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Modified Heparin Inhibits P-selectin-mediated Cell Adhesion of Human Colon Carcinoma Cells to Immobilized Platelets under Dynamic Flow Conditions

2004; Elsevier BV; Volume: 279; Issue: 28 Linguagem: Inglês

10.1074/jbc.m312951200

1083-351X

Min Wei, Guihua Tai, Yanguang Gao, Na Li, Baiqu Huang, Yifa Zhou, Shui Hao, Xianlu Zeng,

Platelet Disorders and Treatments

Accumulating evidence indicates that the formation of tumor cell platelet emboli complexes in the blood stream is a very important step during metastases and that the anti-metastasis effects of heparin are partially due to a blockade of P-selectin on platelets. In this study, heparin and chemically modified heparins were tested as inhibitors of three human colon carcinoma cell lines (COLO320, LS174T, and CW-2) binding to P-selectin, adhering to CHO cells expressing a transfected human P-selectin cDNA, and adhering to surface-anchored platelets expressing P-selectin under static and flow conditions. The aim was to screen for heparin derivatives with high anti-adhesion activity but negligible anticoagulant activity. In this study, four modified heparins with high anti-adhesion activity were identified including RO-heparin, CR-heparin, 2/3ODS-heparin, and N/2/3DS-heparin. NMR analysis proved the reliability of structure of the four modified heparins. Our findings suggested that the 6-O-sulfate group of glucosamine units in heparin is critical for the inhibition of P-selectin-mediated tumor cell adhesion. Heparan sulfate-like proteoglycans on these tumor cell surfaces are implicated in adhesion of the tumor cells to P-selectin. Some chemically modified heparins with low anticoagulant activities, such as 2/3ODS-heparin, may have potential value as therapeutic agents that block P-selectin-mediated cell adhesion and prevent tumor metastasis. Accumulating evidence indicates that the formation of tumor cell platelet emboli complexes in the blood stream is a very important step during metastases and that the anti-metastasis effects of heparin are partially due to a blockade of P-selectin on platelets. In this study, heparin and chemically modified heparins were tested as inhibitors of three human colon carcinoma cell lines (COLO320, LS174T, and CW-2) binding to P-selectin, adhering to CHO cells expressing a transfected human P-selectin cDNA, and adhering to surface-anchored platelets expressing P-selectin under static and flow conditions. The aim was to screen for heparin derivatives with high anti-adhesion activity but negligible anticoagulant activity. In this study, four modified heparins with high anti-adhesion activity were identified including RO-heparin, CR-heparin, 2/3ODS-heparin, and N/2/3DS-heparin. NMR analysis proved the reliability of structure of the four modified heparins. Our findings suggested that the 6-O-sulfate group of glucosamine units in heparin is critical for the inhibition of P-selectin-mediated tumor cell adhesion. Heparan sulfate-like proteoglycans on these tumor cell surfaces are implicated in adhesion of the tumor cells to P-selectin. Some chemically modified heparins with low anticoagulant activities, such as 2/3ODS-heparin, may have potential value as therapeutic agents that block P-selectin-mediated cell adhesion and prevent tumor metastasis. Hematogenous metastasis is a highly regulated and dynamic process in which the cancerous cells separate from the primary tumor, migrate across blood vessel walls into the bloodstream, and disperse throughout the body to generate new colonies. Several lines of experiments have demonstrated that platelets can facilitate hematogenous dissemination of tumor cells (1Karpatkin S. Pearlstein E. Ambrogio C. Coller B.S. J. Clin. Investig. 