Research ArticleExperimental Studies

New Sarcomatoid Cancer Cell Line SAR-HCV Established from a Hepatitis C Virus-related Liver Tumour Lesion

DAR-IN TAI, YA-JU SHEN, WEN-HUI WENG, HUI-WEN CHEN, CHEN-CHEN KANG, TSE-CHING CHEN and SHUEN-KUEI LIAO
Anticancer Research January 2011, 31 (1) 129-137;

Abstract

A sarcomatoid carcinoma cell line (SAR-HCV) was established from a malignant liver lesion of a patient infected with hepatitis C virus. SAR-HCV cells were successfully xenografted in SCID mice. Vimentin was strongly positive in cultured SAR-HCV cells, the primary tumour lesion and the xenografts. Hepatocyte paraffin 1 protein and certain cytokeratin markers, CK8, CK18 and AE1/AE3 were not detected in cultured cells, but were focally positive in the tumour lesion and xenografts, suggesting that this cancer cell line preserves some features of hepatocyte differentiation when grown in vivo. HLA class I, N-cadherin, vascular endothelial growth factor, CD44, and heat-shock protein 70 were moderately expressed in this cell line. Spectral karyotyping analysis revealed a nearly triploid karyotype, 34-63<3n>, XXY[12] with complicated genetic abnormalities of chromosomal structure in all metaphases examined. This cell line will be useful in further studying hepato-sarcomatoid carcinoma cells and in understanding carcinogenesis and epithelial–mesenchymal transition in hepatitis C virus-related liver tumour.

Hepatitis C virus (HCV) is a RNA virus that is distributed worldwide. Chronic infection with HCV is one of the important causes of hepatocellular carcinoma (HCC) (1-3). According to the World Health Organization (WHO), approximately 170 million people are chronically infected with HCV (4). In a nationwide prospective survey of relatives of HCC patients in Taiwan, the annual HCC-related mortality in anti-HCV seropositive relatives was 0.8% after age 55 years, which was higher than that of hepatitis B surface antigen seropositive relatives (0.25% per year after age 35 years) (5). The mechanism of HCV-related hepatocarcinogenesis is not yet clear, but could be related to increased oxidative stress by repeated liver inflammation and disturbance of metabolic pathways (6-10). HCV-related liver tumours may be histologically different from other liver cancer cell types, including HCC, cholangiocarcinoma, and hepatocholangiocarcinoma (11, 12). Some of these neoplasms may show sarcomatous changes (13), which may be closely associated with epithelial–mesenchymal transition (EMT), implicating neoplastic progression (14, 15). In total, only about 40 to 50 patients with sarcomatoid liver tumours have been reported in the English medical literature (16-20). However, most cases of sarcomatoid liver tumours may have gone unreported. The prognosis of patients with these sarcomatoid tumours was generally poor because of their highly invasive nature (19). To improve our understanding of the characteristics of hepato-sarcomatoid tumours, we established and carried out an initial characterization of a sarcomatoid cancer cell line from a liver lesion of a patient with HCV-related liver tumour.

Materials and Methods

Patient history. A 68-year-old man was admitted to Chang Gung Memorial Hospital, Taoyuan, Taiwan, in March 1997, with chief complaints of severe right upper quadrant abdominal pain and yellowish discolouration of the skin for 4 days. Both ultrasonography and computed tomography (Figure 1a) revealed a mass in the liver hilum with dilated intrahepatic bile ducts. Assay for hepatitis B surface antigen (HBsAg) was negative, but was positive for hepatitis C virus antibody (anti-HCV) (Abbott laboratories). Both alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) serum levels were within the upper limit of normal. The CA 19.9 serum level was 55.8 U/ml. A sonographically guided core-needle tumour biopsy was carried out, and pathology showed sarcomatoid carcinoma with features of epithelioid and spindle cells components with focal necrosis (Figure 1b). These tumour cells disclosed a strongly positive vimentin stain (Figure 1c) and weakly positive cytokeratin AE1/AE3 stain (Figure 1d) on immunohistochemistry. Culture of the tumour cells was initiated with a piece of tumour taken by tumour needle biopsy. The patient received percutaneous transhepatic biliary drainage, but died of infection two months later.

