Initial adhesion of B16 melanoma variants to non- activated endothelial cells is mediated through specific interaction between GM3 (NeuAca2~3Ga4314Glc/?l.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 267, No, 24, Issue of A w t 25, pp. 17261-17270.1992 Printed in U.S.A.
Cell Adhesion in a Dynamic Flow Systemas Compared to Static System GLYCOSPHINGOLIPID-GLYCOSPHINGOLIPID INTERACTION IN THE DYNAMIC SYSTEM PREDOMINATES OVER LECTIN- OR INTEGRIN-BASEDMECHANISMS IN ADHESION OF B16 MELANOMA CELLS TO NON-ACTIVATED ENDOTHELIAL CELLS* (Received for publication, March 12, 1992)
Naoya Kojima,Mitsuru Shiota, Yoshito Sadahira, Kazuko Handa, and Sen-itirohHakomori From the The Biomembrane Institute, Seattle, Washington 98119 and the Department of Pathobwlogy, University of Washington, Seattle, Washington 98195
Initial adhesion of B16 melanoma variants to nonThe molecular basis of specific cell recognition is of central activated endothelial cells is mediated through specific importance in current cell biology. Studies to date suggest interaction between GM3 (NeuAca2~3Ga4314Glc/?l that four combinations of molecular “families” provide the +Cer) expressed on melanoma cells and lactosylcer- basis for most recognition events: (i) various adhesive proteins amide (LacCer, Gal#I1+4Glc@l~Cer)expressed on are recognized by members of the integrin family (1-3); (ii) endothelial cells. This adhesion is predominant over members of the immunoglobulin family interact with each integrin- or lectin-mediated adhesion in a dynamic other orwith integrins (3,4); (iii) carbohydrates (CHOs)’ are flow experimental system employing a parallel plate recognized by members of the lectin family (5-7), particularly laminar flow chamber (Lawrence, M. B., Smith, C. W., selectins (8, 9); (iv) CHOs interact with other CHOs (10). Eskin, S. G., and McIntire, L. V. (1990) Blood 75, These studies were all based on static adhesion systems; i.e. 227-237). In thissystem, a tumor cell suspension flows over aglass plate coated with glycosphingolipid,lectin, adhesion molecules are coated on solid-phase and incubated or fibronectin, and adhesion is recorded on videotape. under static conditions with cells expressing specific receptors These conditions were designed to mimic the micro- or CHO epitopes. Using such a static system, we previously vascular environment in which tumor metastatic dep- reported the specific adhesion, spreading, and enhanced moosition takes place. In contrast,lectin- and fibronectin- tility of GM3-expressing cells on Gg3- or LacCer-coated solidbased mechanisms are predominant in previously used phase, based on GM3/Gg3 or GM3/LacCer interaction (11, static adhesion systems. Under static conditions, the 12). Adhesion of neutrophils and monocytes to endothelial cells relative degree of adhesion of the four B16 variants to endothelial cells or to LacCer-coated plates was the has been observed under dynamic flow conditions, designed same as their relative degree of GM3 expression (i.e. to mimic the microvascular environment (13, 14). SelectinBL6 = F10 > F1 > WA4), and adhesion was inhibited dependent adhesion of neutrophils under dynamic flow conin thepresence of methyl-&lactoside, or liposomes con- ditions appears to occur preferentially over integrin-dependtaining LacCer or GM3. Adhesion was also inhibited ent adhesion (15). Tumor cell adhesion to activated endotheby pretreatment of B16 cells with anti-GM3 antibody lial cells, as mediated by integrin receptorsexpressed on tumor DH2 or sialidase and by pretreatment of endothelial cells, and by ICAMs or selectins expressed on activatedendocells with anti-LacCer antibody T5A7. Under dynamic thelial cells, has received considerable attention recently (16, flow conditions, WA4 cells did not adhere to mouse 17). However, there is still no firm evidence for involvement endothelial cells at high shear stress (>2.5 dynes/cm2) of these adhesion molecules in the very initial interaction but did adhere at lower shear stress. In contrast, BL6 between circulating tumor cells and non-activated endothelial and F10 cells adhered strongly at both low and high cells, particularly under dynamic flow conditions. We now shear stress. BL6 