Vascular Endothelium Sialyl Lewis X to Adhesion of Human Cancer Cells to Contribution of Carbohydrate Antigens Sialyl Lewis A and Updated Version

The carbohydrate antigen, sialyl Le x, is known to be a ligand for the cell adhesion molecule called ELAM-1 (E-selectin, endothelial cell leukocyte adhesion molecule-l), which is present on cytokine-activated human endothelial cells. Recently, we reported that another carbohydrate antigen, sialyl Le a, can also serve as a ligand for ELAM-1 (A. Takada, K. Ohmori, N. Takahashi, K. Tsuyuoka, K. Yago, K. Zenita, A. Hasegawa, and R. Kannagi, Biochem. Biophys. Res. Commun., 179: 713-719, 1991). Both sialyl Le x and sialyl Le a are expressed in many human malignant cells. In order to assess the contribution of these carbohydrate antigens to the adhesion of human malignant cells to vascular endothelium, we selected a panel of 12 cultured human epithelial cancer cell lines and a panel of 12 human leukemia cell lines which express sialyl Le x and/or sialyl Le a antigens. All 12 epithelial cancer cell lines exhibited a clearly ELAM-1dependent adhesion to cytokine-activated human umbilical vein endothelial cells, while only 3 of the 12 leukemia cell lines exhibited significant participation of ELAM-1 in the adhesion. With regard to epithelial cancer cells, the adhesion of 6 cancer cell lines, mostly of colon and pancreas origin, was dependent almost exclusively on sialyl Le a. A significant contribution of the sialyl Le X antigen was noted in the adhesion of the other 6 cell lines, including cancers of lung and liver origin. These results imply that the sialyl Lea/ELAM-1 adhesion system, as well as the sialyl LeX/ELAM-1 adhesion system, plays an important role in the adhesion of human cancer cells to human umbilical vein endothelial cells. With regard to leukemia cells, on the other hand, adhesion of the 3 leukemia cell lines that showed ELAM-l-dependent adhesion was mediated by the sialyi Le x antigen, and none of these leukemia cell lines expressed sialyl Le a or exhibited sialyl Lea-dependent adhesion. I N T R O D U C T I O N The lectin-l ike domain of ELAM-13 is k n o w n to recognize an oncofeta l carbohydrate antigen, sialyl Le x (1-4). Recently, other investigators and our group (5-7) found that another cancer-associated carbohydrate antigen, sialyl Le a, can also serve as a l igand for ELAM-1 . Findings in support of this include: (a) several cul tured h u m a n colon cancer cell lines, for instance, Colo201, exhibit a clearly E L A M 1 d e p e n d e n t adhesion to r iLl /3-act ivated H U V E C s (5, 7); (b) this adhesion is significantly inhibi ted by t reatment with several antisialyl Le a antibodies but not by anti-sialyl Le x ant ibody t reatment (5, 7); (c) pre t reatment of H U V E C s with either sialyl Le x or sialyl Le a glycol ipid results in nearly comple te inhibi t ion of adhesion (5); and (d) synthetic sialyl Lea-protein conjugate can bind to ELAM-1 (6, 8). The ant igen sialyl Le a, as well as sialyl Le x, is known to be expressed frequent ly on hum an cancer cells, (9-12) , and we thought Received 6/29/92; accepted 10/28/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan (03557114 and 03258218). z To whom requests for reprints should be addressed, at Laboratory of Experimental Pathology, Research Institute, Aichi Cancer Center, 1-1 Kanokoden, Chikusaku, Nagoya, 464, Japan. 3 The abbreviations used are: ELAM-I (E-selectin), endothelial-leukocyte adhesion molecule1; ICAM1, intercellular adhesion molecule1 ; VCAM1, vascular cell adhesion molecule-l; LFA, lymphocyte function-a~sociated antigen; HUVEC, human umbilical vein endothelial cell; riLl, recombinant interleukin 1. it wor thy to study the contr ibut ion of these carbohydrate ant igens to the adhes ion of human mal ignant cells. ELAM-1 has been recognized as a molecu le involved in the adhesion of leukocytes to vascular endothe l ium, and several cul tured human leukemia cell lines have been frequent ly used in exper imenta l studies to demonst ra te this. It is well d o c u m e n t e d that, in addi t ion to ELAM-1 , ICAM-1 and VCAM-1 are also involved in the adhesion of leukocytes and leukemia cells to endothel ia l cells (13-15) . However , the molecular m e c h a n i s m involved in the adhesion of huma n epithelial cancer cells to endothel ial cells has not been s tudied extensively. It has been reported that some m e l a n o m a cells interact with endothelial cells via the very-late an t igen-4 /VCAM-1 system, whi le some colon cancer cells involve ELAM-1 (16, 17). This s tudy was undertaken to evaluate the role p layed by the sialyl Le a and sialyl Le x antigens in the adhesion of h u m a n leukemia and cancer cells. For this purpose, we chose a panel of 12 cul tured h u m a n epithelial cancer cell lines, which express sialyl Le x and/or sialyl Le a antigens, and looked at the role of these carbohydrate ant igens in the adhes ion to rlL1/3activated HUVECs . A panel of 12 cul tured l eukemia cell lines was also chosen, to serve as a reference. M A T E R I A L S A N D M E T H O D S Human Cancer and Leukemia Cells Cultured human cancer cell lines, Capan-2, WiDr, and CoR-1, were obtained from the Japanese Cancer Research Resources Bank (Tokyo Japan), and SWI 116 was obtained from American Type Culture Collection (Rockville, MD); QG90 