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.

Fig 1. Dt81Hepa1-6 cell transfer leads to rapid tumor development.

A suspension of 1M Dt81Hepa1-6 cells was injected intrasplenically in C57BL/6 mice and animals were sacrificed after 3.5 to 28 days. (A) Manual count of tumor >0.5mm. (B) Alpha-fetoprotein (AFP) (Afp) mRNA expression in liver homogenates as measured by qPCR. (C) Representative microphotographs at 100x magnification of HPS-stained liver slices obtained after 3.5, 7 and 14 days. Whole-liver photographs obtained after 21 or 28 days. Control mice were injected with 1M Hepa1-6 cells and sacrificed at each time point (3.5 to 28 days) and pooled for analysis (Ctrl). (*P<0.05; **P<0.01; ***P<0.001).

Fig 2. Dose-dependent tumor development following transfer of Dt81Hepa1-6 cells.

Suspensions of Dt81Hepa1-6 cells [1K to 1M] were injected intrasplenically in C57BL/6 mice and animals were sacrificed after 28 days. (A) Manual count of tumor >0.5mm. (B) AFP (Afp) mRNA expression in liver homogenates by qPCR. (C) Representative microphotographs at 100x magnification of HPS-stained liver slice obtained after injection with 1K and 10K cells. Whole liver photographs of mice injected with 0.1M, 0.5M or 1M cells. Control mice were injected with 1M Hepa1-6 cells. (*P<0.05; **P<0.01; ***P<0.001).

Fig 3. Dt81Hepa1-6 cells are specific to the liver.

A suspension of 1M Dt81Hepa1-6 cells was injected subcutaneously (SC) in the left flank of C57BL/6 mice and animals were sacrificed after 28 days. (A) AFP mRNA relative expression in control and SC injected C57BL/6 mice. (B) Whole liver photographs at time of sacrifice. HPS-stained slices representative microphotographs of (C) lungs and spleen after SC injection and (D) Lungs and spleen after IS injection. Representative microphotographs were obtained at 100x magnification. (*P<0.05).

Fig 4. Dt81Hepa1-6 in vitro neoplastic profile.

A cell aggregation assay was performed and (A) the number of cells per aggregate was calculated 24h after seeding both cell lines in non-adherent agar-coated wells. Representative microphotographs are shown. In order to evaluate anchorage-independent growth, a soft-agar colony formation assay was performed; (B) cell lines were seeded at 10K-cell concentration in soft-agar gel [0.3%] in order to form visible colonies and were counted 5 weeks later. Representative microphotographs of cells after 5 weeks are shown. The ability of independent cells to form colonies without cell-to-cell contact was assessed by a proliferative clonal colony assay; (C) cell lines were seeded at 3K/mL concentration to obtain isolated cells and left to grow as colonies over a 4 weeks period and counted. The potential for invasiveness was evaluated with a double-layer droplet cell invasion assay; (D) Cell lines [20K cells] were embedded in COL1 [3mg/mL] to form a double layered droplet and cells penetrating the outer COL1 layer were counted every 12h for 8 days. Area under the curves (AUC) of the time-dependent invasion was calculated and used for statistical analysis. Representative microphotographs are shown. For the wound-healing assays (WHA); (E) normal and COL1-embedded WHA were performed on confluent cells for 24h and length traveled was evaluated using ImageJ. For the cell-spreading assay, (F) Cell lines were seeded for 6h on plastic and COL1 and cells showing an elongated morphology were counted manually. (G) Cell doubling time for Dt81Hepa1-6 and parental Hepa1-6 cells. (H) Viability of Dt81Hepa1-6 and Hepa1-6 cells following in vitro exposure to cisplatin [25ug/mL] for 24h. (*P<0.05; **P<0.01; ***P<0.001).

Fig 5. EpCAM+ Dt81Hepa1-6 cells are highly tumorigenic.

Dt81Hepa1-6 cells were incubated with APC-tagged anti-CD326 (EpCAM) antibody and sorted by FACS as EpCAM+high and EpCAM+low Dt81Hepa1-6 cells. Hepa1-6, Dt81Hepa1-6, EpCAM+high and EpCAM+low were analyzed by (A) qPCR for EpCAM mRNA expression and (B) flow cytometry after labeling with APC-tagged anti-CD326 (EpCAM). Dt81Hepa1-6, EpCAM+low and EpCAM+high were injected intrasplenically in C57BL/6 mice and sacrificed after 21 days. (C) Tumor load (>0.5mm) was assessed and (D) AFP mRNA expression was compared to healthy liver controls. (*P<0.05; **P<0.01; ***P<0.001).

Fig 6. Expression of β-Catenin, Cyclin D1 and Integrins by Dt81Hepa1-6 cells.

All experiments were performed on freshly trypsinized cells prior to their injection in vivo. Genes or proteins of interest were analyzed in Hepa1-6, Dt81Hepa1-6, EpCAM+low and EpCAM+high cell lines by qRT-PCR and/or western blot. (A) β-Catenin (Ctnnb1) gene and (B) protein expression shown on immunoblot (C) Cyclin D1 (Ccnd1), (D) Integrin-alpha1 (Itga1) and (E) Integrin-β1 (Itgb1) mRNA expression. (*P<0.05; **P<0.01).