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Possible Regulation of Migration of Intrahepatic Cholangiocarcinoma Cells by Interaction of CXCR4 Expressed in Carcinoma Cells with Tumor Necrosis Factor-α and Stromal-Derived Factor-1 Released in Stroma

2006; Elsevier BV; Volume: 168; Issue: 4 Linguagem: Inglês

10.2353/ajpath.2006.050204

ISSN

1525-2191

Autores

Shusaku Ohira, Motoko Sasaki, Kenichi Harada, Yasunori Sato, Yoh Zen, Kumiko Isse, Kazuto Kozaka, Akira Ishikawa, Koji Oda, Yuji Nimura, Yasuni Nakanuma,

Tópico(s)

Peptidase Inhibition and Analysis

Resumo

Intrahepatic cholangiocarcinoma (ICC) is highly fatal because of early invasion, widespread metastasis, and lack of an effective therapy. We examined roles of CXCR4 and its ligand, stromal cell-derived factor (SDF)-1, in migration of ICC with respect to tumor-stromal interaction by using two ICC cell lines, a fibroblast cell line (WI-38), and 28 human ICC tissues. The two ICC cell lines expressed CXCR4 mRNA and protein, and WI-38 fibroblasts expressed SDF-1 mRNA and protein. Migration of cultured ICC cells in Matrigel was induced by co-culture with WI-38 fibroblasts and by incubation with SDF-1. Anti-SDF-1 antibody suppressed migration, demonstrating that SDF-1 released from WI-38 fibroblasts was responsible for this migration. Tumor necrosis factor (TNF)-α pretreatment of ICC cells up-regulated CXCR4 mRNA and protein expression in a concentration-dependent manner. Administration of SDF-1 and TNF-α increased synergistically ICC cell migration, which was suppressed by the CXCR4 antagonist AMD3100. In ICC tissue, TNF-α was mainly expressed in infiltrated macrophages, CXCR4 in ICC cells, and SDF-1 in stromal fibroblasts. In conclusion, the interaction of SDF-1 released from fibroblasts and CXCR4 expressed on ICC cells may be actively involved in ICC migration, and TNF-α may enhance ICC cell migration by increasing CXCR4 expression. CXCR4 could be a therapeutic target to prevent ICC invasion. Intrahepatic cholangiocarcinoma (ICC) is highly fatal because of early invasion, widespread metastasis, and lack of an effective therapy. We examined roles of CXCR4 and its ligand, stromal cell-derived factor (SDF)-1, in migration of ICC with respect to tumor-stromal interaction by using two ICC cell lines, a fibroblast cell line (WI-38), and 28 human ICC tissues. The two ICC cell lines expressed CXCR4 mRNA and protein, and WI-38 fibroblasts expressed SDF-1 mRNA and protein. Migration of cultured ICC cells in Matrigel was induced by co-culture with WI-38 fibroblasts and by incubation with SDF-1. Anti-SDF-1 antibody suppressed migration, demonstrating that SDF-1 released from WI-38 fibroblasts was responsible for this migration. Tumor necrosis factor (TNF)-α pretreatment of ICC cells up-regulated CXCR4 mRNA and protein expression in a concentration-dependent manner. Administration of SDF-1 and TNF-α increased synergistically ICC cell migration, which was suppressed by the CXCR4 antagonist AMD3100. In ICC tissue, TNF-α was mainly expressed in infiltrated macrophages, CXCR4 in ICC cells, and SDF-1 in stromal fibroblasts. In conclusion, the interaction of SDF-1 released from fibroblasts and CXCR4 expressed on ICC cells may be actively involved in ICC migration, and TNF-α may enhance ICC cell migration by increasing CXCR4 expression. CXCR4 could be a therapeutic target to prevent ICC invasion. Intrahepatic cholangiocarcinoma (ICC) is the most frequent primary malignant liver tumor next to hepatocellular carcinoma and is highly fatal because of early invasion, widespread metastasis, and the lack of an effective therapy.1Okuda K Nakanuma Y Miyazaki M Cholangiocarcinoma: recent progress. Part 2: molecular pathology and treatment.J Gastroenterol Hepatol. 2002; 17: 1056-1063Crossref PubMed Scopus (66) Google Scholar, 2Okuda K Nakanuma Y Miyazaki M Cholangiocarcinoma: recent progress. Part 1: epidemiology and etiology.J Gastroenterol Hepatol. 