1988; 81: 1012-1019Crossref PubMed Scopus (303) Google Scholar). In this process, platelets act mainly through emboli complexes formed by the interaction of tumor cells and platelets. The formation of tumor cell-platelet emboli complexes in the blood stream not only provides a protective shield that masks them from the cytotoxic activity of natural killer cells, but also favors tumor cell adhesion to vascular endothelial cells (2Borsig L. Wong R. Hynes R.O. Varki N.M. Varki A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2193-2198Crossref PubMed Scopus (354) Google Scholar, 3Nieswandt B. Hafner M. Echtenacher B. Mannel D.N. Cancer Res. 1999; 59: 1295-1300PubMed Google Scholar, 4Honn K.V. Tang D.G. Crissman J.D. Cancer Metast. Rev. 1992; 11: 325-351Crossref PubMed Scopus (283) Google Scholar). The interaction between tumor cells and platelets may involve several kinds of adhesion molecules. Selectins are a family of intercellular adhesion molecules that mediate the initial adhesion of leukocytes to the endothelia of blood vessels during inflammation (5Wei M. Zeng X.L. Prog. Biochem. Biophys. 2003; 30: 185-189Google Scholar, 6Vestweber D. Blanks J.E. Physiol. Rev. 1999; 79: 181-213Crossref PubMed Scopus (826) Google Scholar, 7Kansas G.S. Blood. 1996; 88: 3259-3287Crossref PubMed Google Scholar, 8Lasky L.A. Annu. Rev. Biochem. 1995; 64: 113-139Crossref PubMed Google Scholar, 9Krause T. Turner G.A. Clin. Exp. Metast. 1999; 17: 183-192Crossref PubMed Scopus (112) Google Scholar). The family includes E-, P-, and L-selectin, and all three selectins can bind to sialylated, fucosylated, or, in some cases, sulfated glycans on glycoproteins, glycolipids, or proteoglycans (10McEver R.P. Glycoconj. J. 1997; 14: 585-591Crossref PubMed Scopus (240) Google Scholar). The tetrasaccharides [Neu5Acα2,3Galβ1,4-(Fucα1,3)GlcNAc] (sLex) 1The abbreviations used are: sLex, sialyl Lewis X; sLea, sialyl Lewis A; PSGL-1, P-selectin glycoprotein ligand-1; P-Fc, recombinant human P-selectin/Fc chimera protein; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; CHO, chinese hamster ovary; CHO-P cells, CHO cells expressing a transfected human P-selectin cDNA; aPTT, activated partial thromboplastin time; FBS, fetal bovine serum; PBS, phosphate-buffered saline; BSA, bovine serum albumin. and [Neu5Acα2,3Galβ1,3(Fucα1,4)-GlcNAc] (sLea) have been identified as the minimal ligands for all three types of selectins. Recently, accumulating evidence indicates that P-selectin plays a crucial role during hematogenous metastasis, and P-selectin has been shown to bind to several human cancers and human cancer-derived cell lines, such as colon cancer, lung cancer, breast cancer, malignant melanoma, gastric cancer, tongue squamous cancer, and neuroblastoma (11.Mannori, G., Crottet, P., Cecconi, O., Hanasaki, K., Aruffo, A., Nelson, R. M., Varki, A., and Bevilacqua, M. P. Cancer Res. 55, 4425–4431Google Scholar, 12Stone J.P. Wagner D.D. J. Clin. Investig. 1993; 92: 804-813Crossref PubMed Scopus (160) Google Scholar, 13Aigner S. Ramos C.L. Hafezi-Moghadam A. Lawrence M.B. Friederichs J. Altevogt P. Ley K. FASEB J. 1998; 12: 1241-1251Crossref PubMed Scopus (234) Google Scholar, 14Goetz D.J. Ding H. Atkinson W.J. Vachino G. Camphausen R.T. Cumming D.A. Luscinskas F.W. Am. J. Pathol. 1996; 149: 1661-1673PubMed Google Scholar, 15Ma Y.Q. Geng J.G. J. Immunol. 2000; 165: 558-565Crossref PubMed Scopus (62) Google Scholar, 16Giavazzi R. Foppolo M. Dossi R. Remuzzi A. J. Clin. Investig. 1993; 92: 3038-3044Crossref PubMed Scopus (193) Google Scholar, 17Aruffo A. Dietsch M.T. Wan H. Hellstrom K.E. Hellstrom I. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2292-2296Crossref PubMed Scopus (83) Google Scholar, 18Pottratz S.T. Hall T.D. Scribner W.M. Jayaram H.N. Natarajan V. Am. J. Physiol. 