated the ability to detect heterogeneity in liver fibrosis.