The DEN and CCl₄-Induced Mouse Model of Fibrosis and Inflammation-Associated Hepatocellular Carcinoma

Abstract

Human hepatocellular carcinoma (HCC) develops most often as a complication of fibrosis or cirrhosis. While most human studies of HCC provide crucial insights into the molecular signatures of HCC, seldom do they address the etiology of HCC. Mouse models are essential tools for investigating the pathogenesis of HCC; however, the majority of cancer models in rodents do not feature liver fibrosis. Detailed here is a protocol for an experimental mouse model of HCC that arises in association with advanced liver fibrosis. The disease model is induced by a single injection of N-nitrosodiethylamine (DEN) followed by repeated administration of carbon tetrachloride (CCl₄). A dramatic potentiation of liver tumor incidence is observed following administration of DEN and CCl₄, with100% of mice developing liver tumors at 5 months of age. This model has been employed for studying the molecular mechanisms of fibrogenesis and HCC development, and in cancer hazard/chemotherapy testing of drug candidates.

BASIC PROTOCOL

Hepatocellular carcinoma (HCC) is a common human cancer with a high mortality rate (Center and Jemal, 2011). Although overall cancer incidence and death rates have steadily declined over the past decade (AACR Cancer Progress Report Writing Committee et al., 2013), the incidence of HCC continues to increase. It is known that HCC is a multistep pathological process characterized by the progressive, sequential evolution of liver disease stages from chronic liver injury, to inflammation, hepatocellular degeneration, and necrosis, to hepatocellular regeneration and small cell dysplasia, followed by the appearance of low- and high-grade dysplastic nodules, which eventually manifest as HCC (Farazi and DePinho, 2006; Aravalli et al., 2013).

The etiology of HCC in humans is complex, with many causative factors identified. These include viral infections, consumption of alcoholic beverages, environmental chemicals, hemochromatosis, and metabolic diseases related to insulin resistance collectively identified as non-alcoholic steatohepatitis (El Serag and Rudolph, 2007; Della Corte and Colombo, 2012). Because the diagnosis of HCC most often occurs at the late stages of the disease, rodent models are invaluable for the understanding of both the causality and pathogenesis of this condition (Fausto and Campbell, 2010; Vucur et al., 2010). In this regard, an excellent summary of chemical-induced, xenograft, and genetically induced experimental mouse liver tumor models, and their relevance to human HCC, has been published (Heindryckx et al., 2009). Importantly, while in humans 70% to 90% of HCC cases are associated with advanced liver fibrosis or cirrhosis (Alkofer et al., 2011), the vast majority of chemical-induced rodent hepatic adenomas and carcinomas arise in fibrosis-free liver. Thus, among many limitations, chronic rodent cancer bioassays do not involve the key features of human HCC, namely chronic liver inflammation and fibrosis/cirrhosis.

The protocol described in this unit addresses this major limitation of rodent HCC models by involving a two-stage application of chemicals to the mouse liver for the initiation and promotion of hepatocellular tumors. The initiator in this protocol is a single injection of a low dose of DEN (1 mg/kg, i.p.) into 14-day-old male mice, a well-known model of genotoxic liver carcinogenesis (Druckrey et al., 1964; Vesselinovitch and Mihailovich, 1983). Repeated administration of a low dose of CCl₄ (0.2 ml/kg, i.p.), a pro-fibrogenic agent, beginning at 8 weeks of age for up to 14 consecutive weeks, is the promoter. It is well established that continuous exposure to CCl₄ leads to the development of hepatic fibrosis and compensatory cell proliferation (Stowell et al., 1951). In addition to advanced liver fibrosis, repeated administration of CCl₄ causes progressively worsening anisonucleosis, a morphological manifestation of nuclear injury characterized by variation in the size of the hepatocyte nuclei, a condition associated with hepatic oxidative stress (Guzman et al., 2011). This DEN/CCl₄ mouse model differs from animal models utilizing either agent alone in that the resultant chronic liver fibrosis is accompanied by a dramatic increase in the liver tumor incidence, with 100% of the mice in the co-treatment group developing liver tumors by 5 months of age (Uehara et al., 2013).