1996; 271: L918-L923PubMed Google Scholar). The role of P-selectin on both endothelium and platelets is to mediate the tumor cell adhesion to vascular endothelial cells and the interaction of activated platelets and tumor cells during metastasis. According to recent reports, P-selectin plays roles mainly in the initial process of tumor cell adhesion to platelets (19McCarty O.J. Mousa S.A. Bray P.F. Konstantopoulos K. Blood. 2000; 96: 1789-1797Crossref PubMed Google Scholar). Therefore, blocking P-selectin on platelets with antagonists can effectively inhibit the formation of tumor cell-platelet emboli complexes and successfully prevent hematogenous metastasis. Various P-selectin antagonists have been discovered at present, such as mAbs, sLex, sLea, and its mimetics, as well as recombinant PSGL-1 (20Wang L. Brown J.R. Varki A. Esko J.D. J. Clin. Investig. 2002; 110: 127-136Crossref PubMed Scopus (328) Google Scholar). As these selectin inhibitors have various drawbacks, such as narrow cross-reactivity, weak affinity, relatively low selectivity among the selectins, short circulating half-life, great expense to generate, potential antigenicity, and a very limited track record as intravenous therapeutic agents, the development of these compounds into effective drugs for clinical use has been greatly limited. Heparin has been used clinically as an anticoagulant for over 60 years. Heparin and heparan sulfate are linear polysaccharides consisting of uronic acid-(1→4)-d-glucosamine-repeating disaccharide subunits. In addition to its effects on anticoagulant, heparin exhibits many other activities (21Rabenstein D.L. Nat. Prod. Rep. 2002; 19: 312-331Crossref PubMed Scopus (709) Google Scholar). Several studies have demonstrated that heparin and heparan sulfate are ligands for P-selectin and can block its binding to carbohydrate ligand (22Varki N.M. Varki A. Semin. Thromb. Hemost. 2002; 28: 53-66Crossref PubMed Scopus (140) Google Scholar). Therefore, one of the proposed mechanisms by which heparin can inhibit metastasis is that heparin may block the P-selectin-based interaction of platelets and tumor cells during metastasis. It is reasonable that heparin should be a candidate for a clinical trial in the immediate future. However, the kind of native human tumors that actually use the P-selectin-based ligands to interact with platelets is unknown, and little information is available about the specific oligosaccharide structures in heparin that are recognized by P-selectin. In addition, heparin's significant anticoagulant properties and its potential for bleeding complications may contraindicate its use as an anti-metastasis compound. The anticoagulant activity of heparin depends on its unique antithrombin binding pentasaccharide sequence (23Petitou M. Casu B. Lindahl U. Biochimie (Paris). 2003; 85: 83-89Crossref PubMed Scopus (203) Google Scholar). Chemical modifications, which are directed against the unique structure, can effectively reduce the anticoagulant activities of heparin. In this study, we prepared eight chemically modified heparins and analyzed whether they could still effectively reduce tumor cell adhesion. The results demonstrated that heparin and four modified heparins could significantly inhibit P-selectin-mediated tumor cell adhesion. The sulfate groups at C6 on the glucosamine residues in heparin play a critical role in the inhibition. Our results also showed that three human colon carcinoma cell lines strongly expressed heparan sulfate proteoglycans, which are involved in adhesion of these tumor cells as a ligand of P-selectin. Our data suggest that some chemically modified heparins with low anticoagulant activities, such as 2/3ODS-heparin, have potential value for preventing tumor hematogenous metastasis. Heparin and Its Derivatives—Porcine intestinal heparin (Mr = 18,000–20,000) was purchased from Sigma-Aldrich Inc. Periodate-oxidized, borohydride-reduced heparin (RO-heparin) was prepared according to the method of Casu et al. (24Casu B. Diamantini G. Fedeli G. Mantovani M. Oreste P. Pescador R. Porta R. Prino G. Torri G. Zoppetti G. Arzneimittelforschung. 