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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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Xenograft assay of the CAF. A. Tissue mass in the liver and lung of the NOD/SCID mouse following orthotopic injection of the CAF from HCC. Arrow indicates nodules. B. Primary cells of the nodules in the orthotopic xenografts. Scale bar, 100 μm. C. The positivity of genes in the xenograft primary cells. H = hepatic, L = lung, C + H = positive control human (human liver), C + M = positive control mouse (mouse liver). Gene 18S is homolog for human and mouse species. D. DNA sequencing of human AFP of primary cells obtained from the lung of xenograft. REF_AFP = DNA sequences of AFP genes from injected CAF, XG_AFP = DNA sequences of AFP genes from primary cultures of xenograft, NM_001134.1 = reference DNA sequences from GenBank.
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Fig4: Xenograft assay of the CAF. A. Tissue mass in the liver and lung of the NOD/SCID mouse following orthotopic injection of the CAF from HCC. Arrow indicates nodules. B. Primary cells of the nodules in the orthotopic xenografts. Scale bar, 100 μm. C. The positivity of genes in the xenograft primary cells. H = hepatic, L = lung, C + H = positive control human (human liver), C + M = positive control mouse (mouse liver). Gene 18S is homolog for human and mouse species. D. DNA sequencing of human AFP of primary cells obtained from the lung of xenograft. REF_AFP = DNA sequences of AFP genes from injected CAF, XG_AFP = DNA sequences of AFP genes from primary cultures of xenograft, NM_001134.1 = reference DNA sequences from GenBank.

Mentions: Two CAFs were subjected to xenograft assay. The viability of the cells was more than 95%. Four months after subcutaneous injection in nude mice, the serum level of ALT and AST of xenografts were slightly increased compared to control (102 ± 23 IU vs. 77 ± 9 IU for ALT and 23 ± 2 IU vs. 17 ± 1 IU for AST) even though no tumor nodules were observed. Orthotopic injection in NOD/SCID mice resulted in the appearance of nodules in the liver as well as in the lung and thymus. Cultured primary cells of the nodules and the injected sites expressed liver-specific markers ALB and AFP, including those of human, as confirmed by DNA sequencing with reference to DNA sequences of the CAF (Figure 4). However, we could not notice the human CD90 protein. The positivity of human and murine genes of xenograft models is listed on Table 2. No difference in body weight of all animals was noticed (data not shown).Figure 4


The role of multipotent cancer associated fibroblasts in hepatocarcinogenesis.

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

Xenograft assay of the CAF. A. Tissue mass in the liver and lung of the NOD/SCID mouse following orthotopic injection of the CAF from HCC. Arrow indicates nodules. B. Primary cells of the nodules in the orthotopic xenografts. Scale bar, 100 μm. C. The positivity of genes in the xenograft primary cells. H = hepatic, L = lung, C + H = positive control human (human liver), C + M = positive control mouse (mouse liver). Gene 18S is homolog for human and mouse species. D. DNA sequencing of human AFP of primary cells obtained from the lung of xenograft. REF_AFP = DNA sequences of AFP genes from injected CAF, XG_AFP = DNA sequences of AFP genes from primary cultures of xenograft, NM_001134.1 = reference DNA sequences from GenBank.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4389787&req=5

Fig4: Xenograft assay of the CAF. A. Tissue mass in the liver and lung of the NOD/SCID mouse following orthotopic injection of the CAF from HCC. Arrow indicates nodules. B. Primary cells of the nodules in the orthotopic xenografts. Scale bar, 100 μm. C. The positivity of genes in the xenograft primary cells. H = hepatic, L = lung, C + H = positive control human (human liver), C + M = positive control mouse (mouse liver). Gene 18S is homolog for human and mouse species. D. DNA sequencing of human AFP of primary cells obtained from the lung of xenograft. REF_AFP = DNA sequences of AFP genes from injected CAF, XG_AFP = DNA sequences of AFP genes from primary cultures of xenograft, NM_001134.1 = reference DNA sequences from GenBank.
Mentions: Two CAFs were subjected to xenograft assay. The viability of the cells was more than 95%. Four months after subcutaneous injection in nude mice, the serum level of ALT and AST of xenografts were slightly increased compared to control (102 ± 23 IU vs. 77 ± 9 IU for ALT and 23 ± 2 IU vs. 17 ± 1 IU for AST) even though no tumor nodules were observed. Orthotopic injection in NOD/SCID mice resulted in the appearance of nodules in the liver as well as in the lung and thymus. Cultured primary cells of the nodules and the injected sites expressed liver-specific markers ALB and AFP, including those of human, as confirmed by DNA sequencing with reference to DNA sequences of the CAF (Figure 4). However, we could not notice the human CD90 protein. The positivity of human and murine genes of xenograft models is listed on Table 2. No difference in body weight of all animals was noticed (data not shown).Figure 4

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