Expression and Regulation of CD97 in Colorectal Carcinoma Cell Lines and Tumor Tissues
2002; Elsevier BV; Volume: 161; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)64443-4
ISSN1525-2191
AutoresMatthias Steinert, Manja Wobus, Carsten Boltze, Alexander Schütz, Mandy Wahlbuhl, Jörg Hamann, Gabriela Aust,
Tópico(s)Signaling Pathways in Disease
ResumoThe expression of CD97, a member of the EGF-TM7 family with adhesive properties, is proportional to the aggressiveness and lymph node involvement in thyroid tumors. CD97 has never been systematically investigated in other tumors. First, we examined colorectal carcinoma cell lines (n = 18) for CD97 expression and regulation. All cell lines were CD97-positive. The level of CD97 in each line correlated with migration and invasion in vitro. This result was confirmed in CD97-inducible Tet-off HT1080 cells. Transforming growth factor-β, which inhibits proliferation in transforming growth factor-β-sensitive LS513 and LS1034 cells, down-regulated CD97 in these cell lines. Examining CD97 during sodium butyrate-induced cell differentiation of Caco-2 cells, we could demonstrate a CD97-decreasing effect. Second, we screened 81 colorectal adenocarcinomas by immunohistology for expression of CD97. Normal colorectal epithelium is CD97-negative. Seventy-five of 81 of the carcinomas expressed CD97. The strongest staining for CD97 occurred in scattered tumor cells at the invasion front compared to cells located within solid tumor formations of the same tumor. Carcinomas with more strongly CD97-stained scattered tumor cells showed a poorer clinical stage as well as increased lymph vessel invasion compared to cases with uniform CD97 staining. In summary, CD97 expression correlates with dedifferentiation, migration, and invasion in colorectal tumor cell lines. Moreover, more strongly CD97-stained tumor cells at the invasion front of colorectal carcinomas indicate the involvement of the molecule in tumor migration and invasion. The expression of CD97, a member of the EGF-TM7 family with adhesive properties, is proportional to the aggressiveness and lymph node involvement in thyroid tumors. CD97 has never been systematically investigated in other tumors. First, we examined colorectal carcinoma cell lines (n = 18) for CD97 expression and regulation. All cell lines were CD97-positive. The level of CD97 in each line correlated with migration and invasion in vitro. This result was confirmed in CD97-inducible Tet-off HT1080 cells. Transforming growth factor-β, which inhibits proliferation in transforming growth factor-β-sensitive LS513 and LS1034 cells, down-regulated CD97 in these cell lines. Examining CD97 during sodium butyrate-induced cell differentiation of Caco-2 cells, we could demonstrate a CD97-decreasing effect. Second, we screened 81 colorectal adenocarcinomas by immunohistology for expression of CD97. Normal colorectal epithelium is CD97-negative. Seventy-five of 81 of the carcinomas expressed CD97. The strongest staining for CD97 occurred in scattered tumor cells at the invasion front compared to cells located within solid tumor formations of the same tumor. Carcinomas with more strongly CD97-stained scattered tumor cells showed a poorer clinical stage as well as increased lymph vessel invasion compared to cases with uniform CD97 staining. In summary, CD97 expression correlates with dedifferentiation, migration, and invasion in colorectal tumor cell lines. Moreover, more strongly CD97-stained tumor cells at the invasion front of colorectal carcinomas indicate the involvement of the molecule in tumor migration and invasion. CD97 is a member of a subfamily of class II G-protein-coupled receptors referred to as EGF-TM7, which is expressed predominantly in leukocytes.1McKnight AJ Gordon S The