1986; 36: 637-642PubMed Google Scholar). Carboxyl-reduced heparin (CR-heparin) and carboxyl-reduced sulfated heparin (CRS-heparin) were prepared as described by Wessel et al. (25Wessel H.P. Hosang M. Tschopp T.B. Weimann B.J. Carbohydr. Res. 1990; 204: 131-139Crossref PubMed Scopus (30) Google Scholar). 2-O, 3-O-desulfated heparin (2/3ODS-heparin) was prepared essentially according to the method of Fryer et al. (26Fryer A. Huang Y.C. Rao G. Jacoby D. Mancilla E. Whorton R. Piantadosi C.A. Kennedy T. Hoidal J. J. Pharmacol. Exp. Ther. 1997; 282: 208-219PubMed Google Scholar). A modified procedure of Nagasawa et al. (27Nagasawa K. Inoue Y. Kamata T. Carbohydr. Res. 1977; 58: 47-55Crossref PubMed Scopus (264) Google Scholar) was employed to prepare N-desulfated/N-acetylated heparin (NDS-heparin). N-Desulfated/2-O, 3-O-desulfated heparin (N/2/3DS-heparin) was typically prepared by 2-O, 3-O-desulfation of N-desulfated heparin as described for 2-O, 3-O-desulfated heparin. 6-O-Desulfated heparin (6ODS-heparin) and N-desulfated 6-O-desulfated heparin (N/6DS-heparin) was prepared as recently described (28Baumann H. Scheen H. Huppertz B. Keller R. Carbohydr. Res. 1998; 308: 381-388Crossref PubMed Scopus (44) Google Scholar, 29Kariya Y. Kyogashima M. Suzuki K. Isomura T. Sakamoto T. Horie K. Ishihara M. Takano R. Kamei K. Hara S. J. Biol. Chem. 2000; 275: 25949-25958Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The preparation contained about 10% fewer 2-O-sulfate groups. The anticoagulant activity of heparin and modified heparinoids was analyzed by aPTT assay. Clotting times were determined using an ACL200 Automated Coagulation Laboratory (Japan) and lyophilized silica aPTT kit. Protein and Antibodies—Recombinant human P-selectin/Fc chimera protein (P-Fc), 9E1 (a leukocyte adhesion blocking IgG mAb against P-selectin), and P1 (a leukocyte adhesion nonblocking IgG mAb against P-selectin) were purchased from R&D Systems, Inc. CSLEX-1, an IgM mAb to sLex, was a gift from O. Spertini. 10E4, an IgM mAb to the native heparan sulfate chains of proteoglycans, was generously provided by Dr. Guido David. KPL1, an IgG mAb to PSGL-1 was purchased from Santa Cruz Biotechnology, Inc. Mouse IgM isotype control and FITC-conjugated Ab against mouse IgM were purchased from Sigma-Aldrich Inc. Human IgG and mouse IgG isotype control, FITC-conjugated Ab against human and mouse IgG were purchased from Jackson ImmunoResearch Laboratories, Inc. Cells—The human colon carcinoma cells (COLO320, LS174T, and CW-2), CHO cells, and U937 cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Science (Shanghai, China). They were cultured in IMDM (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37 °C in the presence of 5% CO2. COLO320 and CHO cells were harvested with 0.02% EDTA. LS174T and CW-2 cells were detached by mild trypsinization (0.25% trypsin/EDTA for 2 min at 37 °C; Invitrogen), and subsequently incubated at 37 °C for 2 h to regenerate surface glycoproteins, as previously described (11.Mannori, G., Crottet, P., Cecconi, O., Hanasaki, K., Aruffo, A., Nelson, R. M., Varki, A., and Bevilacqua, M. P. Cancer Res. 55, 4425–4431Google Scholar). Subsequently, tumor cells were washed once with PBS (10 mm HEPES, pH 7.4), resuspended in serum-free media containing 0.1% bovine serum albumin (BSA). CHO cells expressing a transfected human P-selectin cDNA (CHO-P