Abstract 4426: Is development of hepatocellular carcinoma driven by liver homing signals?
Authors: Bilodeau Marc , Lacoste Benoit , Raymond Valérie-Ann 
Published: August 2012

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