Liver Cancer Modeling

Liver cancer can be classified into primary liver cancer and secondary liver cancer. Secondary liver cancer is the metastatic liver cancer. Primary liver cancer involves hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and fibrolamellar HCC. Noticeably, HCC is the most common form, accounting for approximately 90% of primary liver cancers^[1].

There are many risk factors that induce HCC, such as alcohol, hepatitis B virus (HBV) or hepatitis C virus (HCV), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH)^[1]. HCC usually evolves from chronic liver disease, following the pathological process of "normal liver-liver injury-chronic hepatitis-liver fibrosis-liver cirrhosis-liver cancer"^[2].

Mouse models of HCC involve chemotoxic agents-induced models, implantation models, and engineered models.

• 1. Chemotoxic agents-induced models

The chemotoxic agents induce HCC tumorigenesis generally by two mechanisms: through direct DNA damage or the promotion of preneoplastic cell growth. Chemotoxic agents are administered by intraperitoneal injection (i.p.) or oral gavage. Diethylnitrosamine (HY-N7434) (DEN), Aflatoxin B1 (HY-N6615), CCl₄ are common chemotoxic agents to induce HCC^[3]. Among these chemotoxic agents, DEN has been used to induce HCC in mice for several decades. The DEN-induced HCC model develops from basophilic foci, hyperplasic nodules, hepatocellular adenoma and finally HCC^[4]. DEN can be given alone or in combination with other chemotoxic agents.

DEN-induced HCC model can refer as below^[5]。

1. Swiss albino mice (3-week old) are provided feed and water ad libitum.

2. DEN at a dose of 15 mg/kg  (in normal saline, i.p., once) is injected to initiate hepatic carcinogenesis.

3. CCl₄ at a dose of 1.6 g/kg (in 1:1 dilution with corn oil, oral gavage, 3 times per week for the entire experimental period) is administered to stimulate liver cell proliferation and regeneration.

4. Control group is given a single i.p. injection of normal saline, followed by gavage of corn oil three times a week.

Advantages: Mimic cancer progress, tumors grow in a natural microenvironment.

Disadvantages: Difficult to monitor the tumor. Time for inducing the model after the injection varies on the dose, sex, age and strain of mice.

• 2. Implantation models

The implantation models are usually established through subcutaneous injection of human cancer cell lines such as HepG2, Hep3B and Huh7 cells (cell-line-derived xenograft, CDX), or through implantation of human tumor tissue (patient-derived xenograft, PDX) into the liver of immunodeficient mice. CDX and PDX models can both be established by xenograft and orthotopic transplantation. Xenograft transplantation is established by subcutaneously injecting human HCC cell lines or tumor tissue into mice. Orthotopic transplantation is established by injecting HCC tumor cells or tissue into the liver of mice by direct intrahepatic or intrasplenic injection, which usually requires surgical expertise in performing the procedures^[4].

PDX model (xenograft transplantation) can refer as below^[6]。

1. Remove necrotic tissue: Place the fresh tumor tissues in ice-chilled high-glucose DMEM containing 10% FBS, 100 U/mL penicillin and 100 U/mL streptomycin rapidly.

2. After removing necrotic tissue, partition the tumor specimens into 2×1×1 mm³ sections with a No.10 scalpel blade under aseptic conditions.

3. Wash the sections three times in ice-cold PBS.

4. Incubate the tissue fragments in DMEM medium supplemented with 50% Matrigel, 10 ng/mL epidermal growth factor, 10 ng/mL basic fibroblast growth factor, 100 U/mL penicillin and 100 U/mL streptomycin for 30 min.

5. Transplant the cultured tumor tissues into the right flanks of male nonobese, diabetic, severe combined immunodeficiency (NOD/SCID) mice (4-5 weeks old) subcutaneously with a No. 20 trocar.

Record tumor growth three times per week by measuring the length (L) and width (W) with a caliper. Tumor volume (TV, mm³) is calculated as TV = 0.5×L×W².

CDX model (xenograft transplantation) can refer as below^[7]。

1. Prepare four-week-old female athymic BALB/c nude mice with an average weight of approximately 22 g. All mice have free access to a commercial diet and water in animal cages under a pathogen-free condition at 25 °C and a humidity of 55 % in a 12-h dark and light cycles.

2. Culture the human HCC HepG2 cell line in RPMI 1640 medium supplemented with 10% FCS, 100 U/mL penicillin and 100 µg/mL of streptomycin in a humidified 5 % CO₂ incubator at 37 °C.

3. Harvest the cultured HepG2 cells with cold PBS and suspend the cells into cold matrigel quickly.

4. Subcutaneously inject cells (1.0×10⁷ cells, 200 μL) into the left inguinal area of the mice to establish a solid tumor model.

5. Monitor the tumor volumes every 3 days by measuring the length (L) and width (W) with a caliper. Tumor volume (TV, mm³) is calculated as TV = 0.5×L×W².

Advantages:

Xenograft transplantation: Easily induce tumor, short time period, easily control tumor size and location.

Orthotopic transplantation: Tumors occurs in the natural liver microenvironment, able to mimic the metastatic of HCC.

Disadvantages:

Xenograft transplantation: Lack of tumor microenvironment.

Orthotopic transplantation: Complicated and expensive surgical procedures, requires surgical expertise in performing the procedures.

• 3. Engineered models

Engineered models can result in the activation of oncogenes or inactivation of tumor suppressor genes, and thereby inducing tumor formation. The most commonly used and well-characterized genetic models are listed as below^[8]。

c-Myc overexpression

The activation c-Myc is highly associated with carcinogenesis in HCC. Transgenic mice that are c-Myc-overexpressed specifically in the liver can develop hepatic tumors with a relatively long latency of 12-15 months. Tumor incidence is of about 40% at 45 weeks of age, 60% at 55 weeks, and 80% at 65 weeks. HCC phenotype can be showed by HE staining and MR imaging.

P53 knockout

p53 is a key role in the control of genomic integrity and homeostasis. Liver-specific P53 knockout increases proliferation rate of mice hepatocyte and liver progenitor cells, and shows LPC-like cells in periportal liver regions.

Pten knockout

The phosphatase activity of Pten inhibits PI3K and consequently suppresses downstream PKB/Akt and mTOR signaling. Pten knockout mice progresses from hepatomegaly and steatosis to tumorigenesis. The incidence of hepatic adenomas is 47% at 44 weeks, and by 74-78 weeks, 66% of animals displayed HCC.

Advantages: Suitable for studying biological development of HCC tumorigenesis.

Disadvantages: Procedure can be dramatically affected by experimental expertise, embryo development may be affected, long latency, and lower incidence for tumor formation.

1. In DEN-induced HCC model, usually younger mice have faster HCC development because of the higher hepatocyte proliferation rates. If DEN is injected into mice younger than 2 weeks old, DEN alone will result in HCC development. If given to older mice, an additional stimulus, such as phenobarbital, is probably required^[4].

2. In xenografts, immunocompromised mice is required to avoid rejection of the foreign tissue. Besides, mouse-derived cell lines or tumors might not fully represent human disease^[4].