cells) were generated by transient transfection of CHO cells, which was performed using the Poly-Fect transfection reagent (Qiagen) according to instructions from the manufacturer. Heparinase Treatment—To remove cell surface heparan sulfate proteoglycans, each aliquot (50 μl) of tumor cells (107/ml) was incubated with 5 units/ml heparinases I, II, and III (Sigma), in the presence of a mixture of protease inhibitors (10 mg/ml leupeptin, 20 mg/ml aprotinin, and 10 mm benzamidine, all from Sigma), for 1 h at 37 °C with end-to-end rotation (15Ma Y.Q. Geng J.G. J. Immunol. 2000; 165: 558-565Crossref PubMed Scopus (62) Google Scholar). Following the enzyme treatment, tumor cells were washed twice and resuspended in PBS/FBS (PBS supplemented with 1 mm CaCl2, 1 mm MgCl2, and 1% heat-inactivated FBS; 5 × 106 cells/ml) for further experimentation. Immobilization of Platelet Layers on Glass Slides—To provide a substrate that readily binds platelets, circular glass slides were coated with 3-aminopropyltriethoxysilane (APES; Sigma). Human blood was drawn by venipuncture from healthy volunteers into sodium citrate (0.38% w/v) anticoagulant. Platelet-rich plasma (PRP) was prepared by centrifugation of whole blood at 200 × g for 15 min. The PRP count was adjusted to 2 × 108/ml before being bound to APES-treated circular glass slides for 60 min (30Buttrum S.M. Hatton R. Nash G.B. Blood. 1993; 82: 1165-1174Crossref PubMed Google Scholar). Nonspecific binding was blocked with 0.1% BSA for 10 min at 37 °C. Under these conditions, a confluent layer of platelets was formed as evaluated by light microscopy for each experiment. The density and confluency of platelet layers were not affected during the flow experiment. Flow Cytometric Assays—All cells were washed once and resuspended in PBS/FBS (5 × 106 cells/ml). For the cell surface P-selectin binding assay, each aliquot (0.1 ml) of cells was incubated with 0.3 μg of human IgG or P-Fc at 22 °C for 20 min, followed by a FITC-conjugated Ab against human IgG at 22 °C for 1 h with end-to-end rotation. Cells were sedimented at 1500 rpm for 5 min in a tabletop centrifuge. Each aliquot was then resuspended in 0.5 ml of PBS/FBS for immediate flow cytometric analysis (FACScan, Beckman-Counter). For antibody or heparin derivative inhibition experiments, 0.3 μg of P-Fc were preincubated with 1.0 μg of 9E1, P1, or heparin derivatives of various concentrations at 22 °C for 30 min. The following experiments were as above. For cell surface P-selectin ligand experiments, each aliquot (0.1 ml) of cells was incubated with 1∼2 μg of mouse IgM, mouse IgG, CSLEX-1 mAb, 10E4 mAb, or KPL1 mAb at 22 °C for 30 min, followed by treatment with a FITC-conjugated Ab against mouse IgM or mouse IgG at 22 °C for 1 h with end-over-end rotation. Laminar Flow Adhesion Assays—Tumor cell adhesion to immobilized platelets (or CHO-P cells) was quantitated under flow conditions using a parallel plate flow chamber (GlycoTech, Rockville, MD). All cells were washed once and resuspended to 1 × 106 cells/ml in serum-free media containing 0.1% BSA. A platelet-coated circular glass slide (or a CHO-P cell-coated 35-mm tissue culture dish) was assembled in a flow chamber and mounted on the stage of an inverted microscope (Olympus Optical, Tokyo, Japan) equipped with a camera (Panasonic, Yokohama, Japan) and connected to a VCR and a TV monitor. Surface-adhered platelets were then incubated with either 1 unit/ml thrombin or Dulbecco's PBS, 0.1% BSA for 10 min at 37 °C. After washing the platelet layer with Dulbecco' s PBS, 0.1% BSA for ∼2 min, tumor cells were perfused through the chamber for 3 min at the appropriate flow rates to obtain wall shear stresses of 0.3–1.2 dyn/cm2 at 22 °C using a syringe pump (Cole-Parmer Instrument