Cell line establishment and culture conditions. For the establishment of the SAR-HCV cell line, a single-cell suspension was prepared from the tumour tissue by mechanical mincing. Cells were seeded in culture dishes with a 56.7 cm2 culture area (Nunc, Rockilde, Denmark) and incubated at 37°C in a humidified atmosphere containing 5% CO2 and 95% air. A medium previously optimized for the growth of other solid tumours was used, namely RPMI-1640 supplemented with 2 mM L-glutamine, 10 mM HEPES buffer, 10% heat-inactivated fetal bovine serum (FBS), penicillin (50 U/ml) and streptomycin (50 μg/ml). When cultures were subjected to subculturing, attached cells were dissociated by trypsinization and, after washing in complete growth medium, the cells were expanded to T25 or T75 flasks or dishes. Intermittently, cells in some cultures were frozen in 90% FBS and 10% dimethyl sulfoxide in liquid nitrogen.

Growth curves of the SAR-HCV cells. Growth curves, each covering a total of five days of culturing without change of medium, were constructed, from which the population doubling time and saturation density were determined. For each time point, triplicate culture dishes were removed from the incubator for cell counts for both viable and non-viable cells.

Determination of nuclear DNA content by flow cytometry. The DNA contents of nuclei from cultured SAR-HCV cells and normal adult peripheral blood mononuclear cells (PBMCs) were analysed according to a method described previously (21). The DNA index relative to normal PBMCs was calculated.

Protein secretion and measurements. After SAR-HCV cells were maintained in culture for 4 days, CEA, AFP, CA19.9, albumin, hepatoglobulin, transferrin and ferritin, possibly secreted in spent medium by the tumour cells were measured by commercially available enzyme-linked immunosorbent assays (ELISA) using appropriate test kits. These measurements were conducted at the Clinical Chemistry Laboratory, Chang Gung Memorial Hospital, Taoyuan, Taiwan. HCV RNA was studied by reverse transcription-polymerase chain reaction (RT-PCR; AMPLICOR HCV test version 2.0; Roche Diagnostic System Inc., Branchburg, NJ, USA). For each time point, triplicate samples were examined.

Immunophenotyping of SAR-HCV cells. The information of hybridoma clones and sources of murine monoclonal antibodies (mAbs) is listed in Table I. Most of these mAbs in conjunction with cytofluorometric analysis were used for immunophenotyping of the SAR-HCV cells. Some mAbs were also used in immunocytochemistry on chamber slide cell culture and immunocytochemistry on tumour tissue sections.

For surface or cytoplasmic immunofluorescence and flow cytometric analysis, monodispersed cells harvested from cell cultures were dispensed into test tubes (5×106 cells/ml PBS/tube) and centrifuged at 400× g to pellet the cells. Test antibodies (5 μg/ml) were added (100 μl/tube) and the mixture incubated at 4°C for 30 min. Immunofluorescence-based detection of surface antigen (use of unfixed live cells) was performed with various antibodies, including appropriate positive and negative controls, followed by flow cytometric analysis using a method described previously (22). For cytoplasmic antigens, the cell pellets were first treated with 1% paraformaldehyde at 4°C for 20 min and then with cold acetone for 3 min (23). This step allowed the immunoreagents and washing solution to penetrate through the cell membrane. Positive (anti-HLA-A,B,C W6/32) and negative (PBS in place of primary antibody, isotype-matched irrelevant mAb, or polyclonal goat anti-mouse IgG antibodies with prior addition of test primary antibody) controls were included in each test.

Northern blot analysis of L-ferritin and H-ferritin mRNAs in SAR-HCV cells. Total RNAs were extracted from SAR-HCV and HepG2 cells using TRI Reagent® (Molecular Research Center, Cincinnati, USA), and the isolated RNAs were subjected to electrophoresis on 1% agarose gel containing formaldehyde. After transferring onto nylon membranes followed by fixation by ultraviolet irradiation, the RNAs were glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (as an internal loading control) for at least 1 h at 42°C overnight, according to the method described by Pang et al. (24). Blots were then washed with buffer containing 2× SSC (1× SSC consisted of 150 mM NaCl, 15 mM Na-citrate, pH 7.0) and 1% sodium dodecyl sulfate (SDS) for 30 min at 50°C followed by wash with buffer containing 0.2× SSC and 1% SDS at 50°C for another 30 min. The blot was covered by Saran wrap to prevent drying and exposed to Kodak XAR-5 film at −70°C using a Kodak intensifying screen.

Chromosome preparation and spectral karyotyping (SKY). SKY analysis was performed according to the manufacturer's instructions with modification as previously described (Applied Spectral Imaging, Migdal Haemek, Israel) (25). In brief, after standard culturing of cell lines, chromosome spreads were prepared for performing SKY analysis. The chromosome-specific painting probes were generated using a mixture of five different fluorochromes and excess Cot-1 DNA. After hybridization, all images were analyzed using a SD200 Spectracube system (Applied Spectral Imaging) mounted on a Leica DM2500 microscope with a custom-designed optical filter (SKY-1; Chroma Technology, Brattleboro, VT, USA). After capture, twenty-four unique colours for 22+X,Y pair chromosomes are provided by fluorochromes spectral viewer. The clonality criteria and the karyotype description follow the recommendations of the International System for Human Cytogenetic Nomenclature (ISCN 2005) (26).

