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

Abstract

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.

Metabolic reprogramming enables hepatocarcinoma cells to efficiently adapt and survive to a nutrient-restricted microenvironment

“…Herein, we found that HCC Dt81Hepa1-6 cells, a highly tumorigenic derivative of Hepa1-6 cells [16], displayed higher metabolic plasticity than their less tumorigenic parental cell line. This metabolic versatility manifested itself through a higher rate of aerobic glycolysis in presence of glucose, but also by their ability to degrade and use fatty acids for energy in glucose-deficient microenvironments.…”

“…Authenticated Hepa1-6 murine, Huh7 and HepG2 human hepatoma cell lines were bought from the American Type Culture Collection (Manassas, Virginia, USA). Dt81Hepa1-6 cell line was derived from Hepa1-6 cells through in vivo passage in C57BL/6 mice [16]. All cultures were maintained at 37 C and 5% CO 2 .…”

“…All culture medium contained penicillin [100units/ml] and streptomycin [100mg/ml]. If not stated otherwise, cells were seeded at 0.125M cells/cm 2 for Hepa1-6, 0.25M cells/cm 2 for Dt81Hepa1-6, 0.0625M cells/ cm 2 for Huh7 and 0.185M cells/cm 2 for HepG2 to achieve 70% of cell confluence [16]. Etomoxir [40 mM] and sodium oxamate [100 mM] were used to inhibit fatty acid oxidation and glycolysis respectively [17,18].…”

“…To assess if the enhanced tumorigenicity displayed by Dt81Hepa1-6 cells could be explained by an increased glucose-related metabolic activity [16], the rate of glucose cell uptake (a prerequisite for increased glucose utilization) was evaluated using the 2-NBDG fluorescent glucose analog. At all doses of 2-NBDG tested [0 to 100 mM], Dt81Hepa1-6 showed increased capacity to uptake glucose compared to Hepa1-6 (P<0.0001) ( Fig.…”

Tumor Microenvironment: A Metabolic Player that Shapes the Immune Response

“…For several decades, neoplastic cells revealed their capacity to exploit, hijack, and disrupt cellular programs that regulate cell division, survival, and growth, leading to tumor formation and dissemination. The best-known causes of malignant transformation are the genetic and epigenetic modifications that induce stem-cell-like properties, such as unlimited cell division and blocked differentiation [2][3][4]. Metabolism and bioenergetics are central to satisfy the multiple nutrient needs for anabolism and biomass production of malignant proliferating cells [5][6][7][8].…”