Co.), thereby mimicking the fluid mechanical environment of the microcirculation and postcapillary venules. Interactions between tumor cells and surface-adherent platelets (or CHO-P cells) were visualized in real time by phase-contrast videomicroscopy. The numbers of bound cells were quantified from videotape recordings of 10–20 fields of view obtained (3 min after flowing cells through the chamber) while scanning the lower plate of the flow chamber using a ×10 objective lens. Their number was manually determined by reviewing the videotapes. For some inhibition experiments, immobilized platelets (or CHO-P cells) were preincubated with mAb (20 μg/ml) or heparin derivatives with various concentrations at 22 °C for 30 min. Statistics—Data were expressed as the mean ± S.D. Statistical significance of differences between means was determined by analysis of variance. If means were shown to be significantly different, multiple comparisons by pairs were performed with the Tukey test. Probability values of p < .05 or p < .01 were selected to be statistically significant. COLO320 Cells Express P-selectin Ligand—P-selectin has been shown to bind to several human cancers and human cancer-derived cell lines, although which kind of native human tumors use P-selectin-based mechanisms to successfully metastasize is still not clear. In this assay, COLO320, a human colon carcinoma cell line, was first examined for the interaction with P-Fc using a static cell surface binding assay, and a FITC-conjugated Ab to human IgG was used to reveal the binding of P-Fc to tumor cells by flow cytometry. As shown in Fig. 1, P-Fc, but not human IgG, bound to COLO320 cells. The percentage of positive cells binding to P-selectins was 90.3%. Preincubation of P-Fc with 9E1 (a leukocyte adhesion-blocking mAb to P-selectin), but not P1 (a leukocyte adhesion nonblocking mAb to P-selectin), inhibited this binding, confirming the binding specificity of P-selectin to COLO320 cells. Modified Heparins Inhibit P-selectin Binding to COLO320 Cells under Static Conditions—Using various well established methods, we prepared a series of chemically modified heparins, including RO-heparin, CR-heparin, CRS-heparin, 2/3ODS-heparin, NDS-heparin, N/2/3DS-heparin, 6ODS-heparin, and N/6DS-heparin. Representative saccharide units of each type of heparinoid are given in Fig. 2. The Sepharose CL-6B gel chromatographic analysis and the ion chromatographic analysis indicated that the integrity and the molecular weights (data not show) of the main structures of the eight chemically modified heparins remained whereas the sulfate content changed as expected (Table I).Table ISulfate content of modified heparinsModified heparinsSulfate contentaDetermined by ion chromatography. Values are mean ± S.D. of three separate experiments.Modified heparinsSulfate contentaDetermined by ion chromatography. Values are mean ± S.D. of three separate experiments.%%Heparin26.76 ± 0.02NDS-heparin18.90 ± 0.04RO-heparin25.75 ± 0.13N/2/3DS-heparin14.15 ± 0.04CR-heparin24.34 ± 0.056ODS-heparin16.22 ± 0.11CRS-heparin30.16 ± 0.02N/6DS-heparin7.36 ± 0.192/3ODS-heparin18.19 ± 0.04a Determined by ion chromatography. Values are mean ± S.D. of three separate experiments. Open table in a new tab To determine the in vitro anticoagulant activities of these chemically modified heparins, we measured the aPTT values. As shown in Table II, all modified heparins had reduced their anticoagulant activities to some extent when measured with fresh human plasmas. The eight heparin derivatives can be ranked according to their anticoagulant activities as: heparin > RO-heparin > 2/3ODS-heparin > NDS-heparin > CR-heparin > CRS-heparin > 6ODS-heparin > N/2/3DS-heparin > N/6DS-heparin. These results indicate that the more the heparins