EGF-TM7 family: unusual structures at the leukocyte surface.J Leukoc Biol. 1998; 63: 271-280Crossref PubMed Scopus (122) Google Scholar EGF-TM7 proteins possess a unique hybrid structure that consists of varying numbers of N-terminal EGF-like domains coupled to a seven-span transmembrane (TM7) domain by a mucin-like spacer.1McKnight AJ Gordon S The EGF-TM7 family: unusual structures at the leukocyte surface.J Leukoc Biol. 1998; 63: 271-280Crossref PubMed Scopus (122) Google Scholar To date, six EGF-TM7 molecules have been described, namely CD97,2Hamann J Eichler W Hamann D Kerstens HM Poddighe PJ Hoovers JM Hartmann E Strauss M van Lier RA Expression cloning and chromosomal mapping of the leukocyte activation antigen CD97, a new seven-span transmembrane molecule of the secretion receptor superfamily with an unusual extracellular domain.J Immunol. 1995; 155: 1942-1950PubMed Google Scholar, 3Gray JX Haino M Roth MJ Maguire JE Jensen PN Yarme A Stetler-Stevenson MA Siebenlist U Kelly K CD97 is a processed, seven-transmembrane, heterodimeric receptor associated with inflammation.J Immunol. 1996; 157: 5438-5447PubMed Google Scholar EMR1,4Baud V Chissoe SL Viegas-Pequignot E Diriong S N'Guyen VC Roe BA Lipinski M EMR1, an unusual member in the family of hormone receptors with seven transmembrane segments.Genomics. 1995; 26: 334-344Crossref PubMed Scopus (99) Google Scholar EMR2,5Lin HH Stacey M Hamann J Gordon S McKnight AJ Human EMR2, a novel EGF-TM7 molecule on chromosome 19p13.1, is closely related to CD97.Genomics. 2000; 67: 188-200Crossref PubMed Scopus (85) Google Scholar EMR3,6Stacey M Lin HH Hilyard KL Gordon S McKnight AJ Human epidermal growth factor (EGF) module-containing mucin-like hormone receptor 3 is a new member of the EGF-TM7 family that recognizes a ligand on human macrophages and activated neutrophils.J Biol Chem. 2001; 276: 18863-18870Crossref PubMed Scopus (87) Google Scholar EMR4,7Caminschi I Lucas KM O'Keeffe MA Hochrein H Laabi Y Kontgen F Lew AM Shortman K Wright MD Molecular cloning of F4/80-like-receptor, a seven-span membrane protein expressed differentially by dendritic cell and monocyte-macrophage subpopulations.J Immunol. 2001; 167: 3570-3576Crossref PubMed Scopus (44) Google Scholar, 8Stacey M Chang GW Sanos SL Chittenden LR Stubbs L Gordon S Lin HH EMR4, a novel EGF-TM7 molecule up-regulated in activated mouse macrophages, binds to a putative cellular ligand on B lymphoma cell line, A20.J Biol Chem. 2002; 277: 29283-29293Crossref PubMed Scopus (82) Google Scholar and ETL.9Nechiporuk T Urness LD Keating MT ETL, a novel seven transmembrane receptor that is developmentally regulated in the heart. ETL is a member of the secretin family and belongs to the EGF-TM7 subfamily.J Biol Chem. 2001; 276: 4150-4157Crossref PubMed Scopus (81) Google Scholar The varying numbers of EGF-like domains in CD97 result from alternative splicing of the precursor transcript. Various CD97 isoforms have been detected possessing three (EGF 1,2,5), four (EGF 1,2,3,5), or five (EGF 1,2,3,4,5) EGF-like domains. Co-expression and co-regulation of these three isoforms occurs in normal lymphoid cells.10Eichler W CD97 isoform expression in leukocytes.J Leukoc Biol. 2000; 68: 561-567PubMed Google Scholar Based on its structure and expression pattern, CD97 has been implicated in cellular adhesion by interacting with other cell surface proteins or extracellular matrix proteins. Adhesion studies demonstrated that both lymphocytes and erythrocytes specifically bind to CD97 transfectants through interactions with CD55,11Hamann J Vogel B van Schijndel GM van Lier RA The seven-span transmembrane receptor CD97 has a cellular ligand (CD55, DAF).J Exp Med. 1996; 184: 1185-1189Crossref PubMed Scopus (297) Google Scholar a membrane-bound molecule acting as a regulatory protein of the complement cascade. We previously found that CD97 is present at low levels on resting lymphocytes, but is strongly up-regulated within a few hours after lymphocyte activation,12Eichler W Aust G Hamann D Characterization of the early activation-dependent antigen on lymphocytes defined by the monoclonal antibody BL-Ac(F2).Scand J Immunol. 