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. 2017 Aug;98(4):221-233.
doi: 10.1111/iep.12240. Epub 2017 Sep 12.

Hepatocellular carcinoma in a mouse model fed a choline-deficient, L-amino acid-defined, high-fat diet

Ayae Ikawa-Yoshida  1 , Saori Matsuo  2 , Atsuhiko Kato  2 , Yusuke Ohmori  2 , Atsuko Higashida  1 , Eiji Kaneko  1 , Masahiko Matsumoto  2
Affiliations

Hepatocellular carcinoma in a mouse model fed a choline-deficient, L-amino acid-defined, high-fat diet

Ayae Ikawa-Yoshida et al. Int J Exp Pathol. 2017 Aug.

Abstract

Hepatocellular carcinoma (HCC) is a common cancer worldwide and represents the outcome of the natural history of chronic liver disease. The growing rates of HCC may be partially attributable to increased numbers of people with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). However, details of the liver-specific molecular mechanisms responsible for the NAFLD-NASH-HCC progression remain unclear, and mouse models that can be used to explore the exact factors that influence the progression of NAFLD/NASH to the more chronic stages of liver disease and subsequent HCC are not yet fully established. We have previously reported a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) as a dietary NASH model with rapidly progressive liver fibrosis in mice. The current study in C57BL/6J mice fed CDAHFD provided evidence for the chronic persistence of advanced hepatic fibrosis in NASH and disease progression towards HCC in a period of 36 weeks. When mice fed CDAHFD were switched back to a standard diet, hepatic steatosis was normalized and NAFLD activity score improved, but HCC incidence increased and the phenotype of fibrosis-associated HCC development was observed. Moreover, when mice continued to be fed CDAHFD for 60 weeks, HCC further developed without severe body weight loss or carcinogenesis in other organs. The autochthonous tumours showed a variety of histological features and architectural patterns including trabecular, pseudoglandular and solid growth. The CDAHFD mouse model might be a useful tool for studying the development of HCC from NAFLD/NASH, and potentially useful for better understanding pathological changes during hepatocarcinogenesis.

Keywords: choline deficiency; fibrosis; hepatocarcinogenesis; non-alcoholic steatohepatitis; spontaneous liver tumour model.

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Figures

Figure 1
Figure 1
Nodule formation in C57BL/6J mice fed standard diet (SD) or choline‐deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) for 12, 24, 36, 48 or 60 weeks. (a) Representative macroscopic appearance of liver of each nodule stage. Stage 0, no nodules visible on the liver; stage 1, one to three nodules; stage 2, four to six nodules; and stage 3: seven or more nodules (yellow arrows indicate visible nodules). (b) Incidence of nodule stage by number of weeks on each diet. Numbers in parentheses indicate No. of tested mice. [Colour figure can be viewed at wileyonlinelibrary.com].
Figure 2
Figure 2
CDAHFD feeding for 24 weeks resulted in NASH histopathological changes and changes in gene expression. Representative liver histopathology in C57BL/6J mice fed standard diet (SD) or choline‐deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) for 24 weeks. (a‐1 and b‐1) Haematoxylin–eosin (HE) staining; (a‐2 and b‐2) Masson's trichrome (MT) staining. Mice fed CDAHFD showed steatosis, lobular inflammation, hepatocyte ballooning and fibrosis. Scale bars = 100 μm. (c) Histopathological assessment of non‐alcoholic fatty liver in C57BL/6J mice fed CDAHFD for 24 weeks. The morphological features of steatosis (stage 0–3), lobular inflammation (stage 0–3), ballooning (stage 0–2) and fibrosis (stage 0–3) were semiquantitatively evaluated. Subsequently, the NAFLD activity score was applied (<3, non‐NASH; 3–4, possible NASH; and >5, NASH). (d) Assessment of mRNA expression levels of the indicated genes in the nodule‐free areas of liver at 24 weeks by qRT‐PCR normalized to the expression level of Gapdh mRNA. Data are means ± SEM (n = 6). *P < 0.05, **P < 0.01 and ***P < 0.001 vs. SD, t‐test. [Colour figure can be viewed at wileyonlinelibrary.com].
Figure 3
Figure 3
Representative examples of changed liver histopathology (haematoxylin–eosin staining) in mice fed the choline‐deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) for 60 weeks. (a) Area of regenerative hyperplasia (RH). (b) Area of hepatocellular adenoma (HCA). (c, d) Representative liver that shows hepatocellular carcinoma (HCC) and micrograph of its area of HCC. Scale bars = 100 μm (scale bar in panel c‐2 and d‐2 = 1 mm). [Colour figure can be viewed at wileyonlinelibrary.com].
Figure 4
Figure 4
Switching back to standard diet at 37 weeks can improve NASH pathology but cannot prevent the occurrence of tumours. Representative liver histopathology in C57BL/6J mice fed a standard diet (SD) for 48 weeks; a choline‐deficient, L‐amino acid‐defined, high‐ fat diet (CDAHFD) for 48 weeks; or CDAHFD for 36 weeks and then SD until 48 weeks (CDAHFD/SD). (a‐1 and a‐2) Haematoxylin–eosin (HE) staining and Masson's trichrome (MT) staining of SD fed mice. (b‐1 and b‐2) Mice fed CDAHFD for 48 weeks showed steatosis, lobular inflammation, hepatocyte ballooning and bridging fibrosis. (c‐1 and c‐2) Mice switched back to SD showed that steatosis, lobular inflammation and fibrosis were reduced when compared with mice fed CDAHFD for 48 weeks. (d) Micrograph of a nodule in a mouse in the CDAHFD/SD group in which HCA and HCC existed next to each other. (e‐1 and e‐2) HCA region boxed in (d). An irregular hepatocyte growth pattern was observed, but fibrosis was not. (f‐1 and f‐2) HCC region boxed in (d). A pseudoglandular structure was observed and fibrosis penetrates into the gap that is causing structural atypia. Scale bars = 100 μm (scale bar in panel d = 1 mm). [Colour figure can be viewed at wileyonlinelibrary.com].
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