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Establishment and characterization of a novel primary hepatocellular carcinoma cell line with metastatic ability in vivo.

Cheung PF, Yip CW, Ng LW, Lo KW, Wong N, Choy KW, Chow C, Chan KF, Cheung TT, Poon RT, Fan ST, Cheung ST - Cancer Cell Int. (2014)

Bottom Line: The cell line was authenticated by short tandem repeat analysis.Sequence analysis revealed p53 gene mutation.The newly established cell line could serve as useful in vitro and in vivo models for studying primary HCC that possess metastasis ability.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, The University of Hong Kong, Hong Kong, China ; Center for Cancer Research, The University of Hong Kong, Hong Kong, China.

ABSTRACT

Background: Hepatocellular carcinoma (HCC) is a highly aggressive and heterogeneous disease. HCC cell lines established from different patients would be useful in elucidating the molecular pathogenesis. However, success of HCC primary culture establishment remains at low rate. We aim to establish and characterize HCC primary culture and the derived cell line.

Methods: Fresh tumor tissues were collected from 30 HCC patients. Culture conditions were optimized for the attachment and growth of the isolated hepatocytes. Granulin-epithelin precursor (GEP), a growth factor reported to associate with cancer stem cell properties, was examined by flow cytometry to elucidate its role on primary culture establishment. The primary cell line was characterized in detail.

Results: Cells isolated from 16 out of 30 HCC cases (53%) had viability more than 70% and were subject to subsequent in vitro culture. 7 out of 16 cases (44%) could give rise to cells that were able to attach and grow in culture. GEP expression levels significantly correlated with the viability of isolated hepatocytes and success rate of subsequent primary culture establishment. Cells from HCC patient 21 grew and expanded rapidly in vitro and was selected to be further characterized. The line, designated HCC21, derived from a Hong Kong Chinese female patient with HCC at Stage II. The cells exhibited typical epithelial morphology and expressed albumin, AFP and HBV antigens. The cell line was authenticated by short tandem repeat analysis. Comparative genome hybridization analysis revealed chromosomal loss at 1p35-p36, 1q44, 2q11.2-q24.3, 2q37, 4q12-q13.3, 4q21.21-q35.2, 8p12-p23, 15q11.2-q14, 15q24-q26, 16p12.1-p13.3, 16q, 17p, 22q and gain at 1q21-q43 in both HCC21 cells and the original clinical tumor specimen. Sequence analysis revealed p53 gene mutation. Subcutaneous injection of HCC21 cells into immunodeficient mice showed that the cells were able to form tumors at the primary injection sites and metastatic tumors in the peritoneal cavity.

Conclusions: The newly established cell line could serve as useful in vitro and in vivo models for studying primary HCC that possess metastasis ability.

No MeSH data available.


Related in: MedlinePlus

DNA fingerprinting of HCC21 cells. Comparison of genotyping results in adjacent non-tumor liver tissue and tumor specimen from patient, and HCC21 cells at passage 11 and 50. Patient’s tumor specimen and cultured HCC21 cells demonstrated loss of heterozygosity in (A) D16S539 and (B) FGA when compared to adjacent non-tumor liver tissue.
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Fig4: DNA fingerprinting of HCC21 cells. Comparison of genotyping results in adjacent non-tumor liver tissue and tumor specimen from patient, and HCC21 cells at passage 11 and 50. Patient’s tumor specimen and cultured HCC21 cells demonstrated loss of heterozygosity in (A) D16S539 and (B) FGA when compared to adjacent non-tumor liver tissue.

Mentions: The DNA samples of HCC21 early and late passages (11 and 50, respectively), original tumor and adjacent non-tumor liver tissue were subjected to DNA fingerprinting analysis. A total of 15 STR loci (CSF1P0, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPOX, vWA) were co-amplified in each sample. AMLEO locus at the sex chromosomes was also examined. The data were analyzed and allele(s) of each locus were determined (Table 1). The STR profiles of HCC21 cells and the original tumor were identical, suggesting that they came from the same person. Besides, loss of heterozygosity (LOH) was observed in 2 loci, D16S539 and FGA in original tumor, and the aberrations were retained in HCC21 cells (Figure 4).Table 1


Establishment and characterization of a novel primary hepatocellular carcinoma cell line with metastatic ability in vivo.