1994; 39: 111-115Crossref PubMed Scopus (68) Google Scholar which is consistent with CD97 playing a role in adhesion. CD97 is constitutively expressed by monocytes and granulocytes.12Eichler W Aust G Hamann D Characterization of the early activation-dependent antigen on lymphocytes defined by the monoclonal antibody BL-Ac(F2).Scand J Immunol. 1994; 39: 111-115Crossref PubMed Scopus (68) Google Scholar In normal tissues, abundant expression of CD97 is only detected in macrophages and dendritic cells except for glial cells, and in some T and B cells.13Jaspars LH Vos W Aust G van Lier RA Hamann J Tissue distribution of the human CD97 EGF-TM7 receptor.Tissue Antigens. 2001; 57: 325-331Crossref PubMed Scopus (71) Google Scholar CD97 is strongly expressed in dedifferentiated thyroid carcinomas.14Aust G Eichler W Laue S Lehmann I Heldin N-E Lotz O Scherbaum WA Dralle H Hoang-Vu C CD97: a dedifferentiation marker in human thyroid carcinomas.Cancer Res. 1997; 57: 1798-1806PubMed Google Scholar, 15Hoang-Vu C Bull K Schwarz I Krause G Schmutzler C Aust G Köhrle J Dralle H Regulation of CD97 protein in thyroid carcinoma.J Clin Endocrinol Metab. 1999; 84: 1104-1109PubMed Google Scholar Immunostaining for CD97 in thyroid cancer and adjacent thyroid tissue suggests that CD97 expression parallels the aggressiveness and lymph node involvement of thyroid tumors. Epidermal growth factor (EGF), which stimulates the growth and invasion of differentiated thyroid carcinoma cells,16Holting T Siperstein AE Clark OH Duh QY Epidermal growth factor (EGF)- and transforming growth factor alpha-stimulated invasion and growth of follicular thyroid cancer cells can be blocked by antagonism to the EGF receptor and tyrosine kinase in vitro.Eur J Endocrinol. 1995; 132: 229-235Crossref PubMed Scopus (54) Google Scholar up-regulated CD97 in these cells. All-trans retinoic acid treatment redifferentiates thyroid carcinomas17Schmutzler C Kohrle J Retinoic acid redifferentiation therapy for thyroid cancer.Thyroid. 2000; 10: 393-406Crossref PubMed Scopus (117) Google Scholar and leads to a decrease in total cell number and in CD97-positive thyroid carcinoma cells.15Hoang-Vu C Bull K Schwarz I Krause G Schmutzler C Aust G Köhrle J Dralle H Regulation of CD97 protein in thyroid carcinoma.J Clin Endocrinol Metab. 1999; 84: 1104-1109PubMed Google Scholar These data suggest that CD97 expression may play an important role in the dedifferentiation of thyroid tumors. CD97 has never been systematically investigated in other types of tumors. In this study, we first examined CD97 in colorectal carcinoma cell lines by flow cytometry and investigated whether the expression level of CD97 correlates with the migratory and invasive capacity of these cell lines. To confirm the results obtained, stable transfected, CD97 inducible Tet-off HT1080 cells were generated and inserted into the same tests. We further studied whether induction of differentiation influences CD97 expression in the cell line Caco-2 that undergoes enterocytic differentiation in culture after treatment with sodium-butyrate (NaBT)18Souleimani A Asselin C Regulation of C-fos expression by sodium butyrate in the human colon carcinoma cell line Caco-2.Biochem Biophys Res Commun. 