Tumourigenicity in immunodeficient mice. Tumourigenicity assay of the SAR-HCV cell line was carried out in mice with severe combined immunodeficiency (SCID, CB.17) (n=6). Six-week-old female SCID mice were purchased from the Central Animal Facility of the National Taiwan University Hospital, Taipei, Taiwan. The experiments were conducted upon approval from the Animal Ethics Committee, Chang Gung University, Taoyuan, Taiwan. Single-cell suspension of SAR-HCV cells were collected by light trypsinization and washing in phosphate-buffered saline (PBS). The populations of cells were counted and 5×106 or 5×107 cells/0.1 ml PBS were injected subcutaneously at a site above the hind leg of each mouse. The animals were examined twice per week for a period of 80 days to estimate the growth of tumours. The volume of palpable tumour nodules was estimated according to the formula: volume (mm3)=0.4×a×b2, where a is the major tumour diameter and b is the minor diameter perpendicular to the major one (27).

View this table:
Table I.

Monoclonal antibodies used in this study.

Results

Morphology and in vitro growth of the cell line. Representative morphology of SAR-HCV cells grown as monolayers in culture is depicted in Figure 2 left panel. The cells were mostly spindle and elongated in shape, with occasional flat protruding polygonal parts and individually gave a typical fibroblastic morphology. However, unlike normal fibroblast growth in culture, SAR-HCV cells neither contacted closely one another, nor grew in a swirling pattern. They were all adherent but scattered, a cellular morphology being very different from that of normal hepatocytes and hepatocellular carcinoma, whether in an exponential or a saturated phase of monolayer culture conditions.

In vitro growth curve of SAR-HCV cells. A growth curve was constructed from SAR-HCV cells seeded at day 0 at 105/well of a 6-well dish (area/well 7.1 cm2) in triplicate per time point. Cell numbers increased progressively after seeding, which reached a plateau at day 5. The in vitro growth of SAR-HCV cells was characterized by a population doubling time of 33.2 h and a saturation density of 6.8×105/cm2 (Figure 2, right panel). It was noted that from day 2, the cell viability dropped gradually as the cell number increased as a function of time. At day 5, cell viability dropped to around 60%. Similar results were obtained with a repeated experiment.

Figure 1.
Figure 1.

Characteristics of the primary liver sarcomatoid carcinoma under investigation. a: A computed tomography of the malignant liver revealed a mass in the liver hilum with dilated intrahepatic bile ducts. b: Many spindle tumour cells were found in tumour sections following H&E staining (×400). c: Vimentin staining was weakly positive in most tumour cells (×400). d: Cytokeratin (AE1/AE3) staining was focally positive (×400).

Figure 2.
Figure 2.

Morphology and growth curve for the SAR-HCV cell line. Left panel: Morphology of SAR-HCV cells in monolayer culture. The cells attach to the flask with star-like protrusions. Right panel: A typical in vitro growth curve of SAR-HCV cells in which the total viable cell number/dish and % cell viability at each time point are indicated.

View this table:
Table II.

Immunophenotyping results of the SAR-HCV cell line as determined by cytofluorometric analysis.

Cell/tumour markers and virology studies. CEA, AFP, CA19.9, albumin, hepatoglobin and transferrin protein were absent from culture supernatants of SAR-HCV cells, but ferritin was detected with a concentration up to 117.2 μg/ml. By Northern blot analysis, the presence of ferritin transcript in SAR-HCV cells was also demonstrated by showing their ferritin subunits, heavy and light chain-mRNAs, using the human hepatoblastoma HepG2 cell line, which is known to express ferritin, as a positive control cell line (Figure 3). However, HCV RNA was not detected in the spent medium and cell lyses of cultured SAR-HCV cells.

Figure 3.
Figure 3.

Ferritin transcript of SAR-HCV cells as revealed by Northern blot analysis. The expression of both heavy (left panel) and light (right panel) chain mRNAs from SAR-HCV cells and HepG2 cells, a human hepatoblastoma cell line known to express ferritin (left), is illustrated.