The molecular pathways (Uehara et al., 2013) and epigenetic alterations (Chappell et al., 2014) observed in fibrosis-associated mouse liver tumors indicate important features involved in the development of human liver tumors that arise from fibrosis and cirrhosis, a common progression according to human clinico-pathological evidence. This two-stage model of chemical liver carcinogenesis yields animals useful for studying the molecular events associated with early and late events in HCC development and progression, with a particular focus on a combination of etiological factors, such as genotoxicity and advanced liver fibrosis. In addition, because human HCC requires liver cirrhosis, the model described in this protocol is more likely, as compared with traditional 2-year cancer bioassays or studies in mouse transgenic models (Bucher, 1998), to recapitulate the fibrotic phenotype seen in humans and therefore to be more useful as a test system for probing potential cancer hazards associated with xenobiotics.

Synopsis:

Male B6C3F1 mice are administered a single i.p. injection of 1 mg/kg DEN at 14 days of age. Beginning at 8 weeks of age the animals are administered 0.2 ml/kg CCl₄ i.p. two times per week for up to 14 weeks, at which time a 100% incidence of liver adenomas is expected (Uehara et al., 2013). Time-course evaluation of histopathological features of underlying liver disease, including single cell necrosis, ballooning degeneration and hypertrophy of hepatocytes, and fibrosis with inflammatory cell infiltration, as well as the incidence of precancerous lesions (foci) and tumors (adenomas and carcinomas) are described in detail by Uehara et al. (2013). The tumors are determined at sacrifice by examining the liver macroscopically and microscopically. The degree of liver fibrosis is evaluated using the Masson’s trichrome staining technique (Masson, 1929).

NOTE:

As this protocol involves the use of live animals, all experiments must first be reviewed and approved by an Institutional Animal Care and Use Committee and must conform to all government regulations regarding the care and use of laboratory animals. Because the protocol includes the use of chemicals (N-nitrosodiethylamine and carbon tetrachloride) that are regarded as reasonably anticipated to be human carcinogens (National Toxicology Program, 2011), these experiments must be approved by the local environmental health and safety authority and adhere to appropriate best laboratory practices for handling and disposal of all contaminated materials (National Research Council, 2005).

Materials

Female pregnant B6C3F1/J mice (Jackson Laboratory) ~1 week before delivery (the number of animals will depend on the proposed study size; 5–10 pups are expected from each pregnant mouse)

Diethylnitrosamine (see recipe)

Sterile phosphate-buffered saline (PBS; vehicle)

Carbon tetrachloride (CCl₄; see recipe)

Olive oil (vehicle; Sigma-Aldrich or any other supplier)

5-Bromo-2′-deoxyuridine (BrDU; see recipe)

Nembutal (Oak Pharmaceutical) for anesthesia or other approved anesthetic

Neutral buffered formalin (10%)

Paraffin

Hematoxylin and eosin

Animal scale

Disposable plastic syringes (1.0 ml) with needles for i.p. injection

Sharp dissecting scissors

Heparin-containing serum gel Z/1.1-ml centrifuge tubes (Sardstedt)

0.9% NaCl-moistened filter paper

Razor blades

Tissue processing/embedding cassettes (any commercial supplier)

Screw-lid containers

Light microscope

Additional reagents and equipment for the Masson’s trichrome procedure (Masson, 1929) – description of the reagents and the procedure are available from Sigma-Aldrich (Sigma-Aldrich, 2021).

Prepare the animals

• 1
  Acclimate timed pregnant B6C3F1/J mice (Jackson) to housing conditions for ~1 week before delivery of offspring.

  Animals are acclimated under standard lighting and temperature conditions to eliminate the effect of stress. Food and water are available ad libitum. While other strains may be used, it should be noted that both spontaneous and chemical-induced liver cancer incidence vary greatly among mouse strains (Bannasch, 1983).

• 2
  Immediately after birth, randomly assign litters of male pups to treatment groups.

  Male mice are selected because male gender is a risk factor for human HCC (Jepsen et al., 2007). However, females may be used should the research question require it. Approximately three to eight male pups can be expected from one pregnant B6C3F1/J mouse, although the number and gender of pups in a litter may vary greatly among animals.