1993; 193: 330-336Crossref PubMed Scopus (51) Google Scholar, 19Basson MD Turowski GA Rashid Z Hong F Madri JA Regulation of human colonic cell line proliferation and phenotype by sodium butyrate.Dig Dis Sci. 1996; 41: 1989-1993Crossref PubMed Scopus (66) Google Scholar or by blocking phosphatidylinositol 3-kinase (PI3K), an important mediator of intracellular signal transduction.20Wang Q Wang X Hernandez A Kim S Evers BM Inhibition of the phosphatidylinositol 3-kinase pathway contributes to HT29 and Caco-2 intestinal cell differentiation.Gastroenterology. 2001; 120: 1381-1392Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar We assessed the relationship of the proliferating inhibiting effect of transforming growth factor-β (TGF-β) to CD97 expression in both TGF-β-sensitive and -insensitive cell lines. In a second part, colorectal carcinomas and the corresponding normal tissue of the patients were screened for CD97 expression by immunohistology to gain an insight into the distribution pattern of the molecule. The human colorectal cancer cell lines Caco-2, Co115, Colo 201, Colo 205, DLD-1, HCT 116, HCT-15, HT-29, Lisp-1, LoVo, LS1034, LS174T, LS411N, LS513, SW 1116, SW 480, SW 837, and WiDr were obtained from the American Type Culture Collection (ATCC, Rockville, MD) or the European Collection of Cell Cultures (ECACC, Salisbury, UK). The cells were maintained in Dulbecco's modified minimal essential medium or Dulbecco's modified minimal essential medium/Ham's F12 (Life Technologies GmbH, Karlsruhe, Germany) containing 10% fetal calf serum except for Colo 201, Colo 205, and HCT-15 that were grown in RPMI 1640/10% fetal calf serum. CD97(EGF 1,2,5) cDNA was amplified by polymerase chain reaction (PCR) from the vector pcDNA3.1/Zeo+ containing the desired cDNA.2Hamann J Eichler W Hamann D Kerstens HM Poddighe PJ Hoovers JM Hartmann E Strauss M van Lier RA Expression cloning and chromosomal mapping of the leukocyte activation antigen CD97, a new seven-span transmembrane molecule of the secretion receptor superfamily with an unusual extracellular domain.J Immunol. 1995; 155: 1942-1950PubMed Google Scholar Primers generating MluI and NheI sites during amplification were used (PrCD97 MluI: 5′-cga cgc gtg cca cca tgg gag gcc gcg-3′; PrCD97NheI: 5′-aac get agc cct tga tat gcc gga-3′). The cDNA was inserted into the MluI and NheI sites of pBI-EGFP (BD Clontech, Heidelberg, Germany). HT1080 Tet-off cells (Clontech) were grown in Dulbecco's modified minimal essential medium, supplemented with 10% Tet system-approved fetal calf serum (Life Technologies), 4 mmol/L l-glutamine, and 100 μg/ml G418 (Roche Diagnostics GmbH, Mannheim, Germany). The cells were co-transfected with 5 μg of pBI-EGFP-CD97 (EGF 1,2,5) and 250 ng pWE-4 (Clontech) by electroporation. Selection of stable cell lines was initiated 2 days after transfection using 100 μg/ml of hygromycin (Clontech). Fourteen days after transfection, several colonies were isolated and maintained in medium supplemented with 1 μg/ml of doxycycline (Clontech) in 24-well plates. To induce the expression of CD97, cells were cultured without doxycycline. Experiments were performed on cells cultured for 72 hours with or without the antibiotic. Expression of CD97 was verified by flow cytometry. The migration and invasion capacity of colon carcinoma cell lines were evaluated in 24-well transwell chambers (Costar, Bodenheim, Germany) or 6-well Biocoat Matrigel invasion chambers (BD Biosciences, Heidelberg, Germany) containing Matrigel-precoated filters, respectively. In both assays, the upper and lower culture compartments were separated by polycarbonate filters with a pore size of 8 μm. Five μg/ml of collagen I (Sigma-Aldrich Chemie GmbH; Taufkirchen, Germany) was used as a chemoattractant in the lower chamber compartments. Before starting the invasion assay, the Matrigel matrix of the chambers was reconstituted by adding serum-free RPMI 1640 for 2 