and QG56 were gifts from the Kyushu Cancer Center; MKN74 and Colo201 were gifts from Dr. Shoichi Oboshi, Niigata University; C-1 was a gift from Dr. Yoshihiro Hayata, Tokyo Medical University. All cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Cultured human leukemia cell lines, HPB-ALL, U937, NALM-6, MOLT-3, CMK, TK1B and THPI-0, were obtained from the First Division of the Department of Internal Medicine, Kyoto University. Jurkat, P12/Ichikawa, and MOLT-15 were kindly supplied by Dr. Jun Minowada, Fujisaki Cell Center, Hayashibara Biology Research Institute, Okayama, Japan. These cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (18). Monolayer Cell Adhesion Assay Using HUVECs HUVECs (2-6 passages after isolation, obtained from Kurabou Co. Ltd., Osaka, Japan) were stimulated with 1.0 ng/ml of riLl/3 for 4 h in 96-well plates (5, 19, 20). To these plates the SlCr-labeled cultured human cancer cells or leukemia cells (1 • 105/60 lal/well) were added and incubated for 30 min at room temperature with rotation (120 rpm) (J). A short incubation time and continuous rotation were applied to minimize possible nonspecific bindings of epithelial cancer cells to the plates. In preliminary experiments, these conditions were shown to be suitable for the evaluation of the initial phase of cell adhesion, in which adhesion molecules of the selectin family are known to play important roles, and thus allowed the detection of the maximum contribution of the selectin family adhesion molecules. After the adherent cells were washed three times, they were treated with 1% Triton X-100. The lysate was collected using a Skatron supematant collection system (Skatron As, Lier, Norway), and the radioactivity in each well was measured with a gammacounter. The number of attached cells was calculated from the specific radioactivity of the labeled cells. Adherence of cancer or leukemia cells was corrected for binding to unstimulated HUVECs. The riLl/3 was obtained from the 354 American Association for Cancer Research Copyright © 1993 on February 23, 2013 cancerres.aacrjournals.org Downloaded from CANCER CELL ADHESION MEDIATED BY SIAI.YI. LEWIS A ANTIGEN Central Research Laboratory of Otsuka Pharmaceutical Co., Tokushima, Japan, and the recombinant basic fibroblast growth factor used for the in vitro culture of HUVECs was from the Central Research Laboratory of Takeda Pharmaceutical Co., Juso, Japan. Monoclonal Antibodies Used for Inhibition of Cell Adhesion Monoclonal anti-ELAM-1, anti-ICAM-1, and anti-VCAM-I antibodies (BBA2, BBA4, and BBA6, all murine IgG1) were obtained from British Biotechnology Ltd., Abington, Oxon, United Kingdom. These antibodies were preincubated with HUVECs at 50 /ag/ml for 30 min at 37~ prior to the adhesion experiments with cancer or leukemia cells for inhibition experiments (5). When a mixture of these monoclonal antibodies was used for preincubation of HUVECs, the final concentration of each antibody in the preincubation period was adjusted to 50 pg/ml. Monoclonal anti-LFA1/3 (CBL5, IgGt) was obtained from Immunotechs S.A. (Marseille, France), and this antibody was preincubated with cultured leukemia cells at 50 pg/ml for 30 min at room temperature prior to application to the monolayer of HUVECs. Monoclonal antibodies SNH3 (specific to sialyl Lewis X, supplied by Dr. Sen-itiroh Hakomori, Biomembrane Institute, Seattle, WA) and 2D3 (specific to sialyl Lewis A, established in our laboratory) are both murine IgM and were purified from ascitic fluids as described previously (2, 5). The carbohydrate structures of the sialyl Lewis X and sialyl Lewis A antigens and the specificities of the monoclonal antibodies are summarized in Table 1. For inhibition of cell adhesion, these monoclonal antibodies were preincubated with cultured cancer cells or leukemia cells at 25-100 lag/ml for 30 min at room temperature prior to application to the monolayer of HUVECs (5). Inhibition of Cell Adhesion with Liposomes containing the Ligand Glycolipid The pure synthetic sialyl Le • glycolipid used for the inhibition experiment had the structure, NeuAc~2 ---> 3Gal/31 ---> 4(Fuccd --> 3)GIcNAc/31 ---> 3 Gal/31 --> 4Glc131 ---> ICer (5, 7, 21). The anomeric structures and linkages of the terminal four sugar residues of this synthetic glycolipid are identical with those in the sialyl SSEA-1 epitope as shown in Table 1. The sialyl Le x liposome contained 40 pg/well of the glycolipid, 20 pg/well of cholesterol, and 40 pg/well of phosphatidylcholine (5). The control liposome contained the same amount of cholesterol and phosphatidylcholine but no glycolipid. The liposome suspensions were preincubated with HUVECs for 30 min at room temperature prior to the addition of lymphocytes. Fluorescence-activated Flow Cytometry Flow cytometric analysis was performed using FACScan (Becton Dickinson Immunocytometry Systems, Mountain View, CA). The indirect immunofluorescence method was applied for staining of cultured cells with the antibodies SNH3 and 2D3 as the first antibody (stained for 30 rain at room temperature at the concentration of 1.0 Iag/ml/1 • 10 6 cells), followed by the addition of the fluorescein isothiocyanate-labeled rabbit anti-murine IgM (Ia chain specific) antibody as the second antibody (Cappel Inc., Malvern, CA), as was described previously (22).