Multiomics characterization of an alcohol-induced hepatocellular carcinoma mouse model

lab animal Article https://doi.org/10.1038/s41684-026-01724-7 Multiomics characterization of an alcohol-induced hepatocellular carcinoma mouse model Check for updates Seol Hee Park1,2,6, Seo Bhin Park3,6, Jisoo Kang3, Sumin Shin3, Ga-Young Kim3,4, Yunseo Bong3,4, Minkyoung Gu3,4, Young-Sun Lee    5 , Gao Bin    1 & Wonhyo Seo    1,3,4 Hepatocellular carcinoma (HCC) is a major global health problem, ranking as the sixth most frequently diagnosed cancer and the third leading cause of cancer-related mortality worldwide. Although the incidence of viral infection-mediated HCC has decreased in recent years, the incidence of alcohol- and metabolic dysfunction-associated HCC has increased, driven by changes in lifestyle and diet. Excessive alcohol consumption contributes to advanced liver diseases, including liver fibrosis, cirrhosis and HCC. Despite the clinical relevance of alcohol-associated HCC, there are no suitable animal models that adequately reflect the pathophysiological features of alcohol-associated HCC in humans. Here, to address this limitation, we established a mouse model of alcohol-associated HCC through the combined administration of N-diethylnitrosamine and carbon tetrachloride (CCl4), followed by administration of an alcohol-containing Lieber–DeCarli diet. The results indicated that chronic alcohol exposure in the presence of N-diethylnitrosamine and CCl4 substantially accelerated HCC development, which was characterized by increased oxidative stress, inflammation and severe fibrosis. Furthermore, we found that chronic ethanol consumption disrupted hepatic immunity, characterized by natural killer/natural killer T cell depletion, increased PD1+CD8+ cells, reduced cytotoxicity and elevated inflammation. We also observed marked alterations in the gut microbiome following chronic alcohol administration. These immunological and microbiome alterations fostered an immunosuppressive microenvironment that accelerated HCC progression. Our newly developed mouse model induced liver tumorigenesis within a relatively short timeframe and recapitulated the clinical and pathological features of alcohol-associated HCC. The model therefore represents a valuable tool for studying the mechanisms underlying alcohol-associated HCC and related chronic liver diseases. Liver cancer is a growing global health concern due to its increasing prevalence and high mortality rate. In 2022, liver cancer caused over 750,000 deaths worldwide, making it the third leading cause of cancer-related mortality after lung and colorectal cancers. It is also the sixth most frequently diagnosed cancer, with an estimated 865,000 new cases reported in 20221. Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma, which are characterized by hepatocellular and bile ductular differentiation, respectively, are the most prevalent primary liver cancer types2,3. HCC is the most common form of liver cancer, accounting for ~90% of all cases4. Major risk factors for HCC include viral infection, diabetes, obesity and A full list of affiliations appears at the end of the paper. Lab Animal chronic alcohol consumption5–9. Notably, more than 90% of HCC cases are associated with chronic liver disease, with liver cirrhosis being one of the most important risk factors. In cirrhosis, HCC is a leading cause of liver-related mortality primarily in compensated disease, whereas in decompensated cirrhosis, mortality is usually driven by complications such as portal hypertension or liver function impairment (for example, ascites, variceal bleeding, encephalopathy and jaundice)10,11. Chronic liver disease can also be induced by viral infection, where the hepatitis B and C viruses have long been recognized as substantial contributors to HCC development12. Although the incidence of viral infection-mediated HCC e-mail: ; ; https://doi.org/10.1038/s41684-026-01724-7 has decreased in recent years, the prevalence of HCC has increased among patients with steatohepatitis related to metabolic syndrome or excessive alcohol consumption13–15. Alcohol is classified as a group 1 carcinogen by the International Agency for Research on Cancer. It is both toxic and cocarcinogenic, substantially increasing the risk of HCC development16,17. Alcohol-associated liver disease (ALD), the most prevalent type of liver disease in the USA and Europe, is characterized by progressive chronic liver damage, inflammation, fibrosis, cirrhosis and, ultimately, HCC18–26. Patients with metabolic dysfunction-associated steatohepatitis experience more rapid progression to HCC, and this risk is markedly increased when excessive alcohol consumption is involved27. Although excessive alcohol exposure is widely recognized as a primary cause of ALD, a recent meta-analysis revealed that the chronic consumption of even low quantities of alcohol can substantially increase the risk of ALD and HCC20. Despite the increasing burden of alcohol-associated HCC, current animal models fail to reflect human alcohol-associated HCC28. To model chronic alcohol consumption, the Lieber–DeCarli diet has the advantage that rodents consume a liquid-only diet containing ethanol (EtOH) and can overcome their aversion to alcohol29. However, long-term administration of the Lieber–DeCarli diet is not sufficient to induce cirrhosis and HCC in mice owing to the notably faster metabolic rate of mice compared with humans30. Combining chronic carbon tetrachloride (CCl4) administration with the Lieber–DeCarli diet successfully mimics advanced liver pathology (advanced stage of HCC); however, it fails to induce the early and intermediate stages of HCC31. Therefore, we co-administered N-diethylnitrosamine (DEN), a highly potent carcinogen, to chemically induce an HCC model32,33. Despite the potent carcinogenicity of DEN, repeated administration of DEN followed by 7 weeks of a 4% alcohol-containing Lieber–DeCarli diet resulted in limited induction of inflammation, fibrosis and tumor progression28,34. To overcome these experimental limitations, we developed a novel alcohol-associated HCC mouse model by combining DEN and CCl4 administration with a Lieber–DeCarli diet containing 4.5% (v/v) EtOH. This approach enhanced tumor development while recapitulating the pathophysiological features of alcohol-associated HCC in a relatively short timeframe. The new model provides a valuable platform for studying the mechanisms of alcohol-associated HCC and exploring potential therapeutic interventions. Results Experimental induction of alcohol-associated HCC in mice using a combination of DEN, CCl4 and EtOH To develop an alcohol-associated HCC mouse model, we used a combination of carcinogen administration and chronic alcohol feeding. A single intraperitoneal dose of DEN (25 mg/kg) was administered to 14-day-old male pups, which were then housed for 8–10 weeks until they reached a body weight of approximately 23–25 g. The mice were then fed on a Lieber–DeCarli diet containing 4.5% EtOH for 20 week (...truncated)