HCC (Hepatocellular carcinoma) cells exhibit greater metabolic plasticity than normal hepatocytes since they must survive in a dynamic microenvironment where nutrients and oxygen are often scarce. Using a metabolomic approach combined with functional in vitro and in vivo assays, we aimed to identify an HCC metabolic signature associated with increased tumorigenicity and patient mortality. Metabolite profiling of HCC Dt81Hepa1-6 cells revealed that their increased tumorigenicity was associated with elevated levels of glycolytic metabolites. Tumorigenic Dt81Hepa1-6 also had an increased ability to uptake glucose leading to a higher glycolytic flux that stemmed from an increased expression of glucose transporter GLUT-1. Dt81Hepa1-6-derived tumors displayed increased mRNA expressions of glycolytic genes, Hypoxia-inducible factor-1alpha and of Cyclin D1. HCC tumors also displayed increased energy charge, reduced antioxidative metabolites and similar fatty acid biosynthesis compared to healthy liver. Increased tumoral expression of glycolytic and hypoxia signaling pathway mRNAs was associated with decreased survival in HCC patients. In conclusion, HCC cells can rapidly alter their metabolism according to their environment and switch to the use of glucose through aerobic glycolysis to sustain their tumorigenicity and proliferative ability. Therefore, cancer metabolic reprogramming could be essential for the tumorigenicity of HCC cells during cancer initiation and invasion.

There are limited numbers of models to study hepatocellular carcinoma (HCC) in vivo in immunocompetent hosts. In an effort to develop a cell line with improved tumorigenicity, we derived a new cell line from Hepa1-6 cells through an in vivo passage in C57BL/6 mice. The resulting Dt81Hepa1-6 cell line showed enhanced tumorigenicity compared to Hepa1-6 with more frequent (28±12 vs. 0±0 lesions at 21 days) and more rapid tumor development (21 (100%) vs. 70 days (10%)) in C57BL/6 mice. The minimal Dt81Hepa1-6 cell number required to obtain visible tumors was 100,000 cells. The Dt81Hepa1-6 cell line showed high hepatotropism with subcutaneous injection leading to liver tumors without development of tumors in lungs or spleen. In vitro, Dt81Hepa1-6 cells showed increased anchorage-independent growth (34.7±6.8 vs. 12.3±3.3 colonies; P<0.05) and increased EpCAM (8.7±1.1 folds; P<0.01) and β-catenin (5.4±1.0 folds; P<0.01) expression. A significant proportion of Dt81Hepa1-6 cells expressed EpCAM compared to Hepa1-6 (34.8±1.1% vs 0.9±0.13%; P<0.001). Enriched EpCAM+ Dt81Hepa1-6 cells led to higher tumor load than EpCAM- Dt81Hepa1-6 cells (1093±74 vs 473±100 tumors; P<0.01). The in vivo selected Dt81Hepa1-6 cell line shows high liver specificity and increased tumorigenicity compared to Hepa1-6 cells. These properties are associated with increased expression of EpCAM and β-catenin confirming that EpCAM+ HCC cells comprise a subset with characteristics of tumor-initiating cells with stem/progenitor cell features. The Dt81Hepa1-6 cell line with its cancer stem cell-like properties will be a useful tool for the study of hepatocellular carcinoma in vivo.

