Generation and analysis of genetically defined liver carcinomas derived from bipotential liver progenitors

Abstract

Hepatocellular carcinoma is a chemoresistant cancer and a leading cause of cancer mortality; however, the molecular mechanisms responsible for the aggressive nature of this disease are poorly understood. In this study, we developed a new liver cancer mouse model that is based on the ex vivo genetic manipulation of embryonic liver progenitor cells (hepatoblasts). After retroviral gene transfer of oncogenes or short hairpin RNAs targeting tumor suppressor genes, genetically altered liver progenitor cells are seeded into the liver of otherwise normal recipient mice. We show that histopathology of the engineered liver carcinomas reveals features of the human disease. Furthermore, representational oligonucleotide microarray analysis (ROMA) of murine liver tumors initiated by two defined genetic hits revealed spontaneously acquired genetic alterations that are characteristic for human hepatocellular carcinoma. This model provides a powerful platform for applications like cancer gene discovery or high-throughput preclinical drug testing.

Figure 1

(A) Schematic representation showing the embryonic derivation of hepatic cell lineages. (B) E-Cadherin-positive fetal liver progenitor cells (LPCs, also known as hepatoblasts) can be purified by immunomagnetic beads. E-Cadherin⁺ fetal liver progenitor cells are isolated from ED = 12.5–15 fetal mouse livers using the MACS® system with indirect labeling. Freshly harvested hepatoblasts are grown in gelatin-coated plates on irradiated NIH-3T3 feeder layers. Islands of LPCs are growing in close proximity to 3T3 feeder cells (left). E-Cadherin⁺ LPCs show an extensive growth potential in vitro when grown on feeder layers in HGF/EGF-enriched hepatocyte growth medium (middle). E-Cadherin⁺ LPCs can be infected efficiently with standard retroviral vectors as shown by GFP fluorescence (right). (C) Characterization of the E-Cadherin⁺ liver progenitor cells with established hepatoblast/oval cell markers. E-Cadherin⁺ LPCs reveal strong positivity for AFP, Alb, and CKAE1; positivity for CK8; and weak positivity for OV-6.

Figure 2

Generation of genetically defined in situ liver cancers. (A) Technical outline. E-Cadherin-positive fetal liver progenitor cells (hepatoblasts) are purified and cultured as described above. Using the murine stem cell virus (MSCV) optimized to drive long-term gene expression in vivo, the cells are infected with oncogenes and/or shRNAs directed against tumor suppressor genes. After infection and expansion, the cells are transplanted into the spleens of conditioned recipient mice. Because all retroviral vectors used for infections are GFP-tagged, development of in situ HCCs can be monitored by external GFP imaging. (B) Schematic representation of the timeline of the approach. Mice undergo two pretreatments with the liver cell cycle inhibitor retrorsine. After transplantation of the cells into the spleen, the transplanted hepatoblasts are selectively expanded by CCl₄ treatment of the mice. (C) GFP⁺ transplanted hepatoblasts can be detected in the recipient liver by anti-GFP immunofluorescence (right). H&E staining of an adjacent section of the liver (left). (D) External GFP imaging of a p53; c-myc tumor-bearing mouse (left). The GFP-positive spot in the square represents the intrahepatic tumor mass. The two additional spots (marked by asterisks) represent transplanted cells residing in the spleen after transplantation. Mice with advanced intrahepatic tumor growth present with swollen, ascites-containing abdomen allowing detection of tumor burden by palpation. GFP imaging of the explanted liver (bottom panel) reveals an advanced intrahepatic tumor, filling out a whole liver lobe. (E) Primary liver tumors can be outgrown in culture and retransplanted in situ into recipient mice. Shown is a tumor that was retransplanted by direct liver injection of 2 × 10⁶ tumor cells. Note the extensive intrahepatic metastasis of the transplanted cells.

Figure 3

Second-generation, microRNA-based shRNA expression vectors can be used to model hepatocarcinogenesis in vivo. (A) Predicted folding for the pri-microRNA. The predicted mature siRNA duplex is labeled with red bars. (B) Liver carcinomas were generated either by infecting p53^−/− liver progenitor cells (LPCs) with oncogenic Ras or by coinfecting wild-type LPCs with a microRNA-based shRNA construct directed against p53 (MLS.1224) together with oncogenic Ras. (C) To facilitate the exact measurement of tumor growth, “p53^−/−;H-RasV12” and “sh-p53;H-RasV12” liver carcinomas were also grown subcutaneously on NCR nu/nu mice. Shown are representative external GFP imagings of the subcutaneously grown tumors and tumor growth curves for each group. Growth rates of “p53^−/−;H-RasV12” and “sh-p53;HRasV12” liver carcinomas were assessed by caliper measurements (n = 6 in each group).

Figure 4

Mouse HCCs derived from E-Cadherin⁺ liver progenitor cells reveal histopathological subtypes of human HCC. Three common variegations of human HCCs (solid, trabecular, and pseudoglandular growth pattern) are lined up with corresponding mouse HCCs derived from genetically modified hepatoblasts. All shown mouse HCCs are derived from p53^−/− hepatoblasts overexpressing c-myc. Mouse HCCs were stained for cytokeratin 8 as a typical liver cytokeratin.