were modified, the more their anticoagulant activities were reduced.Table IIAnticoagulant activities of modified heparinsaPTTaDetermined by the aPTT method.2 μg/ml4 μg/ml6 μg/ml8 μg/ml0.1 mg/ml1.0 mg/ml5.0 mg/mlControl30.330.330.330.330.330.330.3Heparin72.9>120bValues that are more than 120 seconds cannot be determined.RO-heparin34.539.644.249.3>120CR-heparin31.231.332.332.651.2>120CRS-heparin30.931.231.332.543.9>1202/3ODS-heparin32.934.837.239.463.4>120NDS-heparin31.632.333.134.057.9>120N/2/3DS-heparin30.830.830.931.033.740.168.86ODS-heparin31.931.431.831.136.880.9>120N/6DS-heparin31.331.331.730.630.833.446.2a Determined by the aPTT method.b Values that are more than 120 seconds cannot be determined. Open table in a new tab As discussed earlier, heparin can function as a ligand for P-selectin and can block P-selectin binding to its native carbohydrate ligands (22Varki N.M. Varki A. Semin. Thromb. Hemost. 2002; 28: 53-66Crossref PubMed Scopus (140) Google Scholar). In this assay, therefore, we examined whether modified heparins could inhibit the P-selectin binding under static conditions. First, we used heparin and modified heparins with a high concentration of 5.0 mg/ml to conduct the experiment. Fig. 3A shows that heparin, RO-heparin, CR-heparin, 2/3ODS-heparin, and N/2/3DS-heparin, but not CRS-heparin, NDS-heparin, 6ODS-heparin, and N/6DS-heparin, could significantly abolish the binding of P-selectin to COLO320 cells. Compared with the positive control, the preincubation of heparin, RO-heparin, CR-heparin, 2/3ODS-heparin, and N/2/3DS-heparin reduces the percentage of COLO320 cells binding to P-selectin by 92, 81, 80, 80, and 71%, respectively. In the following experiments, we tested the above four modified heparins with low concentrations of 1.0 mg/ml and 0.1 mg/ml to determine their ability to inhibit tumor cells binding to P-selectin. The results showed that the four modified heparins at concentrations of 1.0 mg/ml, to some extent, had an inhibiting effect (Fig. 3B). However, at the concentration of 0.1 mg/ml, the inhibition was faint (Fig. 3C). In brief, the results indicated that some modified heparins remain able to inhibit P-selectin binding to tumor cells, and the ability is proportionally related to their concentration. Modified Heparins Inhibit COLO320 Cells Adhering to CHO-P Cells under Flow Conditions—The previous experiment proved that human colon carcinoma COLO320 cells can bind to P-selectin under static conditions, and the binding can be inhibited by heparin and the four modified heparins. However, in the experiment above, recombinant human P-selectin/Fc chimera protein (P-Fc) was used instead of P-selectin, and the experiment was conducted under static conditions. Therefore, these experiments cannot completely reflect the binding of tumor cells to P-selectin under physiological conditions. To corroborate the above findings, CHO cells expressing a transfected human P-selectin cDNA (CHO-P) was used as the carrier of P-selectin, and the flow adhesion assay, mimicking the fluid mechanical environment of the microcirculation and postcapillary venules in vitro, was performed. In this assay, tumor cells were perfused through a parallel plate flow chamber whose lower plate was coated with a layer of CHO-P cells at the appropriate flow rates to obtain wall shear stresses of 0.3 dyn/cm2. The interaction of tumor cells and CHO-P cells can be observed when the tumor cells run through the surface of CHO-P cells. As shown in Fig. 4, COLO320 cells bound to CHO-P cells, but not to CHO cells. Preincubation of CHO-P cells with 9E1, but not P1, inhibited this binding, indicating the binding specificity of COLO320 cells to CHO-P cells. To investigate whether heparin and the four modified heparins could inhibit