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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

Co-culture study between the primary cells and the HCC cell lines HuH-7 and JHH-6. A. Cross-talk between the HCC cells lines and the CAF. B. Cross-talk between the HCC cells lines and the NTF. Target mRNA expression was normalized to reference genes RNA18S and ACTB. CTRL = cells control without co-culture (=1.00). Target mRNA expressions were normalized to reference genes 18SRNA and ACTB. *p < 0.05 compared to each control.
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Fig3: Co-culture study between the primary cells and the HCC cell lines HuH-7 and JHH-6. A. Cross-talk between the HCC cells lines and the CAF. B. Cross-talk between the HCC cells lines and the NTF. Target mRNA expression was normalized to reference genes RNA18S and ACTB. CTRL = cells control without co-culture (=1.00). Target mRNA expressions were normalized to reference genes 18SRNA and ACTB. *p < 0.05 compared to each control.

Mentions: After co-culture, we observed a significant effect of the presence of both CAF and NTF in HCC cell lines. In the presence of the CAF, the up-regulations of TGFB1 and FAP were observed in both HCC cell lines (p < 0.05), a slight up-regulation of ACTA2 was observed in HuH-7. On the other hand, the presence of HuH-7 cells increased the expressions of ACTA2 and COL1 in CAF, while JHH-6 cells did not (Figure 3A).Figure 3


The role of multipotent cancer associated fibroblasts in hepatocarcinogenesis.

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

Co-culture study between the primary cells and the HCC cell lines HuH-7 and JHH-6. A. Cross-talk between the HCC cells lines and the CAF. B. Cross-talk between the HCC cells lines and the NTF. Target mRNA expression was normalized to reference genes RNA18S and ACTB. CTRL = cells control without co-culture (=1.00). Target mRNA expressions were normalized to reference genes 18SRNA and ACTB. *p < 0.05 compared to each control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Co-culture study between the primary cells and the HCC cell lines HuH-7 and JHH-6. A. Cross-talk between the HCC cells lines and the CAF. B. Cross-talk between the HCC cells lines and the NTF. Target mRNA expression was normalized to reference genes RNA18S and ACTB. CTRL = cells control without co-culture (=1.00). Target mRNA expressions were normalized to reference genes 18SRNA and ACTB. *p < 0.05 compared to each control.
Mentions: After co-culture, we observed a significant effect of the presence of both CAF and NTF in HCC cell lines. In the presence of the CAF, the up-regulations of TGFB1 and FAP were observed in both HCC cell lines (p < 0.05), a slight up-regulation of ACTA2 was observed in HuH-7. On the other hand, the presence of HuH-7 cells increased the expressions of ACTA2 and COL1 in CAF, while JHH-6 cells did not (Figure 3A).Figure 3

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