cell adhesion to endothelial cells at present evidence that initial adhesion of B16 melanoma cells both low and high shear stress was inhibited in the presence of antibody DH2, ethyl-#I-lactoside,or lactose, to non-activated mouse and human endothelial cells is meas well as by pretreatment of BL6 cells with sialidase. diated by interaction between GM3 and LacCer, which are Thus, some clear differences, as well as similarities, in highly expressed on B16 cells and endothelial cells, respeccell adhesion under static versus dynamic conditions tively. This GM3/LacCer interaction apparently occurs prior are demonstrated. These findings suggest that mela- to activation of endothelial cells, and prior to involvement of noma cell adhesionto endothelial cells, based on GM3/ ICAMs or selectins, and predominates over integrin- or lectinLacCer interaction,initiatesmetastatic deposition, mediated cell adhesion in a dynamic flow system. which may trigger a series of “cascade”reactions leading to activation of endothelial cells and expression of The abbreviations used are: CHO, carbohydrate; ConA, concanIg family or selectin receptors, thereby promoting avalin A; Gg3, GalNAc@l+4Gal@l+4Glc@l+Cer; GM3, NeuAccu2SGal@l+4Glc@l+Cer; GSL, glycosphingolipid; ICAM, intercellular adhesion and migration of tumor cells. * The study was supported by National Cancer Institute Outstanding Investigator Grant CA42505 (to S. H.) andfunds from The Biomembrane Institute, in part under a researchcontract with Otsuka Pharmaceutical Co. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
adhesion molecule; IL-1,interleukin-1; LacCer, lactosylceramide (Gal@l+4Glcpl+Cer); mAb, monoclonal antibody; PBS, phosphatebuffered saline containing 0.1%bovine serum albumin; PC, phosphatidylcholine; PG, paragloboside (nLc,); SPG, sialosylparagloboside; TLC, thinlayer chromatography. Glycolipids are abbreviated according to the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature ((1977)Lipids 12,455-463); however, the suffix -0seCer is omitted.
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coated with 1% bovine serum albumin in PBS at 37 "C for at least 1 h. Wells were then washed with PBS and used as fibronectin- or Cells and Reagents-B16 melanoma variant cell lines F10, F1, and laminin-coated surfaces. BL6 were kindly donated by Dr. Isaiah J. Fidler, M. D. Anderson B16 melanoma variants BL6, F10, F1, and WA4 were cultured in Cancer Center, Houston, TX. F10 and F1 show high and low lung 10 ml of RPMI 1640 containing 10%fetal calf serum in 25-cm* colonization potential, respectively (18); BL6 produces lung metasCorning flasks and labeled with [3H]thymidine (2 pCi/ml) for 16 h. tasis from subcutaneously grown tumors (19). Wheat germ agglutininFor measurement of cell adhesion, labeled cells were detached by resistant B16 clone WA4, which shows no lung colonization potential treatment in 0.02% EDTA at 37 "C. Detached cells were collected (20), was donated by Dr. Max Burger, BioCenter, University of Basel, and washed two times with PBS. Cells were suspended in PBS at a Switzerland. All these cell lines were characterized by surface expres- density of 2 X lo5 cells/ml, and 100-p1 aliquots of suspension were sion of GM3 as defined by mAb DH2 (see "Results"). Human umbil- added to each well of 96-well plates co-coated with GSL and fibroical vein endothelial cells (referred to hereafter simply as "human nectin or laminin as described above. After addition of cells, plates endothelial cells") were purchased from Cell Systems, Kirkland, WA. were centrifuged at 100 X g for 1 min and incubated for 30 min at Mouse splenic endothelial cells were established by one of the authors 37 "C. Plates were washed with PBS, and remaining adherent cells (Y.S.) at the Department of Pathology, Kawasaki Medical School, were collected by cell harvester and counted by scintillation counter. Kurashiki,Okayama. The cell line was characterized by typical Suspensions of human and mouse endothelial cells (8 X 10' cells/ endothelial cell morphology distinct from that of fibroblasts and ml) were placed in 96- or 48-well flat-bottomplates (purchased macrophages (i.e. contact-inhibited, extremely flattened, polygonal