Oncogene-Driven Induction of Orthotopic Cholangiocarcinoma in Mice.

Cholangiocarcinoma (CCA) is a malignancy affecting the epithelial cells that line the bile ducts. This cancer shows a poor prognosis and current treatments remain inefficient. Orthotopic CCA mouse models are useful for the development of innovative therapeutic strategies. Here, we describe an orthotopic model of intrahepatic CCA that can be easily induced in mice within 5 weeks at a high incidence. It is achieved by expressing two oncogenes, namely, (i) the intracellular domain of the Notch1 receptor (NICD) and (ii) AKT, in hepatocytes by means of the sleeping beauty transposon system. These plasmid vectors are delivered by hydrodynamic injection into the tail vein. The present chapter also describes how to perform magnetic resonance imaging (MRI) of the livers to visualize intrahepatic CCA nodules.

Orthotopic Model of Hepatocellular Carcinoma in Mice.

Orthotopic models of hepatocellular carcinoma (HCC) consist in the implantation of tumor cells into the liver by direct intrahepatic injection. In this model, tumorigenesis is triggered within the hepatic microenvironment, thus mimicking the metastatic behavior of HCC. Herein, we detail a surgically mediated methodology that allows the reproducible and effective induction of liver-sessile tumors in mice. We enumerate the steps to be followed before and after the surgical procedure, including HCC cell preparation, the quantity of cancer cells to be injected, presurgical preparation of the mice, and finally, postoperative care. The surgical procedure involves laparotomy to expose the liver, injection of cells into the left-lateral hepatic lobe, and closure of the incision with sutures followed by wound clips. We also provide information concerning the subsequent tumor growth follow-up, as well as the application of bioluminescence imaging to monitor tumor development.

A Mouse Model of Non-Alcoholic Steatohepatitis and Hepatocellular Carcinoma Induced by Western Diet and Carbon Tetrachloride.

Non-alcoholic steatohepatitis (NASH) is a severe form of non-alcoholic fatty liver disease (NAFLD). Obesity is a known risk factor of NASH, which, in turn, increases the risk of developing cirrhosis (liver scarring) and hepatocellular carcinoma (HCC). In addition to being a potentially life-threatening condition, public health concerns surrounding NASH are amplified by the lack of FDA-approved treatments. Although various preclinical models reflecting both the histopathology and the pathophysiological progression of human NASH exist, most of these models are diet-based and require 6-13 months for NASH symptom manifestation. Here, we describe a simple and rapid-progression model of NASH and NASH-driven HCC in mice. Mice received a western diet equivalent (WD; i.e., a high-fat, high-fructose, and high-cholesterol diet), high-sugar water (23.1 g/L fructose and 18.9 g/L glucose), and weekly intraperitoneal injections of carbon tetrachloride (CCl4) at a dose of 0.2 µL/g of body weight. The resulting phenotype, consisting in liver fibrosis and HCC, appeared within 24 weeks of diet/treatment initiation and presented similar histological and transcriptomic features as human NASH and NASH-driven HCC, thereby supporting the adequacy of this preclinical model for the development and evaluation of drugs that can prevent or reverse these diseases.

Flow Cytometry Assessment of Lymphocyte Populations Infiltrating Liver Tumors.

Tissue-resident and recruited immune cells are essential mediators of natural and therapy-induced immunosurveillance of liver neoplasia. This idea has been recently reinforced by the clinical approval of immune checkpoint inhibitors for the immunotherapy of hepatocellular carcinoma and cholangiocarcinoma. Such research progress relies on the in-depth characterization of the immune populations that are present in pre-neoplastic and neoplastic hepatic lesions. A convenient technology for advancing along this path is high-dimensional cytometry.In this chapter, we present a protocol to assess the subtype and differentiation state of hepatic lymphocyte populations by multicolor immunofluorescence staining and flow cytometry. We detail the steps required for viability assessment and immune cell phenotyping of single-cell suspensions of liver cells by means of surface and intracellular staining of more than a dozen markers of interest. This protocol does not require prior removal of debris and dead cells and allows to process multiple samples in parallel. The procedure includes the use of a fixative-resistant viability dye that allows cell fixation and permeabilization after cell surface staining and before intracellular staining and data acquisition on a flow cytometer. Moreover, we provide a panel of fluorochrome-labeled antibodies designed for the characterization of lymphocytic subsets that can be adapted to distinct experimental settings. Finally, we present an overview of the post-staining pipeline, including data acquisition on a flow cytometer and tools for post-acquisition analyses.

