Abstract 87: Development of a novel six-month experimental mouse model of hepatocellular carcinoma with nonalcoholic steatohepatitis
Authors: Kishida Norihiro , Matsuda Sachiko , Aiura Koichi , Itano Osamu , Kitagawa Yuko 
Published: May 2015

Background: Hepatocellular carcinoma (HCC) with nonalcoholic steatohepatitis (NASH) is increasing in incidence, but its clinicopathological features are still obscure. Several animal models of HCC with NASH have been developed to facilitate the study; however, these models require relatively long periods of time (usually &gt;1 year) to reliably produce HCC, and none possess all the clinical features, such as insulin resistance, inflammation, fibrosis, and carcinogenesis. In this study, we developed a novel 6-month experimental mouse model of HCC with NASH, which shows clinical features in 6 months, through feeding of a high-fat, choline-deficient diet to C57BL/6J mice combined with diethylnitrosamine (DEN) exposure.

Methods: Three-week-old C57BL/6J mice were randomly divided into four groups: the control diet group (MF), the high-fat, choline-deficient diet group (HF), the control diet with DEN exposure group (DEN), and the high-fat, choline-deficient diet with DEN exposure group (HFDEN). The mice in the DEN and HFDEN groups received a one-time intraperitoneal injection of DEN at the start of the respective feeding protocols. Each group of mice (N = 4 or 5) were sacrificed every 4 weeks, and body weight and liver weight were measured. Plasma alanine aminotransferase (ALT), triglyceride (TG), fasting glucose (FBS), and insulin were measured by ELISA-based methods. The liver tissues were fixed with 10% formalin and embedded in paraffin. The specimens were stained with hematoxylin and eosin to assess the NAFLD activity score (NAS), with Sirius Red to evaluate fibrosis, and with Sudan III to evaluate steatosis. Contrast computed tomography (CT) was performed to evaluate tumor development before sacrifice.

Results: At 6 months of feeding, the MF, HF, DEN, and HFDEN mice had a mean body weight of 31.7 g, 54.5 g, 32.6 g, and 49.5 g, respectively, and a mean liver weight of 1.2 g, 4.0 g, 1.3 g, and 2.9 g, respectively. The TG level peaked at 2 months for all four groups and decreased thereafter. The ALT level increased monthly, with the HF and HFDEN groups showing remarkably high levels. All HF and HFDEN mice showed insulin resistance since month 3. In contrast, the MF and DEN groups had insulin resistance from month 6. The NAS of the HFDEN group was the highest among the four groups. Fibrosis and steatosis were varied, however, with those of the HF and HFDEN groups tending to increase monthly. CT scans showed that all HFDEN mice developed hepatic tumors from month 5. Microscopically, these tumors were precancerous lesions or HCCs.

Conclusion: Our HFDEN model showed insulin resistance at 3 months and NASH at 4 months. All HFDEN mice developed precancerous lesions or HCCs in 5 months and HCCs in 6 months, whereas the HF group showed simple steatosis features even at 6 months and developed insulin resistance at 6 months. We have thus established a novel 6-month mouse model of HCC with NASH.

Citation Format: Norihiro Kishida, Sachiko Matsuda, Koichi Aiura, Osamu Itano, Yuko Kitagawa. Development of a novel six-month experimental mouse model of hepatocellular carcinoma with nonalcoholic steatohepatitis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 87. doi:10.1158/1538-7445.AM2014-87