2002; 17: 1049-1055Crossref PubMed Scopus (190) Google Scholar Whereas several molecules and histological features of ICC are reported to relate to the prognosis of the patients and to other features such as metastasis,3Harada K Masuda S Hirano M Nakanuma Y Reduced expression of syndecan-1 correlates with histologic dedifferentiation, lymph node metastasis, and poor prognosis in intrahepatic cholangiocarcinoma.Hum Pathol. 2003; 34: 857-863Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 4Shimonishi T Miyazaki K Kono N Sabit H Tuneyama K Harada K Hirabayashi J Kasai K Nakanuma Y Expression of endogenous galectin-1 and galectin-3 in intrahepatic cholangiocarcinoma.Hum Pathol. 2001; 32: 302-310Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar the genetic and molecular aspects of its biological behavior, particularly information regarding the mechanisms regulating invasion or migration, remain poor. 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CXCR4 is also involved in the migration and spread of ovarian carcinoma cells.13Scotton CJ Wilson JL Milliken D Stamp G Balkwill FR Epithelial cancer cell migration: a role for chemokine receptors?.Cancer Res. 2001; 61: 4961-4965PubMed Google Scholar Cytokines secreted from malignant cells and mesenchymal/inflammatory cells are also known to regulate the biological activities of malignant cells.11Muller A Homey B Soto H Ge N Catron D Buchanan ME McClanahan T Murphy E Yuan W Wagner SN Barrera JL Mohar A Verastegui E Zlotnik A Involvement of chemokine receptors in breast cancer metastasis.Nature. 2001; 410: 50-56Crossref PubMed Scopus (4568) Google Scholar, 12Wang JM Deng X Gong W Su S Chemokines and their role in tumor growth and metastasis.J Immunol Methods. 1998; 220: 1-17Crossref PubMed Scopus (284) Google Scholar, 14Csiszar A Szentes T Haraszti B Balazs A Petranyi GG Pocsik E The pattern of cytokine gene expression in human colorectal carcinoma.Pathol Oncol Res. 2004; 10: 109-116Crossref PubMed Scopus (41) Google Scholar, 17Scotton CJ Wilson JL Scott K Stamp G Wilbanks GD Fricker S Bridger G Balkwill FR Multiple actions of the chemokine CXCL12 on epithelial tumor cells in human ovarian cancer.Cancer Res. 2002; 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10: 109-116Crossref PubMed Scopus (41) Google Scholar, 24Balkwill F Tumor necrosis factor or tumor promoting factor?.Cytokine Growth Factor Rev. 2002; 13: 135-141Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 25Rosen EM Goldberg ID Liu D Setter E Donovan MA Bhargava M Reiss M Kacinski BM Tumor necrosis factor stimulates epithelial tumor cell motility.Cancer Res. 1991; 51: 5315-5321PubMed Google Scholar, 26Katerinaki E Evans GS Lorigan PC MacNeil S TNF-alpha increases human melanoma cell invasion and migration in vitro: the role of proteolytic enzymes.Br J Cancer. 2003; 89: 1123-1129Crossref PubMed Scopus (77) Google Scholar In nonneoplastic, inflamed intrahepatic bile ducts, SDF-1 is expressed in biliary epithelial cells (BECs).29Terada R Yamamoto K Hakoda T Shimada N Okano N Baba N Ninomiya Y Gershwin ME Shiratori Y Stromal cell-derived factor-1 from biliary epithelial cells recruits CXCR4-positive cells: implications for inflammatory liver diseases.Lab Invest. 2003; 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64: 3517-3524Crossref PubMed Scopus (198) Google Scholar Recently, Park and colleagues33Park J Tadlock L Gores GJ Patel T Inhibition of interleukin 6-mediated mitogen-activated protein kinase activation attenuates growth of a cholangiocarcinoma cell line.Hepatology. 1999; 30: 1128-1133Crossref PubMed Scopus (175) Google Scholar reported that ICC cell lines increased IL-6 secretion in response to TNF-α, and IL-6 is known to induce the proliferation of ICC cells and is a marker of poor prognosis among ICC patients.34Sugawara H Yasoshima M Katayanagi K Kono N Watanabe Y Harada K Nakanuma Y Relationship between interleukin-6 and proliferation and differentiation in cholangiocarcinoma.Histopathology. 