Basic Study
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Apr 28, 2016; 22(16): 4091-4108
Published online Apr 28, 2016. doi: 10.3748/wjg.v22.i16.4091
Adult mouse model of early hepatocellular carcinoma promoted by alcoholic liver disease
Aditya Ambade, Abhishek Satishchandran, Benedek Gyongyosi, Patrick Lowe, Gyongyi Szabo
Aditya Ambade, Abhishek Satishchandran, Benedek Gyongyosi, Patrick Lowe, Gyongyi Szabo, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, United States
Author contributions: Ambade A and Szabo G designed research and wrote the manuscript; Ambade A did the DEN injections and the Lieber DeCarli alcohol diet feeding, analyzed and interpreted all the data; Ambade A, Satishchandran A, Gyongyosi B and Lowe P performed the animal sacrifice; Szabo G obtained the funding and provided overall study supervision.
Supported by NIH/ NIAAA, No. AA011576 to Szabo G.
Institutional review board statement: The study protocol was approved by the Institutional Animal Use and Care Committee of the University of Massachusetts Medical School.
Institutional animal care and use committee statement: All procedures were performed in accordance with the policies of Institutional Animal Use and Care Committee of the University of Massachusetts Medical School. The study protocol was reviewed and approved by the Institutional Animal Use and Care Committee of the University of Massachusetts Medical School.
Conflict-of-interest statement: The authors do not have any conflict-of-interest to declare.
Data sharing statement: No additional data available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Gyongyi Szabo, MD, PhD, Department of Medicine, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, United States. gyongyi.szabo@umassmed.edu
Telephone: +1-508-8565276 Fax: +1-528-8565033
Received: December 16, 2015
Peer-review started: December 17, 2015
First decision: January 13, 2016
Revised: March 9, 2016
Accepted: March 18, 2016
Article in press: March 18, 2016
Published online: April 28, 2016
Processing time: 125 Days and 5.5 Hours
Abstract

AIM: To establish a mouse model of alcohol-driven hepatocellular carcinoma (HCC) that develops in livers with alcoholic liver disease (ALD).

METHODS: Adult C57BL/6 male mice received multiple doses of chemical carcinogen diethyl nitrosamine (DEN) followed by 7 wk of 4% Lieber-DeCarli diet. Serum alanine aminotransferase (ALT), alpha fetoprotein (AFP) and liver Cyp2e1 were assessed. Expression of F4/80, CD68 for macrophages and Ly6G, MPO, E-selectin for neutrophils was measured. Macrophage polarization was determined by IL-1β/iNOS (M1) and Arg-1/IL-10/CD163/CD206 (M2) expression. Liver steatosis and fibrosis were measured by oil-red-O and Sirius red staining respectively. HCC development was monitored by magnetic resonance imaging, confirmed by histology. Cellular proliferation was assessed by proliferating cell nuclear antigen (PCNA).

RESULTS: Alcohol-DEN mice showed higher ALTs than pair fed-DEN mice throughout the alcohol feeding without weight gain. Alcohol feeding resulted in increased ALT, liver steatosis and inflammation compared to pair-fed controls. Alcohol-DEN mice had reduced steatosis and increased fibrosis indicating advanced liver disease. Molecular characterization showed highest levels of both neutrophil and macrophage markers in alcohol-DEN livers. Importantly, M2 macrophages were predominantly higher in alcohol-DEN livers. Magnetic resonance imaging revealed increased numbers of intrahepatic cysts and liver histology confirmed the presence of early HCC in alcohol-DEN mice compared to all other groups. This correlated with increased serum alpha-fetoprotein, a marker of HCC, in alcohol-DEN mice. PCNA immunostaining revealed significantly increased hepatocyte proliferation in livers from alcohol-DEN compared to pair fed-DEN or alcohol-fed mice.

CONCLUSION: We describe a new 12-wk HCC model in adult mice that develops in livers with alcoholic hepatitis and defines ALD as co-factor in HCC.

Keywords: Alpha-fetoprotein; Macrophage polarization; Steatohepatitis; Proliferating cell nuclear antigen; Liver tumor

Core tip: Chronic alcohol consumption leads to broad spectrum of disorders from steatosis to steatohepatitis to cirrhosis and hepatocellular cancer (HCC). Currently there are no animal models of HCC that evaluate effect of alcoholic liver disease (ALD) on HCC acceleration. We describe a new 12-wk HCC model in adult mice that develops in livers with alcoholic hepatitis and defines ALD as co-factor in HCC. Our model involves sequential step-wise progression of ALD to HCC and highlights the role of alcohol as a tumor promoting agent. Importantly, our model shows inflammation, cellular regeneration, and fibrosis, all the features of human HCC pathogenesis.