• 3
  Weigh each pup at 14 days of age to calculate doses to be administered. Inject into the selected subjects either DEN (1 mg/kg i.p. in PBS) or PBS (vehicle) alone in a volume of 15 ml/kg body weight.

  To minimize contamination of each treatment during the lactation period, pups in each litter should be assigned to the same treatment group and treated in an identical manner. Because of its carcinogenicity, DEN should be handled using the “basic prudent practices” and precautions for work with compounds of high chronic toxicity (National Research Council, 2005).

• 4Wean the animals from their mothers at 3 to 4 weeks of age.
• 5
  At 8 weeks of age (6 weeks after the single injection of DEN or vehicle), commence administration of CCl₄ (0.2 ml/kg i.p. in olive oil) or of olive oil alone (vehicle) twice a week for up to 14 additional weeks at a dose volume of 15 ml/kg body weight.

  Treatment with CCl₄ for 9 weeks should yield 100% incidence of preneoplastic liver foci and up to 40% incidence of liver adenomas and 20% for carcinomas, while treatment for 14 weeks will yield 100% incidence of liver adenomas and 50% for carcinomas (Uehara et al., 2013). Tumors observed in this model have not been found to metastasize. No tumors or preneoplastic lesions are anticipated in vehicle-treated mice. Tumors and preneoplastic lesions are not detectable other than by visual and light microscopic examination of the liver after sacrifice.

  Because of its carcinogenicity, CCl₄ should be handled using the “basic prudent practices” and precautions for work with compounds of high chronic toxicity (National Research Council, 2005). Application of a different amount (up to 0.5 ml/kg is used by some) may affect tumor numbers and size but will cause more pronounced liver inflammation and thereby affect the molecular pathogenesis of disease (Dapito et al., 2012).

  Necropsy should be performed to establish the cause of death or illness if animals die or appear sick and need to be euthanized during the experiment. A common cause of premature death is intestinal perforation due to injection error (e.g., peritonitis resulting from an accidental puncture of the intestine).

• 6
  On the day of the final CCl₄ injection, change the water made available to the animals from the regular animal facility-approved source to drinking water containing bromodeoxyuridine (5-bromo-2′-deoxyuridine, BrDU, 0.2 g/liter). Allow free access to this solution for 3 days.

  The BrDU treatment may be omitted if cellular proliferation labeling index is not needed. Regular histopathological evaluation of the tissues will not be affected by this step.

Perform necropsy

• 7At the end of BrDU treatment, record animal body weights.
• 8Inject the animals with Nembutal (120 mg/kg, i.p.) or other anesthetic approved for humane euthanasia by the Institutional Animal Care and Use Committee.
• 9
  After verifying death, place the animal on its back and open the abdomen through vertical incision with a pair of sharp dissecting scissors. Collect blood from the inferior vena cava or a common iliac vein into anticoagulant (e.g., heparin)-containing syringes.

  Avoid injuring major vessels or internal organs to prevent premature bleeding by lifting the abdominal skin with forceps while performing the incision.

• 10Remove the liver using a pair of sharp dissecting scissors.
• 11Record the wet liver weight.
• 12Place the liver on 0.9% NaCl-moistened filter paper.
• 13Reveal macroscopically visible tumors by serial step-sectioning through the entire liver at 5 to 7 mm with a sharp razor blade.
• 14Record the number and size of macroscopically visible tumors.
• 15Excise liver sections (including grossly visible tumors; see Fig. 14.30.1) of 3- to 5-mm thickness, place them into a tissue embedding cassette (one per animal), and immediately place into a screw-lid container with 10% neutral buffered formalin in an amount sufficient to submerge all tissue cassettes.
• 16
  Embed the tissue in paraffin, and cut it into 3- to 5-μm sections, as needed for histopathological evaluation and other special stains.

  Grossly visible large tumors can be separated from noncancerous liver tissue by careful excision. If needed, frozen tissue samples can also be obtained. See step 5 for expected tumor burden.