Cheung PF, Yip CW, Ng LW, Lo KW, Wong N, Choy KW, Chow C, Chan KF, Cheung TT, Poon RT, Fan ST, Cheung ST - Cancer Cell Int. (2014)

DNA fingerprinting of HCC21 cells. Comparison of genotyping results in adjacent non-tumor liver tissue and tumor specimen from patient, and HCC21 cells at passage 11 and 50. Patient’s tumor specimen and cultured HCC21 cells demonstrated loss of heterozygosity in (A) D16S539 and (B) FGA when compared to adjacent non-tumor liver tissue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4209051&req=5

Fig4: DNA fingerprinting of HCC21 cells. Comparison of genotyping results in adjacent non-tumor liver tissue and tumor specimen from patient, and HCC21 cells at passage 11 and 50. Patient’s tumor specimen and cultured HCC21 cells demonstrated loss of heterozygosity in (A) D16S539 and (B) FGA when compared to adjacent non-tumor liver tissue.
Mentions: The DNA samples of HCC21 early and late passages (11 and 50, respectively), original tumor and adjacent non-tumor liver tissue were subjected to DNA fingerprinting analysis. A total of 15 STR loci (CSF1P0, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPOX, vWA) were co-amplified in each sample. AMLEO locus at the sex chromosomes was also examined. The data were analyzed and allele(s) of each locus were determined (Table 1). The STR profiles of HCC21 cells and the original tumor were identical, suggesting that they came from the same person. Besides, loss of heterozygosity (LOH) was observed in 2 loci, D16S539 and FGA in original tumor, and the aberrations were retained in HCC21 cells (Figure 4).Table 1

Bottom Line: The cell line was authenticated by short tandem repeat analysis.Sequence analysis revealed p53 gene mutation.The newly established cell line could serve as useful in vitro and in vivo models for studying primary HCC that possess metastasis ability.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, The University of Hong Kong, Hong Kong, China ; Center for Cancer Research, The University of Hong Kong, Hong Kong, China.

ABSTRACT

Background: Hepatocellular carcinoma (HCC) is a highly aggressive and heterogeneous disease. HCC cell lines established from different patients would be useful in elucidating the molecular pathogenesis. However, success of HCC primary culture establishment remains at low rate. We aim to establish and characterize HCC primary culture and the derived cell line.

Methods: Fresh tumor tissues were collected from 30 HCC patients. Culture conditions were optimized for the attachment and growth of the isolated hepatocytes. Granulin-epithelin precursor (GEP), a growth factor reported to associate with cancer stem cell properties, was examined by flow cytometry to elucidate its role on primary culture establishment. The primary cell line was characterized in detail.

Results: Cells isolated from 16 out of 30 HCC cases (53%) had viability more than 70% and were subject to subsequent in vitro culture. 7 out of 16 cases (44%) could give rise to cells that were able to attach and grow in culture. GEP expression levels significantly correlated with the viability of isolated hepatocytes and success rate of subsequent primary culture establishment. Cells from HCC patient 21 grew and expanded rapidly in vitro and was selected to be further characterized. The line, designated HCC21, derived from a Hong Kong Chinese female patient with HCC at Stage II. The cells exhibited typical epithelial morphology and expressed albumin, AFP and HBV antigens. The cell line was authenticated by short tandem repeat analysis. Comparative genome hybridization analysis revealed chromosomal loss at 1p35-p36, 1q44, 2q11.2-q24.3, 2q37, 4q12-q13.3, 4q21.21-q35.2, 8p12-p23, 15q11.2-q14, 15q24-q26, 16p12.1-p13.3, 16q, 17p, 22q and gain at 1q21-q43 in both HCC21 cells and the original clinical tumor specimen. Sequence analysis revealed p53 gene mutation. Subcutaneous injection of HCC21 cells into immunodeficient mice showed that the cells were able to form tumors at the primary injection sites and metastatic tumors in the peritoneal cavity.

Conclusions: The newly established cell line could serve as useful in vitro and in vivo models for studying primary HCC that possess metastasis ability.

No MeSH data available.


Related in: MedlinePlus