the binding of COLO320 cells to CHO-P cells, we conducted the following experiments. In the experiments, the concentration of the modified heparins was 5.0 mg/ml, and the wall shear stress was 0.3 dyn/cm2. The results showed that heparin, RO-heparin, CR-heparin, 2/3ODS-heparin, and N/2/3DS-heparin could significantly abolish the adhesion of COLO320 cells to CHO-P cells (Fig. 4). Compared with the positive control, the preincubation of heparin, RO-heparin, CR-heparin, 2/3ODS-heparin, and N/2/3DS-heparin reduced the percentage of COLO320 cell adhesion to CHO-P cells by 79, 77, 64, 64, and 66%, respectively. Modified Heparins Inhibit COLO320 Cells Adhering to Surface-immobilized Platelets under Flow Conditions—The interaction between platelets and the tumor cells is crucial to successful metastasis. Previous studies have shown that platelets interact with a number of tumor cell lines, but the cell adhesion experiments were conducted exclusively under static conditions (11.Mannori, G., Crottet, P., Cecconi, O., Hanasaki, K., Aruffo, A., Nelson, R. M., Varki, A., and Bevilacqua, M. P. Cancer Res. 55, 4425–4431Google Scholar, 12Stone J.P. Wagner D.D. J. Clin. Investig. 1993; 92: 804-813Crossref PubMed Scopus (160) Google Scholar, 31Kim Y.J. Borsig L. Han H.L. Varki N.M. Varki A. Am. J. Pathol. 1999; 155: 461-472Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). To determine whether COLO320 cells also adhere to platelets under flow conditions and to determine the role of P-selectin on platelets in the process of cell adhesion, tumor cells were perfused through a parallel plate flow chamber whose lower plate was coated with a layer of platelets. Fig. 5A shows that immobilized platelets supported COLO320 cell adhesion in a shear stress-dependent manner. No interaction was observed at 1.2 dyn/cm2. In the following experiments, using mAb against P-selectin, we examined the potential role of platelet P-selectin in the adhesive interaction under flow conditions. The results showed that incubation of the platelets layer with 9E1 alone resulted in an ∼50–58% reduction of adhesion at 0.3 dyn/cm2 or 0.6 dyn/cm2, and P1 nearly had no effect on the adhesion (Fig. 5, B and C). The results above indicated that COLO320 cells could adhere to platelets under flow, and P-selectin, which expresses on the surface of platelets, plays an important role in the process of the adhesion of tumor cells to platelets. In the following step, we examined the effect of heparin and the four modified heparins on inhibiting the adhesion of COLO320 cells to surface-immobilized platelets under flow conditions. The results showed that heparin and the four modified heparins could significantly inhibit the adhesion of COLO320 cells to surface-immobilized platelets. At all concentrations of modified heparins used in the experiment, 5.0, 1.0, and 0.1 mg/ml, were dramatically effective for blocking the adhesion of the tumor cells to the surface-adhered platelets under the condition of the wall shear stress of 0.3 dyn/cm2 or 0.6 dyn/cm2 (Fig. 5, B and C). Even at a concentration of 0.1 mg/ml, which is the lowest concentration tested, heparin and the four modified heparins could still significantly inhibit the adhesion of COLO320 cells to surface-immobilized platelets. Adhesion of COLO320 Cells to P-selectin Involves Heparan Sulfate-like Proteoglycans—We next attempted to identify the counter receptor for platelet P-selectin on COLO320 cell surface using oligosaccharide-specific mAbs. In this assay, we used U937 cells as a control because U937 cells express PSGL-1, a natural ligand for P-selectin, and it is well known that under flow conditions, U937 cells can roll on and adhere to immobilized P-selectin. Fig. 6A shows that CSLEX-1 (a mAb to sLex) did n