1998; 33: 145-153Crossref PubMed Scopus (82) Google Scholar So far, specific paracrine effects of the CXCR4/SDF-1 system and TNF-α in the biological activities of ICC have not been identified. In this study, we examined the roles of the CXCR4/SDF-1 system in ICC during migration with respect to tumor-stromal interactions by using two ICC cell lines, one fibroblast cell line, and 28 human ICC tissues. A total of 28 ICC specimens with enough marginal nontumoral liver tissue were obtained from 28 patients (Table 1). All of these tumors were peripheral ICCs and presented grossly as mass-forming type.1Okuda K Nakanuma Y Miyazaki M Cholangiocarcinoma: recent progress. Part 2: molecular pathology and treatment.J Gastroenterol Hepatol. 2002; 17: 1056-1063Crossref PubMed Scopus (66) Google Scholar, 2Okuda K Nakanuma Y Miyazaki M Cholangiocarcinoma: recent progress. Part 1: epidemiology and etiology.J Gastroenterol Hepatol. 2002; 17: 1049-1055Crossref PubMed Scopus (190) Google Scholar More than three tissue sections containing both the ICC and surrounding nonneoplastic liver were obtained in each case. As a control, six normal autopsied livers with minimal autolytic changes were used, and more than three sections were obtained from each liver. The age and sex distribution were comparable with those of ICC patients. All of these specimens were obtained from the Liver Disease File of the Department of Human Pathology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan, and were fixed in 10% buffered formalin and embedded in paraffin. More than 20 serial sections, 3 μm in thickness, were cut from each paraffin block.Table 1Main Clinicopathological Features of Intrahepatic Cholangiocarcinoma (ICC) and Control Livers Used in This StudyICC cases (28 cases)Control cases (6 cases)Age (years)Average, 69.4 (range, 49 to 85)Average, 70 (range, 55 to 86)Gender (M:F)18:103:3Tissue specimenSurgically resected, 21; autopsy, 7Autopsy, 6Location of ICC in the liverRight hepatic lobe, 14; left hepatic lobe, 14Histological gradingWell differentiated, 2; moderately differentiated, 15; poorly differentiated, 11Tumor size (cm in diameter)5.7 (range, 2 to 14)Background liver histologyNonspecific reactive changeAlmost normal Open table in a new tab Monoclonal and polyclonal antibodies and other immunological reagents and their sources are shown in Table 2. We used a goat polyclonal antibody against SDF-1 (Santa Cruz Biotechnology, Santa Cruz, CA) for immunohistochemistry. Mouse monoclonal antibody against SDF-1 (clone 79014; R&D Systems, Minneapolis, MN) was used as a neutralization antibody for SDF-1.Table 2Antibodies, Antigens, and Other Immunohistological Materials Used in This StudySourceCloneType of antibodyAnimals immunizedOptimal dilutionAnti-TNF-α*Used for immunohistochemical staining.Santa Cruz BiotechnologyPolyGoat (IgG)1:200Anti-SDF-1Santa Cruz BiotechnologyPolyGoat (IgG)1:50Anti-CXCR4Santa Cruz BiotechnologyPolyGoat (IgG)1:50Anti-CD68DAKOKP1MonoMouse (IgG)1:600Anti-mast cell tryptaseDAKOAA1MonoMouse (IgG)1:100Anti-TNF-α†Used for neutralization study.R&D SystemsPolyGoat (IgG)2 μg/mlAnti-SDF-1R&D Systems79014MonoMouse (IgG)50 μg/mlAnti-TNFR1R&D Systems16805.21MonoMouse (IgG)10 μg/mlAnti-TNFR2R&D Systems22221MonoMouse (IgG)10 μg/mlTNF-αPeproTech, London, UK100, 1000 U/ml (=5, 50 ng/ml)CXCR4Santa Cruz Biotechnology40 μg/mlSDF-1R&D systems100 ng/mlIL-1βPeproTech1000 U/ml (=100 ng/ml)IL-4PeproTech1000 U/ml (=200 ng/ml)IL-6PeproTech1000 U/ml (=100 ng/ml)INF-γPeproTech1000 U/ml (=50 ng/ml)goat IgGDAKOMouse IgGDAKOAMD3100Sigma, St. Louis, MO1 μg/ml* Used for immunohistochemical staining.† Used for neutralization study. Open table in a new tab Two ICC cell lines (HuCCT-1, obtained from Cell Resource Center for Biochemical Research, Tohoku University, Sendai, Japan; and CCKS-1, established in our laboratory),34Sugawara H Yasoshima M Katayanagi K Kono N Watanabe Y Harada K Nakanuma Y Relationship between interleukin-6 and proliferation and differentiation in cholangiocarcinoma.Histopathology. 