Research Article: Highly tumorigenic hepatocellular carcinoma cell line with cancer stem cell-like properties

Date Published: February 2, 2017

Publisher: Public Library of Science

Author(s): Benoit Lacoste, Valérie-Ann Raymond, Shamir Cassim, Pascal Lapierre, Marc Bilodeau, Matias A Avila.


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.

Partial Text

Hepatocellular carcinoma (HCC) is the fifth most frequent cancer and the third most deadly cancer worldwide [1] Overall survival rates are typically less than 5% [2]. It is rarely diagnosed early enough to perform curative therapy which partly explains its poor prognosis [3]. Over the years, several animal models have been developed to study its pathogenesis and evaluate potential therapies. These models are categorized as either chemically-induced, genetically-modified or xenograft models [4]. Cell lines are widely used to study HCC in vitro but they do not systematically give rise to solid tumors when implanted in vivo [5]. This has led to the frequent use of immunodeficient animals as hosts despite the difficult translation of data gained with these models to human HCC.

Thirty congenic C57BL/6 mice were injected intrasplenically with 1M Hepa1-6 cells and one mouse developed visible tumors seventy days later (S1 Fig). Cells from these tumors were isolated and cultured in vitro. These cells had a different morphology from the original Hepa1-6 cells and grew in closer aggregates than Hepa1-6 cells in vitro (S1D Fig). The newly derived Dt81Hepa1-6 cells had a similar level of albumin expression compared to Hepa1-6 cells and primary hepatocytes thus confirming their hepatocyte lineage (S1 Fig).

Using in vivo passage of Hepa1-6 HCC cells in immunocompetent C57BL/6 mice, a different clone, Dt81Hepa1-6, with characteristics of tumor-initiating and stem/progenitor cells was isolated. Dt81Hepa1-6 cells are a homogenous cell population with a distinct morphology, different from that of parental Hepa1-6 cells. The cell line derived from this clone is very well adapted for in vivo growth in immunocompetent C57BL/6 mice showing a quasi-exponential time development curve characteristic of growth usually observed in cancer models. A cell dose of 1M cells resulted in the rapid and reproducible development of visible tumors. Furthermore, doses as low as 1000 cells were sufficient to trigger microscopic tumor development. A low-dose HCC model using these cells could be used to mimic the lengthy and gradual cancer development in immune competent hosts, allowing the testing of drugs designed to control HCC recurrence.




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