hours. Tumor cells were radiolabeled for 12 hours using (methyl-3H)-thymidine (0.1 μCi/ml, 7.4 MBq/mmol) and detached with 0.02% ethylenediaminetetraacetic acid. For the invasion assay, 8 × 105 cells per well were incubated on the reconstituted membrane for 72 hours. For the migration assay, 1 × 105 cells per well were seeded onto the filter for 24 hours. Cells passing the filters and attaching to the lower sites of uncoated or Matrigel-coated membranes were harvested using trypsin/ethylenediaminetetraacetic acid. Cell-bound radioactivity was measured using a liquid scintillation counter. The percentages of migration and invasion were calculated on the number of migrating cells compared to the total number of cells. To specify our results, the highly malignant osteosarcoma cell line MG-63 (ATCC) was used as a positive standard cell line in both assays. All quantifications were done in triplicate. The correlation between percent of migration and invasion to mean fluorescence intensity of CD97 was calculated using Spearman's method. MEM-180 and CLB-CD97/321Kwakkenbos MJ van Lier RA Hamann J Characterization of EGF-TM7 family members by novel monoclonal antibodies. Leucocyte Typing VII.in: Mason D White Cell Differentiation Antigens. Oxford University Press, Oxford, England2002: 381-383Google Scholar bind to the stalk region of CD97 and do not show reactivity to EMR2, another member of the EGF-TM7 family. CLB-CD97/111Hamann J Vogel B van Schijndel GM van Lier RA The seven-span transmembrane receptor CD97 has a cellular ligand (CD55, DAF).J Exp Med. 1996; 184: 1185-1189Crossref PubMed Scopus (297) Google Scholar binds to the N-terminal EGF-domain of CD97 but also detects EMR2. Thus, the mAb CLB-CD97/1 was used in combination with MEM-180 to achieve specificity in the enzyme-linked immunosorbent assay (ELISA) system. The CD55 mAb was purchased from BD Biosciences. Carcinoma cells were phenotyped with the desired mAb by an indirect immunofluorescence method using a F(ab′)2 fragment of goat anti-mouse immunoglobulin (FITC-GAM; DAKO, Hamburg, Germany) with a FACSscan (Becton Dickinson, Mountain View, CA). The levels of CD97 and CD55 were determined as mean fluorescence intensity of stained cells in comparison to cells stained with an isotype-matched but irrelevant mAb. To determine the level of CD97 in transfected Tet-off HT1080 cells, the CD97 mAb (clone MEM-180) was coupled to N-hydroxysuccinimidobiotin (Sigma). After incubation with the biotin-labeled CD97 mAb and washing, the HT1080 cells were stained with streptavidin-phycoerythrin (DAKO). The effect on proliferation was evaluated in parallel using the CellTiter96AQueous one-solution cell proliferation assay (Promega GmbH, Mannheim, Germany) at day 9. Colorectal carcinoma cells were seeded at an initial density of 3 × 103 cells per well in 96-well plates. Wortmannin (0.01 to 1 μmol/L, Sigma), TGF-β (0.01 to 1 ng/ml, R & D systems), and/or NaBT (0.5 to 50 mmol/L, Sigma) were added at day 0. Control cells received the same amount of the solvent of the agents. Fivefold cultures were analyzed in four experiments. To detect apoptosis, cells were treated with the required concentration of TGF-β, wortmannin, and/or NaBT for 48 hours; after that, they were trypsinized, double-stained with propidium iodine and annexin V-FITC using the annexin V-Fluos staining kit (Roche) according to the manufacturer's instructions, and analyzed by flow cytometry. Annexin V binds to phosphatidylserine residues expressed on the outer leaflet of the cell membrane as an early event in apoptosis. Cells that stain for annexin V-FITC but not propidium iodine are considered apoptotic. The loss of cell viability in the detached cells was confirmed by the loss of the ability of the cells to exclude trypan blue. To