Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5+ stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.

We introduce a redox-active iron complex, Fe-PyC3A, as a biochemically responsive MRI contrast agent. Switching between Fe 3+-PyC3A and Fe 2+-PyC3A yields a full order of magnitude relaxivity change that is field-independent between 1.4 and 11.7 T. The oxidation of Fe 2+-PyC3A to Fe 3+-PyC3A by hydrogen peroxide is very rapid, and we capitalized on this behavior for the molecular imaging of acute inflammation, which is characterized by elevated levels of reactive oxygen species. Injection of Fe 2+-PyC3A generates strong, selective contrast enhancement of inflamed pancreatic tissue in a mouse model (caerulein/LPS model). No significant signal enhancement is observed in normal pancreatic tissue (saline-treated mice). Importantly, signal enhancement of the inflamed pancreas correlates strongly and significantly with ex vivo quantitation of the pro-inflammatory biomarker myeloperoxidase. This is the first example of using metal ion redox for the MR imaging of pathologic change in vivo. Redox-active Fe 3+/2+ complexes represent a new design paradigm for biochemically responsive MRI contrast agents.

ObjectiveHepatocellular carcinoma (HCC) is the fastest-growing cause of cancer-related mortality with chronic viral hepatitis and non-alcoholic steatohepatitis (NASH) as major aetiologies. Treatment options for HCC are unsatisfactory and chemopreventive approaches are absent. Chronic hepatitis C (CHC) results in epigenetic alterations driving HCC risk and persisting following cure. Here, we aimed to investigate epigenetic modifications as targets for liver cancer chemoprevention.DesignLiver tissues from patients with NASH and CHC were analysed by ChIP-Seq (H3K27ac) and RNA-Seq. The liver disease-specific epigenetic and transcriptional reprogramming in patients was modelled in a liver cell culture system. Perturbation studies combined with a targeted small molecule screen followed by in vivo and ex vivo validation were used to identify chromatin modifiers and readers for HCC chemoprevention.ResultsIn patients, CHC and NASH share similar epigenetic and transcriptomic modifications driving cancer risk. Using a cell-based system modelling epigenetic modifications in patients, we identified chromatin readers as targets to revert liver gene transcription driving clinical HCC risk. Proof-of-concept studies in a NASH-HCC mouse model showed that the pharmacological inhibition of chromatin reader bromodomain 4 inhibited liver disease progression and hepatocarcinogenesis by restoring transcriptional reprogramming of the genes that were epigenetically altered in patients.ConclusionOur results unravel the functional relevance of metabolic and virus-induced epigenetic alterations for pathogenesis of HCC development and identify chromatin readers as targets for chemoprevention in patients with chronic liver diseases.

Objectives: The goals of this study were to compare the efficacy of the new manganese-based magnetic resonance imaging (MRI) contrast agent Mn-PyC3A to the commercial gadolinium-based agents Gd-DOTA and to Gd-EOB-DTPA to detect tumors in murine models of breast cancer and metastatic liver disease, respectively, and to quantify the fractional excretion and elimination of Mn-PyC3A in rats. Methods: T1-weighted contrast-enhanced MRI with 0.1 mmol/kg Mn-PyC3A was compared with 0.1 mmol/kg Gd-DOTA in a breast cancer mouse model (n = 8) and to 0.025 mmol/kg Gd-EOB-DTPA in a liver metastasis mouse model (n = 6). The fractional excretion, 1-day biodistribution, and 7-day biodistribution in rats after injection of 2.0 mmol/kg [ 52 Mn]Mn-PyC3A or Gd-DOTA were quantified by 52 Mn gamma counting or Gd elemental analysis. Imaging data were compared with a paired t test; biodistribution data were compared with an unpaired t test. Results: The postinjection-preinjection increases in tumor-to-muscle contrastto-noise ratio (ΔCNR) 3 minutes after injection of Mn-PyC3A and Gd-DOTA (mean ± standard deviation) were 17 ± 3.8 and 20 ± 4.4, respectively (P = 0.34). Liver-to-tumor ΔCNR values at 8 minutes postinjection of Mn-PyC3A and Gd-EOB-DTPAwere 28 ± 9.0 and 48 ± 23, respectively (P = 0.11). Mn-PyC3A is eliminated with 85% into the urine and 15% into the feces after administration to rats. The percentage of the injected doses (%ID) of Mn and Gd recovered in tissues after 1 day were 0.32 ± 0.12 and 0.57 ± 0.12, respectively (P = 0.0030), and after 7 days were 0.058 ± 0.051 and 0.19 ± 0.052, respectively (P < 0.0001). Conclusions: Mn-PyC3A provides comparable tumor contrast enhancement to Gd-DOTA in a mouse breast cancer model and is more completely eliminated than Gd-DOTA; partial hepatobiliary elimination of Mn-PyC3A enables conspicuous delayed phase visualization of liver metastases.

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.

Chronic liver disease and hepatocellular carcinoma (HCC) are life-threatening diseases with limited treatment options. The lack of clinically relevant/tractable experimental models hampers therapeutic discovery. Here, we develop a simple and robust human liver cell-based system modeling a clinical prognostic liver signature (PLS) predicting long-term liver disease progression toward HCC. Using the PLS as a readout, followed by validation in nonalcoholic steatohepatitis/fibrosis/HCC animal models and patient-derived liver spheroids, we identify nizatidine, a histamine receptor H2 (HRH2) blocker, for treatment of advanced liver disease and HCC chemoprevention. Moreover, perturbation studies combined with single cell RNA-Seq analyses of patient liver tissues uncover hepatocytes and HRH2+, CLEC5Ahigh, MARCOlow liver macrophages as potential nizatidine targets. The PLS model combined with single cell RNA-Seq of patient tissues enables discovery of urgently needed targets and therapeutics for treatment of advanced liver disease and cancer prevention.

Angiopoietin-2 was associated with mortality and other clinically relevant outcomes in a cohort of decompensated cirrhotic patients with acute kidney injury. Further experimental study of Angiopoietin/Tie2 signaling is warranted to explore its potential mechanistic and therapeutic role in this population. This article is protected by copyright. All rights reserved.

A comparative analysis of mouse and human pancreatic development may reveal common mechanisms that control key steps as organ morphogenesis and cell proliferation and differentiation. More specifically, understanding beta cell development remains an issue, despite recent progress related to their generation from human embryonic and induced pluripotent stem cells. In this study, we use an integrated approach, including prospective isolation, organ culture, and characterization of intermediate stages, and report that cells from human and mouse fetal pancreas can be expanded in the long term and give rise to hollow duct-like structures in 3D cultures. The expanded cells express a combination of markers (E-cadherin, PDX1, NKX6-1, SOX9, and HNF1β) that reveals pancreatic progenitor identity. Proliferation of embryonic progenitors was stimulated by the Wnt agonist R-spondin1 (RSPO1), FGF10, and EGF. This combination of growth factors allowed maintaining human fetal pancreatic progenitors in culture for many passages, a finding not reported previously. Importantly, in the absence of EGF, proliferation was reduced, while endocrine differentiation was significantly enhanced. We conclude that modulation of EGF signaling affects in vitro expansion and differentiation of progenitors from embryonic pancreas of both mice and man.