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Liu et al. Hepatoma Res 2020;6:7 I http://dx.doi.org/10.20517/2394-5079.2019.39 Page 9 of 16

NAFLD-associated HCC models
Many models have been developed to represent NAFLD and non-alcoholic steatohepatitis (NASH),
although, as aforementioned, not all of them exhibit features of metabolic syndrome. This is particularly
important in NAFLD-related HCC since the presence of obesity and/or diabetes are themselves
[54]
independent risk factors for the development of cancer .

Most dietary models of NAFLD (HFD, HFHC, MCD, WD and CD diet) rarely induce HCC development
[22]
alone . If spontaneous HCC does occur, it is time-consuming (e.g., 2.5% for C57BL/6 mice fed HFD for
[55]
12 months) . Combination diets such as CD-HFD and CDAA have been shown to significantly increase
rates of tumour formation, although overall rates are still low: 25% after 12 months and 35% after 9 months,
respectively [55,56] . Indeed, these diets can recapitulate the key features of human NASH (including fibrosis)
and metabolic syndrome more so than single diets. Susceptibility to tumour formation in dietary models
[59]
also appears to be strain-dependent with DBA/2J > C57LBL/6 > A/J [57,58] . Asgharpour et al. generated an
isogenic strain (B6/129) derived from a cross of two common mouse strains, C57BL/6J and 129S1/SvImJ,
and fed them a high-fat-high-carbohydrate diet with high-fructose-glucose water - so-called DIAMOND
mice. This promising model mimicked all the physiological, metabolic, histological and transcriptomic gene
signature and clinical endpoints of human NASH including HCC in 89% at 32-52 weeks. These tumours
had gene signatures which strongly resembled the S1 and S2 human subclasses of HCC. Interestingly,
neither C57BL/6J nor 129S1/SvImJ parent strain mice fed with the same diet developed HCC.

Combining dietary models with a hepatotoxin substantially hastens and increases HCC formation (i.e.,
up to 100% of male C57BL/6 mice fed CDAA, HFD, CD-HFD or WD + intraperitoneal injections of DEN
or CCl at 6-9 months) as well as tumour size [56,60-62] . The addition of cholesterol to a HFD (HFHC) in a
4
[64]
DEN-induced model appears to further increase tumour burden . In another model, Henderson et al. ,
[63]
treated male C57BL/6 mice with DEN (25 mg/kg once at 14 days old), TAA (300 mg/L in drinking water
ad libitum from four weeks old) and HFD. These agents acted synergistically to develop HCCs in 83% of
mice as early as 24 weeks of age, which was significantly more than control mice or those treated with
DEN and TAA only. However, combining with hepatotoxins needs to be tempered by some limitations.
For example, use of CCl can induce liver metabolism enzymes (which may impact the use of this model
4
for drug discovery) and also mitigate metabolic processes involved in NASH, particularly susceptibility to
[65]
diet-induced obesity and insulin resistance . As mentioned above, the STAM mouse was recently shown
to be the mouse model (out of four studied) that most closely resembles human HCC at a molecular level.
Specifically, STAM tumours carried mutations of CTNNB1 at a rate comparable to human tumours, and
(less frequently) mutations of TP53 - the most frequently altered genes in human HCC . In contrast,
[34]
CTNNB1 and TP53 were rarely mutated in DEN-induced tumours, which instead carried Hras, Braf
and APC mutations rarely seen in human HCC. The STAM combination involves first treating neonatal
C57BL/6 male mice with low-dose streptozotocin (STZ) at Day 2, which induces diabetes by causing death
of pancreatic β cells, resulting in lean mice with hypoinsulinaemia and hyperglycaemia, but no insulin
[66]
resistance (the phenotype of type 1 diabetes) . STZ is also a DNA alkylating agent (similar to DEN) with
[67]
potential carcinogenic effects . When these mice are then fed with HFD, they develop weight gain, NASH
[66]
[68]
by eight weeks, cirrhosis and HCC relatively quickly by 16-20 weeks . Takakura et al. characterised
STAM tumours at 20 weeks by clinical parameters used in human liver disease [i.e., Child-Turcotte-Pugh
score and dynamic contrast-enhanced computed tomography (CT) measurements of HCCs]. Interestingly,
the authors deduced that STAM mice had cirrhosis corresponding to Child-Turcotte-Pugh Class B
(significant coagulopathy, occasional ascites, no encephalopathy and normal albumin and bilirubin) and
tumours equivalent to Barcelona Clinic Liver Cancer Stage B (intermediate) or C (advanced) disease in
humans. No HCCs develop when STZ is given alone, again pointing to the need for an additional stimulus.
Female mice treated with the STAM regimen also fail to develop tumours, akin to the gender disparity seen
in other models.