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The role of multipotent cancer associated fibroblasts in hepatocarcinogenesis.

Sukowati CH, Anfuso B, Crocé LS, Tiribelli C - BMC Cancer (2015)

Bottom Line: When co-cultured with human HCC cell lines, CAF up-regulated gene expressions of TGFB1 and FAP of HuH-7 and JHH-6 while NTF did not induced either of the genes.These cells mutually interacts with HCC cells.Their trans-differentiation flexibility may induce a switch from normal to cancerous microenvironment.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine Surgery and Health Sciences, University of Trieste, 34100, Trieste, Italy. caecilia.sukowati@csf.units.it.

ABSTRACT

Background: The presence of tumor supporting cells in various cancer, including in hepatocellular carcinoma (HCC), has become an important target in the study of carcinogenesis. The cancer-associated fibroblast (CAF), one of the most important cellular components in the cancer stroma, might contribute to the progression of the disease due to its plasticity, a behavior of the stem cells. In this study, we investigate the significance of the CAF and its role in the HCC progression and metastasis.

Methods: Primary CAF and non-tumoral fibroblast (NTF) from nine paired HCC and distant non-tumoral liver tissues were isolated and cultured. The cells were characterized by flow cytometry, RT-PCR, anchorage-independent assay and in vitro cells directed trans-differentiation. Co-culture study was performed in Transwell system and xenograft assay was performed in immunodeficient mice.

Results: CAF and NTF were positive for CD90, CD44, αSMA, and vimentin and negative for CD34, CD45, CD117, and CD133. When stimulated, they showed the potential to differentiate into adipocytes, osteoblasts, and pancreatic cells. When co-cultured with human HCC cell lines, CAF up-regulated gene expressions of TGFB1 and FAP of HuH-7 and JHH-6 while NTF did not induced either of the genes. Xenograft assay showed that the CAF had the capacity to enter into circulation as confirmed by RT-PCR and DNA sequencing.

Conclusion: Our data provides evidence of the plasticity of the CAF and the NTF as stem cells in the process of hepatocarcinogenesis and metastasis. These cells mutually interacts with HCC cells. Their trans-differentiation flexibility may induce a switch from normal to cancerous microenvironment.

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Related in: MedlinePlus

The phenotypes of primary cells CAF and NTF. A. Flow cytometric analysis of surface marker proteins showed the positivity for CD90 and CD44, and the negativity for CD133, CD45, and STRO-1. Black graph: control, White graph: positive expression. B. Morphology of the cells and localization of positive proteins CD90, CD44, αSMA, and Vimentin. Scale bar, 100 μm. C. mRNA expressions of vimentin (VIM) and αSMA (ACTA2) in CAF and NTF. VIM and ACTA2 mRNA relative expressions were normalized to reference genes RNA18S and ACTB. D. 3D clonogenic capacity of the CAF in Matrigel. Scale bar, 100 μm.
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Fig1: The phenotypes of primary cells CAF and NTF. A. Flow cytometric analysis of surface marker proteins showed the positivity for CD90 and CD44, and the negativity for CD133, CD45, and STRO-1. Black graph: control, White graph: positive expression. B. Morphology of the cells and localization of positive proteins CD90, CD44, αSMA, and Vimentin. Scale bar, 100 μm. C. mRNA expressions of vimentin (VIM) and αSMA (ACTA2) in CAF and NTF. VIM and ACTA2 mRNA relative expressions were normalized to reference genes RNA18S and ACTB. D. 3D clonogenic capacity of the CAF in Matrigel. Scale bar, 100 μm.

Mentions: Primary cells from HCC (CAF) and from cirrhotic tissue (NTF) were obtained. All cells showed fibroblastic-like morphology and they had the ability to form clonal colonies after plating in low density. The presence of surface markers proteins was detected by flow cytometry and immunofluorescence. CAF showed higher percentages of antigens CD90 and CD44 (52 ± 27% and 59 ± 22%, respectively) as compared to NTF (37 ± 28% and 74 ± 12%), however the differences were not statistically significant. Hematopoietic cells markers CD133, CD45, and STRO-1 were either negative or very low expressed (Figure 1A).Figure 1


The role of multipotent cancer associated fibroblasts in hepatocarcinogenesis.