determine the effect of the different agents on CD97 expression, 1 × 105 cells/well were cultured on 24-well plates for 24 hours. The medium was aspirated and replaced with 500 μl of Opti-Mem (Life Technologies) without fetal calf serum containing the desired concentration of TGF-β, wortmannin, and/or NaBT. The cells were trypsinized and stained with CD97 mAbs for fluorescence-activated cell sorting analysis after 12, 24, 48, 72, and 96 hours. Total cellular RNA was isolated from tissues and cell cultures with the Qiagen total RNA isolation kit (Qiagen) in accordance with the manufacturer's instructions. Five μg of total RNA was taken to synthesize cDNA using a first-strand cDNA synthesis kit from Amersham Pharmacia Biotech (Freiburg, Germany) in a reaction volume of 15 μl. The primer pair PhCD97EGFs1 (5′-tcc tgc cgg cag ctc caa ml-3′)/r1 (5′-ggc agc ggc agc tgt atg aac-3′) spans the alternatively spliced EGF-like domain coding region resulting in a 452 by (EGF 1,2,5), 584 by (EGF 1,2,3,5), or 731 by (EGF 1,2,3,4,5) PCR product. The primer pair PhCD97TM7s1 (5′-ctg gcc gcc ttc tgc tgg atg ag-3′)/r1 (5′-ctg cgc gat ggc cgt gat gg-3′) partly amplifies the transmembrane coding region of CD97 resulting in a 356 by-product in all CD97 isoforms. To correct for variations across different cDNA preparations, all samples were first adjusted to contain equal input of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA concentrations.14Aust G Eichler W Laue S Lehmann I Heldin N-E Lotz O Scherbaum WA Dralle H Hoang-Vu C CD97: a dedifferentiation marker in human thyroid carcinomas.Cancer Res. 1997; 57: 1798-1806PubMed Google Scholar Each 25-μl amplification reaction contained 2.5 μl 10× concentrated PCR buffer (15 mmol/L MgCl2), 0.3 U TaqDNA polymerase, 100 μmol/L dNTPs (all from Roche), 0.1 μmol/L of each primer, and 1 μl cDNA in adjusted dilution. The study comprised a series of 81 sporadic colorectal adenocarcinomas obtained from patients undergoing surgery at the Department of Surgery, University of Leipzig, between October 1999 and July 2001. Fourteen were from the proximal colon (cecum, ascending, and transverse colon), 27 were from the distal colon (descending and sigmoid colon) and 40 were from the rectum. Normal mucosal specimens from at least 5 cm away from colorectal carcinoma lesions were obtained from all patients. Samples were cryopreserved in liquid nitrogen. The study was approved by the local Committees of Medical Ethics; all patients gave written consent. No patient had received chemotherapy or radiotherapy before surgery. Histological diagnosis and staging were established following the TNM classification (stage I, n = 10; II, n = 21; III, n = 27; IV, n = 23) proposed by the International Union Against Cancer as evaluated in the Institute of Pathology, University of Leipzig, according to standard criteria. Every tumor was further examined histologically 1) for differentiation, according to the predominant growth pattern (well differentiated, grade I, n = 3; moderately differentiated, grade II, n = 59; poorly differentiated, grade III, n = 19); 2) for the presence of blood and lymph vessel invasion;22Minsky BD Mies C Recht A Rich TA Chaffey JT Resectable adenocarcinoma of the rectosigmoid and rectum. II. The influence of blood vessel invasion.Cancer. 1988; 61: 1417-1424Crossref PubMed Scopus (118) Google Scholar and 3) for the evidence of tumor budding defined as small clusters of undifferentiated cancer cells ahead of the invasive front of the lesion as described.23Hase K Shatney C Johnson D Trollope M Vierra M Prognostic value of tumor "budding" in patients with colorectal cancer.Dis Colon Rectum. 