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An orthotopic mouse model of hepatocellular carcinoma with underlying liver cirrhosis

Nature Protocols volume 10, pages 1264–1274 (2015)Cite this article

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Abstract

Subcutaneous xenografts have been used for decades to study hepatocellular carcinoma (HCC). These models do not reproduce the specific pathophysiological features of HCCs, which occur in cirrhotic livers that show pronounced necroinflammation, abnormal angiogenesis and extensive fibrosis. As these features are crucial for studying the role of the pathologic host microenvironment in tumor initiation, progression and treatment response, alternative HCC models are desirable. Here we describe a syngeneic orthotopic HCC model in immunocompetent mice with liver cirrhosis induced by carbon tetrachloride (CCl4) that recapitulates key features of human HCC. Induction of substantial hepatic fibrosis requires 12 weeks of CCl4 administration. Intrahepatic implantation of mouse HCC cell lines requires 30 min per mouse. Tumor growth varies by tumor cell line and mouse strain used. Alternatively, tumors can be induced in a genetically engineered mouse model. In this setting, CCl4 is administered for 12 weeks after tail-vein injection of Cre-expressing adenovirus (adeno-Cre) in Stk4−/−Stk3F/− (also known as Mst1−/−Mst2F/−; F indicates a floxed allele) mice, and it results in the development of HCC tumors (hepatocarcinogenesis) concomitantly with liver cirrhosis.

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Figure 1: Experimental design.
Figure 2: Microscopic appearance of hepatocellular carcinoma in mice with cirrhotic liver.
Figure 3: Surgical procedure for intrahepatic HCC graft implantation.
Figure 4: Macroscopic appearance of primary HCC and lung metastases.
Figure 5: Differential tumor growth rates and treatment response in orthotopic HCC growing in normal livers versus fibrotic livers.

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Acknowledgements

The authors thank D. Nguyen, A. Pieters and C. Smith for their outstanding support in establishing this protocol. This study was supported by the NIH grant P01-CA080124, and in part by grants R01-CA159258, R21-CA139168, R01-CA126642 and National Cancer Institute/Proton Beam Federal Share Program awards (to D.G.D. and R.K.J.); by the American Cancer Society grant 120733-RSG-11-073-01-TBG (to D.G.D.); by a Max Kade Fellowship by the Austrian Academy of Science and a Erwin-Schroedinger Fellowship by the Austrian Science Funds (to T.R.); by a Howard Hughes Medical Institute Medical Research Fellowship (to C.F.); and by a Postdoctoral Fellowship from Astellas Foundation for Research on Metabolic Disorders (to T.H.).

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Authors and Affiliations

  1. Department of Radiation Oncology, Steele Laboratories for Tumor Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA

    Thomas Reiberger, Yunching Chen, Rakesh R Ramjiawan, Tai Hato, Christopher Fan, Rekha Samuel, Sylvie Roberge, Peigen Huang, Rakesh K Jain & Dan G Duda

  2. Division of Gastroenterology and Hepatology, Medical University of Vienna, Vienna, Austria

    Thomas Reiberger

  3. Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu, Taiwan

    Yunching Chen

  4. Department of Medical Oncology, Angiogenesis Laboratory, Cancer Center Amsterdam, Vrije Universiteit (VU) University Medical Center, Amsterdam, the Netherlands

    Rakesh R Ramjiawan

  5. Duke University School of Medicine, Durham, North Carolina, USA

    Christopher Fan

  6. Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA

    Gregory Y Lauwers

  7. Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA

    Andrew X Zhu & Nabeel Bardeesy

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  1. Thomas Reiberger
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Contributions

T.R., Y.C., A.X.Z., R.K.J. and D.G.D. contributed to the concept and design of the study. T.R., Y.C., R.R.R., T.H., P.H., S.R., C.F., R.S. and G.Y.L. were responsible for acquisition of the data. T.R., Y.C., R.R.R., T.H., R.S., P.H., G.Y.L., A.X.Z., N.B., R.K.J. and D.G.D. contributed to analysis and interpretation of the data. T.R., Y.C., R.R.R., T.H., C.F., R.S., S.R., P.H., G.Y.L., A.X.Z., N.B., R.K.J. and D.G.D. were involved in drafting of the article and revising it for important intellectual content.

Corresponding author

Correspondence to Dan G Duda.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Imaging of orthotopic hepatocellular carcinoma by using abdominal high-frequency ultrasound.

(A) B-mode image of a HCA-1 Tumor 10 days after orthotopic implantation in a cirrhotic C3H mouse. Scale bar: 2mm. (B) B-mode image of a HCC tumor developed 10 weeks after Adeno-Cre injection and CCl4 administration in a Mst1−/−Mst2F/− mouse. Scale bar: 2mm.

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Reiberger, T., Chen, Y., Ramjiawan, R. et al. An orthotopic mouse model of hepatocellular carcinoma with underlying liver cirrhosis. Nat Protoc 10, 1264–1274 (2015). https://doi.org/10.1038/nprot.2015.080

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