Sukowati CH, Anfuso B, Crocé LS, Tiribelli C - BMC Cancer (2015)

The phenotypes of primary cells CAF and NTF. A. Flow cytometric analysis of surface marker proteins showed the positivity for CD90 and CD44, and the negativity for CD133, CD45, and STRO-1. Black graph: control, White graph: positive expression. B. Morphology of the cells and localization of positive proteins CD90, CD44, αSMA, and Vimentin. Scale bar, 100 μm. C. mRNA expressions of vimentin (VIM) and αSMA (ACTA2) in CAF and NTF. VIM and ACTA2 mRNA relative expressions were normalized to reference genes RNA18S and ACTB. D. 3D clonogenic capacity of the CAF in Matrigel. Scale bar, 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4389787&req=5

Fig1: The phenotypes of primary cells CAF and NTF. A. Flow cytometric analysis of surface marker proteins showed the positivity for CD90 and CD44, and the negativity for CD133, CD45, and STRO-1. Black graph: control, White graph: positive expression. B. Morphology of the cells and localization of positive proteins CD90, CD44, αSMA, and Vimentin. Scale bar, 100 μm. C. mRNA expressions of vimentin (VIM) and αSMA (ACTA2) in CAF and NTF. VIM and ACTA2 mRNA relative expressions were normalized to reference genes RNA18S and ACTB. D. 3D clonogenic capacity of the CAF in Matrigel. Scale bar, 100 μm.
Mentions: Primary cells from HCC (CAF) and from cirrhotic tissue (NTF) were obtained. All cells showed fibroblastic-like morphology and they had the ability to form clonal colonies after plating in low density. The presence of surface markers proteins was detected by flow cytometry and immunofluorescence. CAF showed higher percentages of antigens CD90 and CD44 (52 ± 27% and 59 ± 22%, respectively) as compared to NTF (37 ± 28% and 74 ± 12%), however the differences were not statistically significant. Hematopoietic cells markers CD133, CD45, and STRO-1 were either negative or very low expressed (Figure 1A).Figure 1

Bottom Line: When co-cultured with human HCC cell lines, CAF up-regulated gene expressions of TGFB1 and FAP of HuH-7 and JHH-6 while NTF did not induced either of the genes.These cells mutually interacts with HCC cells.Their trans-differentiation flexibility may induce a switch from normal to cancerous microenvironment.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine Surgery and Health Sciences, University of Trieste, 34100, Trieste, Italy. caecilia.sukowati@csf.units.it.

ABSTRACT

Background: The presence of tumor supporting cells in various cancer, including in hepatocellular carcinoma (HCC), has become an important target in the study of carcinogenesis. The cancer-associated fibroblast (CAF), one of the most important cellular components in the cancer stroma, might contribute to the progression of the disease due to its plasticity, a behavior of the stem cells. In this study, we investigate the significance of the CAF and its role in the HCC progression and metastasis.

Methods: Primary CAF and non-tumoral fibroblast (NTF) from nine paired HCC and distant non-tumoral liver tissues were isolated and cultured. The cells were characterized by flow cytometry, RT-PCR, anchorage-independent assay and in vitro cells directed trans-differentiation. Co-culture study was performed in Transwell system and xenograft assay was performed in immunodeficient mice.

Results: CAF and NTF were positive for CD90, CD44, αSMA, and vimentin and negative for CD34, CD45, CD117, and CD133. When stimulated, they showed the potential to differentiate into adipocytes, osteoblasts, and pancreatic cells. When co-cultured with human HCC cell lines, CAF up-regulated gene expressions of TGFB1 and FAP of HuH-7 and JHH-6 while NTF did not induced either of the genes. Xenograft assay showed that the CAF had the capacity to enter into circulation as confirmed by RT-PCR and DNA sequencing.

Conclusion: Our data provides evidence of the plasticity of the CAF and the NTF as stem cells in the process of hepatocarcinogenesis and metastasis. These cells mutually interacts with HCC cells. Their trans-differentiation flexibility may induce a switch from normal to cancerous microenvironment.

Show MeSH
Related in: MedlinePlus