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Page 4 of 16 Liu et al. Hepatoma Res 2020;6:7 I http://dx.doi.org/10.20517/2394-5079.2019.39

mice. Another method is the use of stem cell transduction, which involves retroviral infection of hepatic
progenitor cells isolated from foetal livers of mice to introduce oncogenes or target tumour suppressors into
[21]
a healthy liver . Manipulation of key genetic pathways in combination with liver injury models described
[26]
below has now been advocated to achieve a more realistic representation of human HCC .
Chemically- or diet-induced models
Several chemotoxins can induce hepatocarcinogenesis by causing direct DNA damage (genotoxic) or
promoting clonal expansion of preneoplastic cells (non-genotoxic). Di-ethyl-nitrosamine (DEN) is the most
widely used genotoxic drug for chemically-induced HCC. Once bioactivated by cytochrome P450, DEN
becomes an alkylating agent leading to the formation of mutagenic DNA adducts while also generating
reactive oxygen species (ROS) which damage DNA, overall resulting in hepatocyte death. Similar to what
occurs in humans, subsequent cycles of necrosis and regeneration in the mouse liver promote mutations,
[33]
neoplastic transformation and eventual HCC development . Indeed, DEN tumours have consistently
[34]
exhibited high mutation rates . DEN is most effective at inducing HCC when injected intraperitoneally
into young male mice (less than two weeks old) when hepatocytes are still proliferating. Commonly used
non-genotoxic carcinogens include carbon tetrachloride (CCl ) and thioacetamide (TAA). These agents act
4
as tumour promoters by damaging cellular structures, increasing the risk of genetic error and stimulating
[24]
cell malignant transformation by affecting proliferation, differentiation and apoptosis processes . CCl is
4
a potent hepatotoxin which causes centrilobular liver damage by the production of ROS and peroxidative
degradation of phospholipids in plasma, lysosomal and mitochondrial membranes. Prolonged exposure
(via oral, intraperitoneal or inhaled routes) leads to liver inflammation, fibrosis, cirrhosis and HCC
development. TAA is another centrilobular hepatotoxin which can be administered via intraperitoneal
injections or adding it to drinking water. It is bioactivated by mixed-function monooxygenases leading to
its S-oxide and highly reactive S,S-dioxide, which modifies amine-lipids and proteins to initiate cellular
[24]
necrosis . The carcinogenic effects of all chemically-induced HCC models vary with age, mouse strain
and sex. Ethanol feeding models are discussed in the ALD-associated HCC models section.

Diet-based models are most commonly used to study fatty liver diseases, particularly NAFLD and less so
ALD. Mice are usually fed ad libitum with one of the following diets: high-fat diet (HFD), high-fat high-
cholesterol (HFHC), methionine and choline-deficient diet (MCD), choline-deficient high-fat diet (CD-
HFD), choline-deficient L-amino acid-defined (CDAA) diet or a Western diet (WD). Although these
models can reliably produce steatosis, inflammation and even fibrosis, not many of them will result in HCC
[22]
development after a prolonged period . Furthermore, not all models reliably reproduce the accompanying
metabolic features of the disease such as obesity and insulin resistance . For example, a major drawback
[21]
of the MCD model is that mice exhibit the opposite of the human metabolic syndrome with weight loss,
no insulin resistance and low serum glucose, triglyceride and cholesterol. The specifics of these diet-based
models and their combinations are further discussed in the NAFLD-associated HCC models section.

Implantation models
Human or murine HCC cell lines can be injected into recipient mice to form orthotopic tumours
(intrahepatic, intrasplenic or intraportal injection) in the liver or heterotopic tumours (subcutaneous
injection) typically in the flank. The main advantages of implantation models are their quick time to
develop visible tumours (weeks to months in spontaneous models) that are easy to measure (especially
subcutaneous heterotopic tumours) and reproducible - making them popular models for drug screening.
This is counterbalanced by disadvantages such as considerable differences between cell lines necessitating
multiple cell lines to be tested, the lack of tumour-liver microenvironment interactions in heterotopic
[21]
models and the need for surgical expertise for orthotopic models . Implantation of human cells (xenograft
models) requires immunocompromised mice to prevent rejection of these foreign cells while murine
cells can be implanted into immunocompetent mice (syngeneic/allograft models). Mouse tumour cell
lines harbour mutations that are neutral or not relevant in human cancer making xenograft models more