1998; 33: 145-153Crossref PubMed Scopus (82) Google Scholar, 35Saito K Minato H Kono N Nakanuma Y Ishida F Kosugi M Establishment of the human cholangiocellular carcinoma cell line (CCKS1).Acta Hepatol Jpn. 1993; 34: 122-129Crossref Scopus (20) Google Scholar one line of nonneoplastic human intrahepatic biliary epithelial cells (HIBECs) from an explanted liver with hepatitis C virus-related cirrhosis,36Kamihira T Shimoda S Harada K Kawano A Handa M Baba E Tsuneyama K Nakamura M Ishibashi H Nakanuma Y Gershwin ME Harada M Distinct costimulation dependent and independent autoreactive T-cell clones in primary biliary cirrhosis.Gastroenterology. 2003; 125: 1379-1387Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar and the embryonic lung fibroblast cell line WI-38 (Cell Resource Center for Biochemical Research) were used. Cultured HuCCT-1, CCKS-1, and WI-38 cells were maintained in RPMI 1640 medium, whereas HIBECs were maintained in Dulbecco's modified Eagle's medium/F12, containing 10% fetal calf serum (Life Technologies, Inc., Grand Island, NY), and penicillin-streptomycin-glutamine (Life Technologies, Inc.). SDF-1 was used at concentration of 0.1, 1, 10, and 100 ng/ml, according to previous reports,15Marchesi F Monti P Leone BE Zerbi A Vecchi A Piemonti L Mantovani A Allavena P Increased survival, proliferation, and migration in metastatic human pancreatic tumor cells expressing functional CXCR4.Cancer Res. 2004; 64: 8420-8427Crossref PubMed Scopus (305) Google Scholar, 17Scotton CJ Wilson JL Scott K Stamp G Wilbanks GD Fricker S Bridger G Balkwill FR Multiple actions of the chemokine CXCL12 on epithelial tumor cells in human ovarian cancer.Cancer Res. 2002; 62: 5930-5938PubMed Google Scholar and TNF-α was used at 100 U/ml and 1000 U/ml. Two receptors for TNF-α, tumor necrosis factor receptors (TNFR) 1 and 2, are well known. For the experiment on the inhibition of TNF-α, TNF-α neutralization antibody and anti-TNFR1 and anti-TNFR2 neutralization antibodies (Table 2) were used. Furthermore, the effects of other inflammatory cytokines (IL-1β, IL-4, IL-6, and interferon (IFN)-γ; each at 1000 U/ml) on the expression of CXCR4 mRNA in ICC cell lines were examined by adding each cytokine in the culture medium and by comparing them with the effect of TNF-α. The expression of TNF-α, SDF-1, CXCR4, mast cell tryptase, and CD68 in ICC and control specimens was examined immunohistochemically using each primary antibody. To unmask the antigen in the tissue, deparaffinized sections were pretreated in a microwave oven in ethylenediaminetetraacetic acid buffer (pH 8.0) at 95°C for detection of SDF-1, CXCR4, and CD68 or in 0.1% trypsin buffer at 37°C for detection of TNF-α. After the blockage of endogenous peroxidase in 1% H2O2 in methanol for 20 minutes and pretreatment with protein block serum (DakoCytomation, Kyoto, Japan) for 15 minutes to block nonspecific reaction, the sections were incubated with each primary antibody at 4°C overnight. The Envision+ solution (DakoCytomation) was then applied for 60 minutes. The reaction products were visualized via a benzidine reaction. The sections were then lightly counterstained with hematoxylin. Negative controls included substituting the primary antibody with similarly diluted goat normal IgG. Our preliminary study showed that in the immunostaining of SDF-1, TNF-α, and CXCR4, mononuclear cells resembling mast cells were clearly positive. These cells were positive when control immunoglobulin was used for the primary antibody and were actually positive for mast cell tryptase. Because mast cells are known to show nonspecific binding to immunoglobulin,37Yamashiro M Kouda W Kono N Tsuneyama K Matsui O Nakanuma Y Distribution of intrahepatic mast cells in various hepatobiliary