Adult mouse model of early hepatocellular carcinoma promoted by alcoholic liver disease.
OBJECTIVE
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.


CONCLUSIONS
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.
Aditya Ambade, Abhishek Satishchandran, Benedek Gyongyosi, Patrick Lowe, Gyongyi Szabo, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, United States.
Aditya
A
Ambade
Aditya Ambade, Abhishek Satishchandran, Benedek Gyongyosi, Patrick Lowe, Gyongyi Szabo, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, United States.
Abhishek
A
Satishchandran
Aditya Ambade, Abhishek Satishchandran, Benedek Gyongyosi, Patrick Lowe, Gyongyi Szabo, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, United States.
Benedek
B
Gyongyosi
Aditya Ambade, Abhishek Satishchandran, Benedek Gyongyosi, Patrick Lowe, Gyongyi Szabo, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, United States.
Patrick
P
Lowe
Aditya Ambade, Abhishek Satishchandran, Benedek Gyongyosi, Patrick Lowe, Gyongyi Szabo, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, United States.
Gyongyi
G
Szabo
D000410:Alanine Transaminase / Q000097:blood; D000818:Animals; D014408:Biomarkers, Tumor / Q000097:blood; D006528:Carcinoma, Hepatocellular / Q000097:blood / Q000209:etiology* / Q000473:pathology; D049109:Cell Proliferation; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism; D004052:Diethylnitrosamine; D004731:Endotoxins / Q000097:blood; D000431:Ethanol; D005235:Fatty Liver, Alcoholic / Q000097:blood / Q000150:complications* / Q000473:pathology; D022781:Hepatocytes / Q000378:metabolism / Q000473:pathology; D007150:Immunohistochemistry; D008104:Liver Cirrhosis, Alcoholic / Q000097:blood / Q000150:complications* / Q000473:pathology; D008114:Liver Neoplasms, Experimental / Q000097:blood / Q000139:chemically induced / Q000209:etiology* / Q000473:pathology; D008262:Macrophage Activation; D008264:Macrophages / Q000378:metabolism / Q000473:pathology; D008279:Magnetic Resonance Imaging; D008297:Male; D008810:Mice, Inbred C57BL; D020556:Neutrophil Infiltration; D009504:Neutrophils / Q000378:metabolism / Q000473:pathology; D010641:Phenotype; D013997:Time Factors; D000509:alpha-Fetoproteins / Q000378:metabolism
D014408:Biomarkers, Tumor; D004731:Endotoxins; D000509:alpha-Fetoproteins; D004052:Diethylnitrosamine; D000431:Ethanol; D019392:Cytochrome P-450 CYP2E1; D000410:Alanine Transaminase
Alpha-fetoprotein; Liver tumor; Macrophage polarization; Proliferating cell nuclear antigen; Steatohepatitis