Murine hepatocellular carcinoma derived stem cells reveal epithelial-to-mesenchymal plasticity

Abstract

Aim: To establish a model to enrich and characterize stem-like cells from murine normal liver and hepatocellular carcinoma (HCC) cell lines and to further investigate stem-like cell association with epithelial-to-mesenchymal transition (EMT).

Methods: In this study, we utilized a stem cell conditioned serum-free medium to enrich stem-like cells from mouse HCC and normal liver cell lines, Hepa 1-6 and AML12, respectively. We isolated the 3-dimensional spheres and assessed their stemness characteristics by evaluating the RNA levels of stemness genes and a cell surface stem cell marker by quantitative reverse transcriptase-PCR (qRT-PCR). Next, we examined the relationship between stem cells and EMT using qRT-PCR.

Results: Three-dimensional spheres were enriched by culturing murine HCC and normal hepatocyte cell lines in stem cell conditioned serum-free medium supplemented with epidermal growth factor, basic fibroblast growth factor and heparin sulfate. The 3-dimensional spheres had enhanced stemness markers such as Klf4 and Bmi1 and hepatic cancer stem cell (CSC) marker Cd44 compared to parental cells grown as adherent cultures. We report that epithelial markers E-cadherin and ZO-1 were downregulated, while mesenchymal markers Vimentin and Fibronectin were upregulated in 3-dimensional spheres. The 3-dimensional spheres also exhibited changes in expression of Snai, Zeb and Twist family of EMT transcription factors.

Conclusion: Our novel method successfully enriched stem-like cells which possessed an EMT phenotype. The isolation and characterization of murine hepatic CSCs could establish a precise target for the development of more effective therapies for HCC.

Keywords: AML12; Cancer initiating cells; Cancer stem cells; Cellular plasticity; Epithelial-to-mesenchymal transition; Epithelial-to-mesenchymal transition transcription factors; Hepa 1-6; Hepatocellular carcinoma.

Figure 1

Enrichment of cancer stem cells using the sphere culture method. A: Photomicrographs of Hepa 1-6 3-dimensional spheres on day 5 cultured in stem cell conditioned serum-free medium; B: Hepa 1-6 sphere sizes and numbers were enumerated; C: Photomicrographs of AML12 3-dimensional spheres on day 5; D: AML12 spheres sizes and numbers were enumerated.

Figure 2

Three-dimensional spheres expressed high stemness and cell surface cancer stem cell markers. qRT-PCR analysis revealed higher expression of A: Klf4, B: Bmi1 and C: Cd44 in Hepa 1-6 3-dimensional spheres compared with Hepa 1-6 grown as adherent cells. qRT-PCR analysis revealed higher expression of D: Klf4, E: Bmi1 and F: Cd44 in AML12 3-dimensional spheres compared with AML12 grown as adherent cells. Values are mean ± SEM of three experiments in triplicate (^aP < 0.05, ^bP < 0.01, ^eP < 0.001). qRT-PCR: Quantitative reverse transcriptase-PCR; ActB: Beta-Actin.

Figure 3

3-dimensional spheres decreased expression of epithelial markers. qRT-PCR analysis revealed downregulation of A: E-cadherin and B: ZO-1 in Hepa 1-6 CSCs compared with Hepa 1-6 grown as adherent cells. qRT-PCR analysis revealed lower expression of C: E-cadherin and D: ZO-1 in AML12 CSCs compared with AML12 grown as adherent cells. Values are mean ± SEM of three experiments in triplicate (^aP < 0.05, ^eP < 0.001). qRT-PCR: Quantitative reverse transcriptase-PCR; ActB: Beta-Actin.

Figure 4

Three-dimensional spheres have higher expression of mesenchymal markers. qRT-PCR analysis revealed upregulation of A: Vimentin and B: Fibronectin in Hepa 1-6 3-dimensional spheres compared with Hepa 1-6 grown as adherent cells. qRT-PCR analysis revealed higher expression of C: Vimentin and D: Fibronectin in AML12 3-dimensional spheres compared with AML12 grown as adherent cells. Values are mean ± SEM of three experiments in triplicate (^aP < 0.05, ^bP < 0.01, ^eP < 0.001). qRT-PCR: Quantitative reverse transcriptase-PCR; ActB: Beta-Actin.

Figure 5

Expression of putative epithelial-to-mesenchymal transition transcription factors in Hepa 1-6 3-dimensional spheres. qRT-PCR analysis revealed upregulation of A: Snai1, B: Snai2, C: Zeb1, D: Zeb2, E: Twist1, and F: Twist2 in Hepa 1-6 3-dimensional spheres compared with Hepa 1-6 grown as adherent cells. Values are mean ± SEM of three experiments in triplicate (^aP < 0.05, ^eP < 0.001). qRT-PCR: Quantitative reverse transcriptase-PCR; ActB: Beta-Actin.

Figure 6

Expression of putative epithelial-to-mesenchymal transition transcription factors in AML12 3-dimensional spheres. qRT-PCR analysis revealed upregulation of A: Snai1, C: Zeb1, D: Zeb2 and downregulation of B: Snai2, E: Twist1, and F: Twist2 in AML12 3-dimensional spheres compared with AML12 grown as adherent cells. Values are mean ± SEM of three experiments in triplicate (^bP < 0.01, ^eP < 0.001). qRT-PCR: Quantitative reverse transcriptase-PCR; ActB: Beta-Actin.