respectively from Falcon, Lincoln Park, NJ, andCostar, Cambridge, appearance at confluence), active uptake of fluorescent-labeled acet- MA) pretreated with 0.5% gelatin at 37 "C for 1 h. Endothelial cells ylated low density lipoprotein, and staining by fluorescent-labeled were cultured until confluence. Medium was removed, endothelial Griffonia simplicifolia lectin known to stain murine endothelial cells cells were washed with PBS, and labeled B16 cells (1 X 10' cells/well (21, 22). While murine endothelialcells used in this experiment were (volume 100 pl) for 96-well plates, 3 X lo5 cells/well (volume 300 pl) not susceptible to stimulation by human IL-10, this cell line was for 48-well plates) in PBSwere placed in each well and incubated at activated by TNFa, andexpression of VCAM-1 was greatly enhanced 37 "C for 30 min. In IL-1 stimulation experiments, endothelial cells upon TNFa stimulation (data not shown). Cell biological properties were treated with 5 units/ml of human recombinant IL-10 (Boehrinof this cell line will be presented elsewhere.* GM3, LacCer, Gg3, Gb4, ger Mannheim, 1000 units/ml) for at least 4 hat 37 "Cbefore addition PG, and otherGSLs were prepared in pure state from organ extracts of B16 cells. After incubation, cells were washed two times with PBS, (23). Methyl-p-lactoside and methyl-P-N-acetyllactosaminide were and adherent cells were detached by trypsinization, collected, and synthesized in this laboratory. counted by scintillation counter. ConA, laminin, G. simplicifolia lectin, and Erythrina corralodenAssay for Inhibition of Cell Adhesion by Oligosaccharides,GSLdron lectin (24) (recognizing N-acetyllactosamine) were purchased liposomes, mAbs, and Enzymatic Pretreatment of Cells-BL6 cells from Sigma. Fibronectin was prepared in this laboratory from human were harvested with 0.02% EDTA, washed with PBS, and suspended plasma by Dr. Mingzhe Zheng. Anti-Gal-binding lectin mAb 5D7 (25) in PBS at a concentration of 1 X lo6 cells/ml. Methyl-0-D-lactoside wasdonated by Dr. AvrahamRaz, Michigan CancerFoundation, and lactose were dissolved in PBS at a concentration of200mM. Detroit, MI. Anti-ELAM-1 mAb 3B7 (26) was donated by Dr. Walter Liposomes (1 ml) were made from 500 nmol of cholesterol, 500 nmol Newman, Otsuka Maryland Research Laboratories, Rockville, MD. of dipalmitoyl PC,and 200 nmol of GSL in PBS as described Other mAbs were prepared in this laboratory, including anti-GM3 previously (11).In this case, GSL concentration was 200 pM. 100 p1 DH2(27), anti-LacCer T5A7 (28),anti-Gg3 2D4 (29),anti-SLe" of 200 mM oligosaccharide or liposomes containing 200 p M of GSL SNH3 (30), and anti-H/Leb/LeYMIA-15-5 (31). Origins, properties, was diluted 2-fold with 100 pl of PBS. 100 pl of BL6 cell suspension and isotypes of these mAbs are described in the references cited. (2 X lo5 cells) was added to 100 pl of oligosaccharide solution or Cell Surface Labeling and Characterization of GSLs in Endothelial liposome suspension and incubated for 30 min at 37 "C. After incuCells (Including Mouse Lung Microvascular Endothelial Cells)-Hubation, mixtures of cells with oligosaccharide or liposome were placed man and mouse endothelial cells were cultured in 25-cm2 Corning on plates coated with LacCer (1pg/well), human endothelial cells, or flasks until confluence, and cell surface CHOs were labeled as previ- mouse endothelial cells, as described above. After incubation at 37 "C ously described (32). Cell layers were washed with PBS (pH 7.0), for 30 min, wells were washed andadherent cells collected and treated with Gal oxidase (10 units/ml) in PBS (pH 7.0) for 1 h at counted as described above. 