Perform histopathological examination

• 17Stain the sections with hematoxylin and eosin (Titford, 2005), and examine the tumors and liver tissues histopathologically under the light microscope (see Tables 1 and 2).
• 18Stain the sections using the Masson’s trichrome procedure (Masson, 1929) and examine liver fibrosis histopathologically under the light microscope (see Tables 1 and 2).
• 19Data analysis and visualizations will depend on study design and research questions. Examples of the expected effects in this model were published elsewhere (Uehara et al., 2013).

Table 1.

Major Diagnostic Criteria for (Pre)neoplastic Lesions in the Mouse Liver

Types of (pre)neoplastic lesions	Characteristics of histopathology
Preneoplastic foci of altered hepatocytes	Normal or minimal compression of the surrounding parenchyma Foci of hepatocytes with increased basophilic staining (basophilic type) Glycogen and/or some clear cells may be present
Hepatocellular adenoma	Relatively uniform hepatocytes accompanied by loss of normal lobular architecture Compression of the surrounding parenchyma
HCC	Broad trabecular growth pattern of atypical hepatocytes with hemorrhage and ischemic necrosis in the central region of tumors Vascular and stromal invasions occasionally

Table 2.

Scoring System for Liver Fibrosis in Mice

Score	Characteristics of histopathology
0	No fibrotic changes
1	Slight fibrotic changes around the central vein and occasionally with thin bridging fibrosis
2	Thick bridging fibrosis and pseudo-lobule formation with dissecting nodules

REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see appendix 2a; for suppliers, see suppliers appendix.

BrDU

Prepare a stock solution by mixing 1 g 5-Bromo-2′-deoxyuridine (BrDU; Sigma, CAS 59-14-3) with 100 ml distilled water. Place the solution in a brown glass water bottle (or wrap in foil) and store up to 1 week at 4°C. Prepare dilutions from the stock solution for administration every 24 to 48 hr by mixing 20 ml of stock solution with 980 ml of tap water. Place the injection solution into brown water bottles that are set into the cages to replace the regular animal facility water. Notify the animal facility staff not to change water bottles during this phase of the study.

CCl₄

Dissolve 1 ml carbon tetrachloride (CCl₄; Sigma, CAS 56-23-5) in olive oil to a final volume of 10 ml (0.01%, v/v). Protect the injection solution from light. Prepare fresh at least once a week and store at 4°C.

DEN

Dissolve 1 mg N-nitrosodiethylamine (DEN; Sigma, CAS 55-18-5) in 15 ml phosphate-buffered saline (PBS; 0.67%, w/v final) on the day of administration.

COMMENTARY

Background Information

While the incidence and lethality of all types of cancer have declined over the past decade, the incidence of liver cancer, including HCC, continues to increase worldwide, but has largely plateaued in the Asian countries (Dasgupta et al., 2020) and the United States (Shiels and O’Brien, 2020). Among the many factors contributing to the global increase are metabolic disorders related to insulin resistance, which are classified as non-alcoholic steatohepatitis (Della Corte and Colombo, 2012). Notably, most HCC cases are diagnosed in subjects with fibrotic or cirrhotic livers (Alkofer et al., 2011), pathological states that are a consequence of chronic liver injury (Fattovich et al., 2004). Advanced liver fibrosis and cirrhosis are known risk factors for HCC; specifically, hepatic stellate cells play a pivotal role in the pathogenesis of liver fibrosis (Dhar et al., 2020). However, the underlying molecular mechanisms that are responsible for the high rate of HCC in the chronically injured and fibrotic liver are subject to active research (Luedde and Schwabe, 2011).

There is growing evidence that chronic inflammatory processes are involved in triggering the molecular and cellular events leading from chronic liver injury to liver fibrosis and ultimately to HCC. Activation of resident and systemic circulation-derived macrophages and neutrophils, as well as the resultant release of pro-inflammatory cytokines and chemokines and other damage-associated molecular patterns, is thought to play an important role in the pathogenesis of many chronic liver diseases, including HCC (Grivennikov et al., 2010). Some of these pathological processes have now been recapitulated in different mouse models (Fausto and Campbell, 2010; Vucur et al., 2010). However, the extent to which these mouse models reflect the clinical realities of human HCC is unclear (Heindryckx et al., 2009). Animal models, most often rat or mouse, are commonly used to test the carcinogenic potential of drugs and other xenobiotics (Wells and Williams, 2009), with the liver being a common target for tumor development in rodent studies involving chronic exposure to such agents (Hoenerhoff et al., 2009). Nonetheless, it is notable that the overwhelming majority of the positive (i.e., significant increases in the incidence of liver adenomas and carcinomas) chronic rodent cancer bioassays fail to produce liver fibrosis or cirrhosis (Huff et al., 1991).