1993; 36: 627-635Crossref PubMed Scopus (468) Google Scholar Tumor budding was divided into two groups according to degree: none or mild (BD-1) and moderate or severe (BD-2). Classification of lesions as BD-1 or BD-2 was based on predominant pattern of tumor budding in the specimen. Sera were obtained from all patients on the day before operation and stored in aliquots at −80°C. Age- and sex-matched healthy blood donors were used as controls (n = 26). Tissues were snap-frozen in liquid nitrogen. Serial frozen sections were cut at 5 μm, fixed in ice-cold methanol for 10 minutes followed by a short rinse in 0.2 mol/L phosphate-buffered saline, and then incubated with the anti-CD97 mAb (clone CLB-CD97/3). The anti-leukocyte common antigen mAb (DAKO) was used to reliably discriminate between tumor cells and tumor-infiltrating leukocytes that are partly CD97+. In negative control sections, an irrelevant mAb of the same isotype was applied at the same concentrations as the primary antibody. After incubation with the mAb (4°C, overnight), bound antibody was detected with a supersensitive detection kit (BioGenex, San Ramon, CA) including biotinylated anti-mouse Ig and horseradish peroxidase-conjugated streptavidin. Immunoreactivity was scored semiquantitatively by two independent investigators who were unaware of the histological results using a light microscope (Axioskop; Carl Zeiss Jena GmbH, Jena, Germany). Tissue from a patient with a dedifferentiated thyroid tumor was used for each staining procedure as a positive control. The percentage of positive cells was judged ranging from 1 to 4 (1, < 10%; 2, 10 to 50%; 3, 51 to 80%; 4, >80% positive tumor cells) and the level of staining intensity was estimated to grades between 0 and 3 (0, negative; 1, weak; 2, moderate; 3, strong staining). The product of positive cells and staining intensity gave the score (0 to 12). We considered samples positive if the score was higher than 2. A score of 3 to 6 was regarded as moderate, and >6 as strong. CD97-positive carcinomas were further examined for the presence of more strongly stained scattered cells or scattered cell groups near or on the invasion front. Staining intensity of these stronger CD97-positive scattered tumor cells was compared to the staining intensity of the tumor cells located within solid tumor formations. The association of CD97 expression and clinicopathological features was assessed with the Fisher's exact test. A level of P < 0.05 was considered significant. The mean nuclear area of more strongly CD97-positive scattered tumor cells was compared to the mean nuclear area of tumor cells located within solid tumor formations by computerized nuclear morphometry24Ikeguchi M Taniguchi T Makino M Kaibara N Reduced E-cadherin expression and enlargement of cancer nuclei strongly correlate with hematogenic metastasis in colorectal adenocarcinoma.Scand J Gastroenterol. 2000; 35: 839-846Crossref PubMed Scopus (39) Google Scholar using a color video camera module and color image freezer (HC300Z; Fuji Photo Film Ltd. Co., Japan). Two hundred cancer nuclei of more strongly CD97-positive scattered tumor cells or tumor cells located within solid tumor formations with complete and clearly identifiable outlines were registered from each patient, and cancer cell nucleus areas were calculated using the Axiovision software (Carl Zeiss Vision GmbH, Hallbergmoos, Germany). The ELISA for CD97 has been described elsewhere.25Hamann J Wishaupt JO van Lier RA Smeets TJ Breedveld FC Tak PP Expression of the activation antigen CD97 and its ligand CD55 in rheumatoid synovial tissue.Arthritis Rheum. 