disorders. An immunohistochemical study.Virchows Arch. 1998; 433: 471-479Crossref PubMed Scopus (55) Google Scholar mast cell-like cells positive for SDF-1, TNF-α, and CXCR4 were not evaluated by immunohistochemistry. Preincubation of anti-SDF-1, anti-TNF-α, or anti-CXCR4 antibodies with SDF-1 or TNF-α or CXCR4 (Table 2) during the immunostaining of SDF-1, TNF-α, or CXCR4 in ICC tissues resulted in a marked reduction in the immunostaining of each protein. To confirm further the specificity of anti-TNF-α and anti-CXCR4 antibodies, the expression of TNF-α or CXCR4 was immunohistochemically examined in the formalin-fixed, paraffin-embedded sections of the ICC cell lines (HuCCT-1 and CCKS-1) cultured in plastic bottles for 3 days, using the same antibodies. It was found that these cultured ICC cells were positive for TNF-α and CXCR4, and the positive staining was diminished by preincubation of each of these antibodies with TNF-α or CXCR4, respectively. Staining of CXCR4 in ICC was classified into two types according to Kato and colleagues38Kato M Kitayama J Kazama S Nagawa H Expression pattern of CXC chemokine receptor-4 is correlated with lymph node metastasis in human invasive ductal carcinoma.Breast Cancer Res. 2003; 5: R144-R150Crossref PubMed Scopus (244) Google Scholar: CXCR4 was expressed homogeneously in almost all ICC cells (diffuse-type) but expressed heterogeneously in carcinoma cells of ICC (focal-type). The latter was found in the background of diffuse immunostaining of CXCR4 of varying degrees. In this sense, focal-type seems to express more CXCR4 than diffuse-type. RNA was isolated from HuCCT-1, CCKS-1, and HIBEC cell lines using the Qiagen RNAeasy kit (Qiagen, Tokyo, Japan). RNA was also isolated from carcinoma tissues from central parts of three surgically resected ICC tissues (ICC 1, 73-year-old male; ICC 2, 71-year-old female; and ICC 3, 72-year-old female) that were included among the ICC cases described above. Then, 2 μg of RNA was used to synthesize the first-strand cDNA with the superscript system (Life Technologies, Inc., Rockville, MD), according to the manufacturer's instructions. RT-PCR reactions for TNF-α, TNFR1, TNFR2, CXCR4, SDF-1, and β-actin were performed as described previously.39Harada K Ohira S Isse K Ozaki S Zen Y Sato Y Nakanuma Y Lipopolysaccharide activates nuclear factor-kappaB through toll-like receptors and related molecules in cultured biliary epithelial cells.Lab Invest. 2003; 83: 1657-1667Crossref PubMed Scopus (139) Google Scholar The oligonucleotide sequences, numbers of cycles, and annealing temperatures of these primers are shown in Table 3. As a quantitative control, primers for the β-actin gene, a housekeeping gene that is considered to be constitutively expressed, were used. After PCR, 5-μl aliquots of the products were subjected to 1.5% or 2.0% agarose gel electrophoresis and stained with ethidium bromide.Table 3Primer Sequences Used in this StudyForwardReversePCR productAnnealingCycle no.HumanTNF-α5′-GCTGTACCTCATCTACTCCCA-3′55′-GCAATTTCTAGGTGAGGTCTTC-3′660 bp55°C21TNFR15′-GAGAGGCCATAGCTGTCTGG-3′5′-GTTTTCTGAAGCGGTGAAGG-3′303 bp60°C28TNFR25′-GGATGAAGCCCAGTTAACCA-3′5′-GTGTCCTGTCTTCATGGGTGA-3′501 bp55°C32CXCR45′-TTCTACCCCAATGACTTGTG-3′5′-ATGTAGTAAGGCAGCCAACA-3′260 bp56°C33SDF-15′-CCGCGCTCTGCCTCAGCGACGGGAAG-3′5′-CTTGTTTAAAGCTTTCTCCAGGTACT-3′227 bp56°C33β-Actin5′-CAAGAGATGGCCACGGCTGCT-3′5′-TCCTTCTGCATCCTGTCGGCA-3′275 bp60°C18 Open table in a new tab ICC cells from both cell lines were cultured in the presence of 100 or 1000 U/ml TNF-α for 48 hours. Multiplex real-time analysis was performed using premade CXCR4 (FAM)- and β-actin (VIC)-specific primers and probes with the ABI Prism 7700 sequence detection system (PE Applied Biosystems, Warrington, UK). RT-PCR was done with the TaqMan Universal PCR Master Mix (PE Applied Biosystems) using 5 μl of cDNA in a 25-μl final reaction mi

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