A Mouse Model of Hepatocellular Carcinoma Induced by Streptozotocin and High-Fat Diet.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the second most common cause of cancer-related death. HCC is associated to chronic diseases such as viral hepatitis, alcoholic, and non-alcoholic fatty liver disease (NAFLD), diabetes mellitus, and obesity, among others. Although pre-clinical models have been investigated to mimic the transition from NAFLD to HCC, they do not accurately reproduce the phenotypic evolution from simple steatosis to steatohepatitis, fibrosis/cirrhosis, and HCC. Hence, these models have failed to demonstrate the influence of diabetes on hepatic carcinogenesis. Here, we report a novel mouse model of HCC triggered by fast-developing diabetes and NAFLD. The first step consists in a single intraperitoneal injection of a low dose of streptozotocin into neonatal C57BL/6J mice to induce type 2 diabetes. In a second step, mice are fed with high-fat diet to accelerate the development of simple steatosis. Continuous high-fat diet exacerbates hepatic fat deposition with increased lobular inflammation (by activation of foam cell-like macrophages) and fibrosis (by activating hepatic stellate cells), two representative pathological traits of steatohepatitis/fibrosis. After 20 weeks, all mice developed multiple HCCs. This model of hepatic carcinogenesis triggered by diabetes mellitus and NAFLD offers the advantage of being rapid and accurately recapitulates the pathogenesis of human HCC without the need of administering hepatic carcinogens.

Isolation of Primary Mouse Hepatocytes and Non-Parenchymal Cells from a Liver with Precancerous Lesions.

In the early stages of liver carcinogenesis, rare hepatocytes and cholangiocytes are transformed into preneoplastic cells, which can progressively acquire a neoplastic phenotype, favored by the failure of natural antitumor immunosurveillance. The detailed study of both hepatic parenchymal (e.g., hepatocytes) and non-parenchymal cells (NPCs), such as immune cells, could help understand the cellular microenvironment surrounding these pre-cancerous and neoplastic lesions.Cultures of primary hepatocytes are of interest in various biomedical research disciplines, serving as an ex vivo model for liver physiology. Obtaining high viability and yield of primary mouse hepatocytes and other liver cell populations is technically challenging, thus limiting their use. In the first section of the current chapter, we introduce a protocol based on the two-step collagenase perfusion technique (by inferior vena cava) to isolate hepatocytes and, to a lower extent, NPCs and detailed the different considerations to take into account for a successful perfusion. The liver is washed by perfusion, hepatocytes are dissociated with collagenase, and different cell populations are separated by centrifugation. Various techniques have been described for the isolation of healthy and malignant hepatocytes; however, the viability and purity of the isolated cells is frequently not satisfactory. Here, we significantly optimized this protocol to reach improved yield and viability of the hepatocytes and concomitantly obtain preserved NPC populations of the liver.Within NPCs, tissue-resident or recruited immune cells are essential actors regulating hepatocarcinogenesis. However, simultaneous isolation of hepatic leukocytes together with other cell types generally yields low immune cell numbers hindering downstream application with these cells. In the second section of this chapter, as opposed to the first section primarily aiming to isolate hepatocytes, we present a tissue dissociation protocol adapted to efficiently recover leukocytes from non-perfused bulk (pre-)cancerous livers. This protocol has been optimized to be operator-friendly and fast compared to other liver processing methods, allowing easy simultaneous sample processing to retrieve hepatic (tumor-infiltrating) immune cells.

Targeted Analysis of Glycerophospholipids and Mono-, Di-, or Tri-Acylglycerides in Liver Cancer.

The metabolic rearrangements of hepatic metabolism associated with liver cancer are still incompletely understood. There is an ongoing need to identify novel and more efficient diagnostic biomarkers and therapeutic targets based on the metabolic mechanisms of these diseases. In comparison to traditional diagnostic biomarkers, metabolomics is a comprehensive technique for discovering chemical signatures for liver cancer screening, prediction, and earlier diagnosis. Lipids are a large and diverse group of complex biomolecules that are at the heart of liver physiology and play an important role in the development and progression of cancer. In this chapter, we described two detailed protocols for targeted lipids analysis: glycerophospholipids and mono, di, tri-acylglycerides, both by Flow Injection Analysis (FIA) HPLC coupled to a SelexIon/QTRAP 6500+ system. These approaches provide a targeted lipidomic metabolomic signature of dissimilar metabolic disorders affecting liver cancers.