Immunophenotyping analysis of SAR-HCV cells. Cytofluorometric study was conducted on SAR-HCV cells in dispersed single-cell suspension (Table II). HLA class I was moderately positive in both cell surface and cytoplasm. Many epithelial markers including AE1/AE3, CK20, EMA, EpCAM, E-cadherin, Sialyl-Tn, Ley, BH8.23 and CEA were not present, whereas two cytoplasmic markers vimentin and N-cadherin, as well as VEGF and HSP70, were moderately positive. Other markers, such as FASL, BCL-2, GRP78 (28), CXCR4 (a receptor for the chemokine CXCL12) and CD44v6 were all negative. CD44s, CD44v5 and CXCL12 were found to be expressed at low levels on the cell surface, but abundantly in the cytoplasm.

SAR-HCV cells grown as monolayers on chamber slides were also tested by immunocytochemistry using some of the mAb panel to the various markers/antigens used in cytofluorometry. Similar results to cytofluorometric analysis in terms of positivity and negativity were obtained. Examples of staining patterns as a result of such analyses are depicted in Figure 4: SAR-HCV cells were stained with mAbs to HLA class I (a), CD54 (ICAM-1) (b), MAK-6 (c) and vimentin (d). Vimentin was observed to be more uniformly distributed in the cytoplasm of most cells tested. A rather heterogeneous staining pattern was seen with HLA class I and CD54 in all cells, while only focal staining was seen with MAK-6. Of note is that the flattened appearance of most tumour cells after staining, which was quite different from that of live fibroblastic cells grown as monolayers (Figure 2 left panel), was likely due to fixation with acetone and the distribution of each specific antigen detected.

Tumourigenicity in SCID mice. Tumour nodules at the injection sites were found 3 months after subcutaneous inoculation in three out of six mice inoculated with 5×106 cells. The tumour volumes of the three resulting tumours 6 months following inoculation were 3322, 3582 and 2415 mm3, respectively. No apparent tumour lesions in the visceral organs were found in these six animals. For an additional three mice subcutaneously inoculated with 1×107 cultured cells of recovered from the xenografts, tumour nodules were developed in all of the three mice with a tumour volume of (3846.7±599.6) mm3 4 months following inoculation.

Figure 4.
Figure 4.

Immunocytochemistry of SAR-HCV cells grown as monolayers in wells of chamber slides. Cells in duplicate wells were stained with monoclonal antibodies to HLA class I (a), CD54 (ICAM-1) (b), MAK-6 (combination of cytokeratins; see Materials and Methods) (c), and vimentin (d).

Figure 5.
Figure 5.

Immunohistochemistry of SAR-HCV xenograft tissue sections. a: The morphology of the tumour in xenograft sections looks like pleomorphic hepatocytes with many mitoses and prominent nucleoli (H&E stain). b: Vimentin staining was diffusely and strongly positive. c: Cytokeratin AE1/AE3 staining was weakly positive. d: Hepatocyte paraffin 1 staining was focally and weakly positive. Magnification for all four figures ×400.

Figure 6.
Figure 6.

Karyotyping of the SAR-HCV cell line analysed in this study. One representative metaphase in chromosomal spread of classification color with the details of the multiple chromosome abnormalities including translocations were found (upper panel). An arranged SKY karyotype in pseudo colour (lower panel).

The tumours were fixed with formalin and subjected to immunohistochemistry studies. In H&E staining, the cells generally appeared to be pleomorphic, with scattered giant cells (Figure 5a). Only a small proportion of tumour cells were found to be spindle-shaped. Multiple mitotic cells and cells with prominent nucleoli were noted. Vimentin stain was strongly positive (Figure 5b), while that for cytokeratin AE1/AE3 and hepatocyte paraffin 1 were focally positive (Figure 5c, d). Staining for CK8 and CK18 was focally positive, while that for CK7 and CK19 was only weakly positive (results not shown).

DNA content. Analysis of the DNA content from the SAR-HCV cell line by flow cytometry indicated that this cell line contained one major aneuploid population of cells with a DNA index of 1.6 relative to the DNA content of human PBMCs from a healthy adult.

Cytogenetic studies. Karyotyping by SKY analysis showed both structural and numerical chromosomal abnormalities (Figure 6). Consistent with the finding of the DNA content, SKY results revealed a nearly triploid karyotype, presenting 34-63<3n>, XXY[12], with complicated genetic abnormalities of chromosomal structure in most metaphases/cells examined. Specifically, of 12 chromosomal spreads examined, at least two clones could be identified in this cell line. The major alterations, including the translocations der(13)t(10;7;13) (q22;p?;q10), der(15)t(15;16)(q10;?), der(2)t(2;?)(q10;?), der(6)t(6;8)(q10;p11.2), were observed in more than 10 metaphase spreads. Furthermore, the additional aberrations, such as der(6)t(6;22)(q10;?), der(9)t(7;9)(p11;q10), der(12) t(6;12)(p12;q10), der(16)t(10;16)(p13;q21) and der(17) t(4;8;17)(?;p?;q10), were also detected in 9 out of 12 metaphases examined. Hence, one copy number gain in chromosomes 11 and 17, and two copy losses on chromosomes 1, 7, 8, 13, 14, 15, 16 and 22 were noted.