Lack of fibrosis and cirrhosis in most positive rodent 2-year cancer bioassays in rats and mice contrasts dramatically with human HCC, wherein liver cirrhosis is both the most common histopathological feature associated with this cancer, and an important mechanism of hepatocarcinogenesis (Farazi and DePinho, 2006). In those few cases where cirrhotic changes are observed in rodent liver in association with chemical-induced hepatocellular neoplasms (e.g., thioacetamide and N-nitrosomorpholine), high necrogenic doses are required (Becker, 1983; Oh et al., 2002). Thus, among their many limitations, chronic rodent cancer bioassays fail to address this key feature of human HCC. Given the importance of liver cirrhosis in the development of human HCC, integrative studies designed to evaluate the mechanisms of fibrogenesis, and how they relate to hepatocyte transformation and modulation of oncogenic signaling, are most relevant for defining the pathogenesis of the human disease.

The protocol detailed herein provides an animal model designed to mimic the pathophysiological features of liver disease leading to HCC in humans, where a combination of etiological factors including genotoxic injury and advanced fibrosis are likely to contribute (Fausto and Campbell, 2010). It was for this reason that a well-known model of liver carcinogenesis induced by a single injection of DEN, a genotoxic agent, into 14-day-old male mice was adapted by adding repeated injections of CCl₄, a pro-fibrogenic agent (Druckrey et al., 1964; Vesselinovitch and Mihailovich, 1983).

Hepatocellular adenomas that develop in this model display proliferation of relatively uniform hepatocytes accompanied with a loss of normal lobular architecture and compression of the surrounding parenchyma. The histopathological features of hepatocellular carcinomas consist of a broad trabecular growth pattern of atypical hepatocytes with hemorrhaging and ischemic necrosis in the center of the tumors. Avascular and stromal invasions are also occasionally present in hepatocellular carcinomas. While the histopathological features of the liver tumors generated in this model are similar to those found with human HCC, the human tumors are particularly heterogeneous, with a wide variety of morphologic features and complex histogenesis (Mitchell, 2013). Thus, the classifications of HCC mainly rely on the clinical and molecular features rather than the histopathology findings (Rebouissou and Nault, 2020). Additional research enabled by the model detailed in this protocol, as well as other mouse models of HCC (Brown et al., 2018), facilitates integration of data obtained from studies in preclinical species and in humans, and helps accelerate the identification of robust predictive biomarkers of response to targeted therapy and immunotherapy of HCC.

This experimental design incorporates chronic exposure to a relatively low dose of CCl₄, which is some 2.5 to 5× lower than that commonly used to induce fibrosis or hepatocarcinogenesis (Dragani et al., 1986; Fujii et al., 2010; Dapito et al., 2012), with a low dose of DEN, which is some 25× lower than that routinely for studies of co-morbidity (Dapito et al., 2012). This model provides a useful experimental pathway for investigating the contributions of inflammation and fibrosis to liver cancer. There is much debate about the relative role of inflammation, growth signals, and cytokines in the progression of HCC, with studies yielding conflicting evidence with respect to the contribution of these factors in murine models of HCC (Maeda et al., 2005; Luedde and Schwabe, 2011; Dapito et al., 2012). One reason for the conflicting results may be the dose of the genotoxic agent and the severity of inflammation caused by a pro-fibrogenic compound. The present model is associated with minimal necrogenic changes and reveals that activation of cancer stem cells is the primary mechanism for a dramatic elevation in the incidence of HCC when both genotoxic and fibrogenic factors are present (Uehara et al., 2013). This model reveals that epigenetic events, rather than mutations in known cancer-related genes, play a prominent role in increased incidence of liver tumors in fibrosis-associated liver cancer (Chappell et al., 2014). In addition, a number of studies used this experimental design to uncover novel molecular signals involved in cell cycle regulation (Preziosi et al., 2018) and cancer cell stemness (Chan et al., 2018), or to test the effectiveness of novel therapeutics (Taha et al., 2021).