Collagen produced during the process of liver fibrosis can induce a hepatocellular protective response through ERK1 signalling. However, the influence of T cells and associated cytokine production on this protection is unknown. In addition, athymic mice are frequently used in hepatocellular carcinoma xenograft experiments but current methods limit our ability to study the impact of liver fibrosis in this setting due to high mortality. Therefore, a mouse model of liver fibrosis lacking T cells was developed using Foxn1 nu/nu mice and progressive oral administration of thioacetamide (TAA) [0.01–0.02%] in drinking water. Fibrosis developed over a period of 16 weeks (alpha-SMA positive area: 20.0 ± 2.2%, preCol1a1 mRNA expression: 11.7 ± 4.1 fold changes, hydroxyproline content: 1041.2 ± 77μg/g of liver) at levels comparable to that of BALB/c mice that received intraperitoneal TAA injections [200 μg/g of body weight (bw)] (alpha-SMA positive area: 20.9 ± 2.9%, preCol1a1 mRNA expression: 13.1 ± 2.3 fold changes, hydroxyproline content: 931.6 ± 14.8μg/g of liver). No mortality was observed. Athymic mice showed phosphorylation of ERK1/2 during fibrogenesis (control 0.03 ± 0.01 vs 16 weeks 0.22 ± 0.06AU; P<0.05). The fibrosis-induced hepatoprotection against cytotoxic agents, as assessed histologically and by serum AST levels, was not affected by the absence of circulating T cells (anti-Fas JO2 [0.5μg/g bw] for 6h (fibrotic 4665 ± 2596 vs non-fibrotic 13953 ± 2260 U/L; P<0.05), APAP [750 mg/kg bw] for 6 hours (fibrotic 292 ± 66 U/L vs non-fibrotic 4086 ± 2205; P<0.01) and CCl4 [0.5mL/Kg bw] for 24h (fibrotic 888 ± 268 vs non-fibrotic 15673 ± 2782 U/L; P<0.001)). In conclusion, liver fibrosis can be induced in athymic Foxn1 nu/nu mice without early mortality. Liver fibrosis leads to ERK1/2 phosphorylation. Finally, circulating T lymphocytes and associated cytokines are not involved in the hepatocellular protection afforded by liver fibrosis.

There are a limited number of models to study hepatocellular carcinoma (HCC). Hepa 1-6 cell line can be used to develop tumors when injected directly in the liver of C57/BL6 mice. We isolated cells from a tumor which developed in the liver following the intrasplenic injection of Hepa 1-6 cells and found that the daughter cells led to more systematic development of tumors. Studies were performed to test the characteristics of the 2 cell lines. In vivo tumorigenicity was assessed by injecting cells in the spleen. Minimal cell dose and liver specificity were assessed at 28 days. Tumors (≥0,5mm) were counted and alpha-fetoprotein (AFP) mRNA measured by relative RT-PCR. Cell lines were compared in vitro for their alpha-1 integrin (ITGa1), beta1-integrin (ITGb1), AFP and Epithelial Cell Adhesion Molecule (EpCAM) mRNA expression. Apoptosis was measured following exposure to cisplatinum [25µg/ml]. Invasiveness and motility were assessed using a modified wound healing assay and a double layered COL1 hemisphere invasion assay. Cell doubling time (CDT) was measured by cell count. Intrahepatic tumours developed more quickly (21d vs. 70d) and more often (66% (4/6) vs. 15% (1/7)) with the daughter cell line. Tumors first appeared 21 days after intrasplenic injection and increased steadily thereafter: this was associated with increased AFP expression in liver homogenates. The minimal cell concentration required to give raise to visible tumors was 10K with the daughter cell line while no tumor was observed at 28d with the parental cell line (1M cells). No extra hepatic tumours were found at any point and with any cell concentration. However, subcutaneous injection induced hepatic tumours in every animal only when using the more tumorigenic daughter cell line (100% 3/3 at 28d). In vitro, the daughter cell line showed increased resistance to apoptosis when exposed to COL1, did not show increase proliferation, but were less motile and less invasive. Cell doubling time was longer with the daughter cell line then with the parental one (45.5h±2.7h vs 34.6h±0.4h; p&lt;0.05). There was no significant difference in the levels of the pro-apoptotic Bid, Bak and Bad proteins and in the Bcl-xL anti-apoptotic protein, ERK1/2 and AKT between the 2 cell lines. On the other hand, the more aggressive cell line showed almost a tenfold increased expression of EpCAM (9.77±2.71 vs 1.05±0.24), and lower expressions of ITGa1 (0.05±0.03 vs 1.06±0.24) and ITGb1 (0.36±0.14 vs 1.03±0.16), all these differences being significant (p&lt;0.05). In conclusion, we observed that HCC cells derived from the same clone can have strikingly different tumorigenic potential. Increased expression of the epithelial cell adhesion molecule was associated with a high potential for intrahepatic tumor development despite a less aggressive phenotype in vitro. These results suggest that liver homing signals might be important for the establishment/survival of HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4426. doi:1538-7445.AM2012-4426