Discussion

In this study, we established the SAR-HCV cancer cell line from an HCV-related sarcomatoid liver tumour. These cancer cells showed sarcomatoid and epithelial features in the primary tumour lesion, as well as in the xenografts in SCID mice resulting from subcutaneous injection of cultured SAR-HCV cells.

Vimentin, one of the characteristic proteins of mesenchymal cells, was stained positively in SAR-HCV cells, in the primary tumour lesion, as well as in the xenografts. Many spindle cells were found in the primary tumour, but were not so obvious in the xenografts, suggesting that this cell line may display different growth behaviours in different environments. The expression of CK8, CK18, AE1/AE3 and hepatocyte paraffin 1 protein tends to suggest that this cell line has preserved some features of hepatocyte differentiation. The fact that AE1/AE3 and many other normal epithelial/ epithelial tumour markers, such as EMA, MAK-6, CK7, CK20, EpCAM, Siayl-Tn and Ley were found to be lacking from SAR-HCV cells in culture (from both before and after SCID mouse-passage) leads us to believe that only those fibroblastic cells from in vivo tumours tend to grow and be maintained in vitro under our current culture conditions. N-Cadherin and vimentin are strongly expressed in SAR-HCV cells and are important mesenchymal markers related to EMT, in which events such as cell migration and metastasis are known to be highly associated (14, 15). Such an expression profile generally suggests a high metastatic potential and poor prognosis for patients with sarcomatoid liver tumour (19).

Our cytogenetic findings of the SAR-HCV cell line revealed highly complicated karyotypes of chromosomal alterations, with the near triploid karyotype being characteristic of all the tumour cells. At least two clones were identified, and such a heterozygous characteristic is consistent with the cancer cell features. Obviously, several translocations occurred in both clones of this cell line, although triploid clones were identical. These two clones tended to show a pattern of chromosome gain with extra copies of chromosomes 11 and 17, and in a few cells there were two copy losses on chromosomes 1, 7, 8, 13, 14, 15, 16 and 22, which resulted in haploidy for the chromosomes. Apparently, the presence of non-random alterations observed in each clone was most likely the primary event, while the change in other random structural abnormalities was probably a secondary event (Figure 5). Although the SKY analysis indicated the presence of several non-random translocations, none of them are as yet known to be related to the HCC prognostic impact. Therefore, to improve the classification of the disease one would need to look at translocation data rather than ploidy patterns in order to assign more correct prognostic implications for the improvement of disease classification. Further cytogenetic studies of the disease stages of additional similar clinical cases are needed in the future.

As yet, HCV-related cancer cell lines are still rarely available (29). HCV is the only known carcinogenetic RNA virus with an exclusively cytoplasmic life cycle (30). The mechanisms by which it causes cancer are unclear, but chronic inflammation and alternated metabolic pathway-associated oxidative chromosomal DNA damage probably play a role (6-10). Multiple and complicated genetic alterations found in this study are consistent with a HCV-related tumour cell line reported by Yoshizawa et al. (29). They also identified many genetic alterations in their HCV-related cancer cell lines. The complicated genetic alternations in HCV-related tumour cell lines may support the oxidative chromosomal DNA damage theory. The extreme chromosomal DNA alterations were also correlated to this poorly differential cell line. The SAR-HCV cell line may be useful in a better understanding of HCV-related hepatocarcinogenesis and further characterization of hepato-sarcomatoid carcinoma.

Acknowledgements

This study was supported in part by grants to D.-I. Tai from the Chang Gung Medical Research Fund (CMRPG380261) and to S.-K. Liao from the National Science Council of Taiwan (NSC96-2314-B-182-017). We wish to thank Hung-Chang Chen and Chin-Hsuan Hsieh (Graduate Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan) for illustrations and editorial work.

  • Received September 10, 2010.
  • Revision received November 23, 2010.
  • Accepted November 24, 2010.

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New Sarcomatoid Cancer Cell Line SAR-HCV Established from a Hepatitis C Virus-related Liver Tumour Lesion
DAR-IN TAI, YA-JU SHEN, WEN-HUI WENG, HUI-WEN CHEN, CHEN-CHEN KANG, TSE-CHING CHEN, SHUEN-KUEI LIAO
Anticancer Research Jan 2011, 31 (1) 129-137;
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