Such “fibrotic” liver murine models may be not only more human-relevant, but also less time-consuming, with the hepatocellular carcinomas and adenomas developing in as few as 17 weeks, as opposed to the more traditional 2-year rodent cancer bioassay for examining carcinogenic potential of new chemical entities. Given the acknowledged need for short-term transgenic mouse assays to replace the 2-year bioassays (Jacobson-Kram, 2010), the model described in this protocol could fulfill this requirement, as well as provide a more clinically relevant laboratory pathology for screening carcinogens or potentially new chemotherapeutic agents. In addition, as it is well recognized that most human HCC result from multiple etiologies and require liver cirrhosis, the model described in this protocol is more likely, as compared to other transgenic models being evaluated (Bucher, 1998), to recapitulate the fibrotic phenotype of HCC that is typically observed in humans.

Critical Parameters and Troubleshooting

Because pregnant mice are more sensitive to noise and smells, it is important to use rubber gloves when moving the cage and pups as a unit. In addition, avoid taking the cage off the shelf repeatedly to view the animals. These procedures will help reduce the incidence of cannibalism that is associated with maternal anxiety.

It is important to keep in mind that pre-weaned pups are very fragile and sensitive. The pups should be weaned at 3 to 4 weeks of age. It is critical that pups be weaned at the appropriate time, as those weaned too early will not survive.

Anticipated Results

In animals that receive only DEN (1 mg/kg), a single nonnecrogenic dose, to study the initiation phase of carcinogenesis, no evidence of injury or gross liver pathology will be observed, although there will be a marked increase in the incidence of liver foci with or without the occurrence of hepatocellular adenomas at 22 weeks of age (Goldsworthy and Fransson-Steen, 2002).

In animals treated with CCl₄ (0.2 ml/kg) alone, there will be a significant increase in liver-body weight ratio and progressive worsening (with time) in liver histopathology. Specifically, single-cell necrosis, ballooning degeneration of hepatocytes, and steatosis will be observed in central and midlobular regions. These are often associated with fibrosis and inflammatory cell infiltration. Nonnecrotic hepatocytes will exhibit hypertrophy with anisonucleosis. Liver foci and adenomas will occur in 12.5% of B6C3F1/J mice at 22 weeks of age, while the incidence of carcinomas will be approximately 25%. Few, if any, foci, adenomas, or carcinomas will be observed at earlier time points.

In contrast, in DEN and CCl₄-treated B6C3F1/J mice, all animals exhibit increases in relative liver weight and a marked elevation of liver injury at 22 weeks of age. Moreover, all animals develop liver adenomas and ~50% will exhibit HCC. It is of note that in CCl₄-treated animals the severity of liver injury in noncancerous tissue (single-cell necrosis, ballooning degeneration and hypertrophy of hepatocytes, and fibrosis with inflammatory cell infiltration) resulting from CCl₄ administration will be of a similar grade regardless of whether the animals were previously injected with DEN.

There should be no liver injury or pre- or neoplastic lesions in the vehicle group at 22 weeks of age.

Time Considerations

Each mouse must be weighed at least once a week before DEN dosing and twice weekly during the CCl₄ promotion period. Depending on the number of animals studied, injections can take up to 2 hr. On the day of necropsy, a significant amount of time is needed for preparation, sampling, and gross examination of liver tumors in the individual animals. The precise amount of time needed depends on the investigator’s skills with these procedures and the number of animals being examined.

Figure 1.

Experimental design, gross pathology, and histopathology of the liver tumors. (A) A single injection of a mutagenic carcinogenic agent N-nitrosodiethylamine (DEN) is followed by repeat dosing with carbon tetrachloride (CCl₄) for up to 14 consecutive weeks. For more detail, see the Basic Protocol. (B) Representative photographs of the livers from the animals in each dosing group. (C) Representative photographs of the histopathology of the livers including fibrosis, hepatocellular adenoma, and carcinoma.