1999; 42: 650-658Crossref PubMed Scopus (113) Google Scholar Briefly, ELISA plates were coated with 5 μg/ml of the mAb MEM-180 at 4°C overnight. After washing the plates, undiluted serum was applied to the plates for 60 minutes at room temperature. After washing, the wells were incubated with 1 μg/ml of biotinylated CLB-CD97/1. Thereafter, streptavidin-peroxidase, and subsequently tetramethylbenzidine were added to the plates and the optical densities were measured in an ELISA reader. Results are expressed as arbitrary units (U/ml) of a standard amount of CD97 (supernatant of COS-7 cells expressing a soluble CD97 construct) in duplicate measurements. CEA (IRMA-coatCEA; Byk-Sangtec Diagnostica, Dietzenbach, Germany), CA15-3 IRMA-matCA15-3; Byk-Sangtec), CA19-9 (IRMA-matCA19-9; Byk-Sangtec), and CA72-4 (ELSA-CA72-4, CISbio international, Gis-Sur-Yvet, France) were determined preoperatively according to the manufacturer's protocol. All colon cell lines examined in this study expressed CD97 with varying intensity (Table 1). CD55, the cellular ligand for CD97, was present on all cell lines except SW480. In Colo 205 cells CD55 expression is rather low. There was no correlation between CD55 and CD97 (r = −0.39, P = 0.11) staining intensity.Table 1Expression of CD97 and CD55 in Colorectal Tumor Cell Lines Determined by Flow Cytometry (Percentage of Cells Expressing the Antigen and Mean Fluorescence Intensity)CD97CD55Cell line%Intensity%Intensity% Migration% InvasionCaco-290–10010.0 ± 4.290–10045.8 ± 14.37.1 ± 2.31.1 ± 0.4Co11590–10015.2 ± 0.490–10040.4 ± 15.46.5 ± 1.91.9 ± 0.3Colo 20190–10046.3 ± 2.390–1006.1 ± 0.555.4 ± 5.320.3 ± 4.2Colo 20590–10056.8 ± 7.05–500.7 ± 0.442.1 ± 3.919.6 ± 2.1DLD-190–10040.6 ± 1.390–10021.6 ± 2.832.0 ± 2.516.3 ± 2.4HCT 11690–10031.4 ± 3.490–10030.4 ± 3.127.1 ± 3.811.2 ± 1.7HCT-1590–10015.9 ± 0.890–10011.2 ± 1.29.4 ± 2.14.9 ± 0.6HT-2990–10020.9 ± 4.990–10022.1 ± 5.19.4 ± 1.93.8 ± 0.5Lisp-190–10016.4 ± 2.450–907.8 ± 3.79.7 ± 2.35.2 ± 1.4LoVo90–10013.0 ± 0.890–10041.9 ± 3.28.3 ± 1.74.9 ± 0.7LS103490–10029.1 ± 9.390–10020.1 ± 8.426.1 ± 2.514.8 ± 2.4LS174T90–10026.4 ± 5.190–1008.5 ± 2.315.8 ± 4.67.2 ± 2.3LS411N90–10014.0 ± 3.190–10025.0 ± 4.97.2 ± 0.44.2 ± 0.4LS51390–10041.9 ± 5.690–10020.1 ± 8.455.2 ± 7.121.7 ± 3.3SW 111690–10045.2 ± 5.690–10017.5 ± 0.533.2 ± 4.319.5 ± 3.4SW 48090–1007.4 ± 0.10–500.7 ± 0.20.2 ± 0.0SW 83790–10025.1 ± 0.390–10012.8 ± 2.112.3 ± 2.07.1 ± 0.9WiDr90–10012.4 ± 0.890–10033.3 ± 4.10.4 ± 0.12.2 ± 0.2The percentage of migration and invasion of the tumor cell lines were calculated from the number of migrating cells compared to the total number of cells in chamber assays. mean ± SD, n = 3. Open table in a new tab The percentage of migration and invasion of the tumor cell lines were calculated from the number of migrating cells compared to the total number of cells in chamber assays. mean ± SD, n = 3. CD97 mRNA analysis by reverse transcriptase-PCR with primers that flank the exons coding the EGF-like domains indicated that all three CD97 isoforms are present in the cell lines (examples are shown in Figure 1). In most cell lines, the shortest CD97 isoform was expressed at the highest level as demonstrated for leukocytes. The relative expression of CD97 (EGF 1,2,3,5) and CD97 (EGF 1,2,3,4,5) mRNA varied between the cell lines. Higher expression of CD97 (EGF 1,2,3,5) mRNA was characteristic for LS513 cells. We found a strong correlation between the expression level of CD97 measured by flow cytometry (Table 1) and migration as well as the invasion capacity of the cell lines (Figure 2). The highly malignant osteosarcoma cell line MG-63, used as a positive control, showed 69% migration and 33% invasiveness. To verify these results, we established stable CD97 (EGF 1,2,5) cDNA-transfected Tet-off HT1080 cells. The expression level of CD97 can be down-regulated in these cells by adding antibiotics. Stable CD97 (EGF 1,2,5) cDNA-transfected cells cultured without doxycycline (CD97on) revealed levels 300 times
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