Biomarker Identification in Liver Cancers Using Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) Imaging: An Approach for Spatially Resolved Metabolomics.

Liver cancers are characterized by interindividual and intratumoral heterogeneity, which makes early diagnosis and the development of therapies challenging. Desorption electrospray ionization mass spectrometry (DESI-MS) imaging is a potent and sensitive MS ionization technique for direct, unaltered 2D and 3D imaging of metabolites in complex biological samples. Indeed, DESI gently desorbs and ionizes analyte molecules from the sample surface using an electrospray source of highly charged aqueous spray droplets in ambient conditions. DESI-MS imaging of biological samples allows untargeted analysis and characterization of metabolites in liver cancers to identify new biomarkers of malignancy. In this chapter, we described a detailed protocol using liver cancer samples collected and stored for histopathology examination, either as frozen or as formalin-fixed, paraffin-embedded specimens. Such hepatocellular carcinoma samples can be subjected to DESI-MS analyses, illustrating the capacity of spatially resolved metabolomics to distinguish malignant lesions from adjacent normal liver tissue.

Inhibition of acyl-CoA binding protein (ACBP) by means of a GABAARγ2-derived peptide.

Acyl-CoA binding protein (ACBP) encoded by diazepam binding inhibitor (DBI) is an extracellular inhibitor of autophagy acting on the gamma-aminobutyric acid A receptor (GABAAR) γ2 subunit (GABAARγ2). Here, we show that lipoanabolic diets cause an upregulation of GABAARγ2 protein in liver hepatocytes but not in other major organs. ACBP/DBI inhibition by systemically injected antibodies has been demonstrated to mediate anorexigenic and organ-protective, autophagy-dependent effects. Here, we set out to develop a new strategy for developing ACBP/DBI antagonists. For this, we built a molecular model of the interaction of ACBP/DBI with peptides derived from GABAARγ2. We then validated the interaction between recombinant and native ACBP/DBI protein and a GABAARγ2-derived eicosapeptide (but not its F77I mutant) by pull down experiments or surface plasmon resonance. The GABAARγ2-derived eicosapeptide inhibited the metabolic activation of hepatocytes by recombinant ACBP/DBI protein in vitro. Moreover, the GABAARγ2-derived eicosapeptide (but not its F77I-mutated control) blocked appetite stimulation by recombinant ACBP/DBI in vivo, induced autophagy in the liver, and protected mice against the hepatotoxin concanavalin A. We conclude that peptidomimetics disrupting the interaction between ACBP/DBI and GABAARγ2 might be used as ACBP/DBI antagonists. This strategy might lead to the future development of clinically relevant small molecules of the ACBP/DBI system.

Acyl-CoA binding protein for the experimental treatment of anorexia.

Extracellular acyl-coenzyme A binding protein [ACBP encoded by diazepam binding inhibitor (DBI)] is a phylogenetically ancient appetite stimulator that is secreted in a nonconventional, autophagy-dependent fashion. Here, we show that low ACBP/DBI plasma concentrations are associated with poor prognosis in patients with anorexia nervosa, a frequent and often intractable eating disorder. In mice, anorexia induced by chronic restraint stress (CRS) is accompanied by a reduction in circulating ACBP/DBI concentrations. We engineered a chemical-genetic system for the secretion of ACBP/DBI through a biotin-activatable, autophagy-independent pathway. In transgenic mice expressing this system in hepatocytes, biotin-induced elevations in plasma ACBP/DBI concentrations prevented anorexia induced by CRS or chemotherapeutic agents including cisplatin, doxorubicin, and paclitaxel. ACBP/DBI reversed the CRS or cisplatin-induced increase in plasma lipocalin-2 concentrations and the hypothalamic activation of anorexigenic melanocortin 4 receptors, for which lipocalin-2 is an agonist. Daily intravenous injections of recombinant ACBP/DBI protein or subcutaneous implantation of osmotic pumps releasing recombinant ACBP/DBI mimicked the orexigenic effects of the chemical-genetic system. In conclusion, the supplementation of extracellular and peripheral ACBP/DBI might constitute a viable strategy for treating anorexia.