37 "C, washed with 20 mM Tris-HC1 (pH 8.4) containing 150 mM BL6 cells (1X 106/ml) were treated with 10 pg/ml of mAbs directed NaC1, and reduced with 0.5 mCi of NaB3H4 in 1 ml of 20 mM Tris- against various GSLs, lectins, or adhesion molecules for l h a t 4 "C, HCI (pH 8.4) for 30 min at room temperature. Next, 1 mM NaBH4 in then washed two times with PBS. mAbs used were DH2 (anti-GM3, 20 mM Tris-HC1 (pH 8.4) was added and incubated for 30 min at IgGs), T5A7 (anti-LacCer, IgM), 2D4 (anti-Gg3, IgM), BE2 (anti-H, room temperature (32). Cells were collected by rubber scraper and IgM), SNH3 (anti-SLe", IgM), 3B7 (anti-ELAM-1, IgG1), and 5D7 washed with PBS. GSLs were extracted two times from labeled cells (anti-Gal-binding lectin, IgG3). In separate experiments, BL6 cells with 3 ml of isopropyl alcohol/hexane/water 55:25:20. The extract were treated with sialidase (0.1 unit/ml) in PBS at 4 "C for 30 min. was dried, acetylated in pyridine/acetic anhydride (l:l), and GSLs After treatment, cell viability was tested, cells counted by using were separated from other neutrallipids and phospholipids by Florisil Trypan Blue, and cell number adjusted to 1 X 106/ml. Endothelial column (33). The separated GSLs were applied to TLC plates and cells were cultured in 48-well plates, and treated BL6 cells (3 X lo'/ developed in chloroform/methanol/water (504010 v/v/v). Plates well) were added. After 30 min, wells were washed with PBS and were dried, treated with enhancer, andautoradiographed. The pattern remaining cells were counted. of 3H-labeled GSLs separatedon TLC was comparedwith those Inother experiments,endothelial cells in 48-well plates were developed with orcinol-HaS04or from immunostaining with various treated with 10 pg/ml of mAbs at 37 "C for 30 min, washed with PBS, anti-GSL mAbs. and untreated BL6 cells (3 X 10'/well) were added to treated endoIn order to studysurfaceexpression of GSLs on mouse lung thelial cells. As controls, BL6 cells or endothelial cells were treated microvascular endothelial cells, lungs were perfused with PBS (pH with a mixture of 10 pg/ml mouse IgG and IgM. 7.0) followed by 10 units of Gal oxidase solution in PBS (pH7.0), via Adhesion Assay in a Dynamic Flow System-A parallel plate lampulmonary artery using a syringe. After 30 min, the lung was perfused inar flow chamber connected to an infusion pump (model 935, Harwith 5 mCi of NaB3H4in PBS (pH 7.4) and then perfused with cold vard Apparatus, Cambridge, MA) was used to simulate the flow shear NaBH4. Thiswas followed by extraction of lung, preparation of GSL stresses present in physiological microvascular environments. The fraction, TLC separation, andautoradiography as described above. flow chamber consists of a glass plate onwhich a parallel,transparent Binding of B16 Cell Variants to GSL-, Lectin-, Fibronectin-, and plastic surface is attached with a Silastic rubber gasket; there is a Laminin-coated Surfaces, and to Endothelial Cells, in a Static Adhesion 114-pm gap between the two surfaces, and this gap is connected to System-For preparation of GSL-coated plastic wells, 100 p1 of GSL an inlet and outlet.A laminar flow with defined rate and wall shear in absolute ethanol was placed on each well of 96-well flat(1 pg) stress is achieved by manipulation of the infusion pump, which is bottom assay plates (Falcon Probind) and dried at 37 'C. 100 p1 of connected to the inlet of the flow chamber.Endothelial cells are the appropriate concentration of fibronectin or laminin was placed grown as a monolayer, or adhesion molecules are coated, on the glass on each well and incubated at 4 "C for 16 h. Wells were washed plate, and a laminar flow of cell suspension is passed through the extensively withPBS, andnonspecific binding sites of each well were chamber. Cell movements are observed under invertedphase-contrast microscope (Diaphot-TMD Nikon) andrecorded by time-lapse videocassette recorder. Adhesion is observed as rolling followed by stopping * Y. Sadahira, N. Kojima, T . Kimoto T, unpublished results. EXPERIMENTALPROCEDURES
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Cell Adhesion through Glycosphingolipids in Dynamic Flow System
of cells. This assembly is essentially the same as that described by Lawrence et al. (13, 15). Number of cellsbound during 3min a t different shear stressesfrom 0.4 to 4.8 dynes/cm2 were counted from several fields recorded on videotape. Wall shear stress (T) was calculated by the equation of Lawrence et al. (13, 14):
T = 3pQ/2ba2 where p = coefficient of viscosity (1.0 cP), Q = volumetric flow rate (cm:'/s), a = half channel height (in this case, 5.7 X lo-" cm), and b = channel width (1.3 cm). Coating of Adhesion Molecules or Endothelial Cells on Glass Plates in the Dynamic Flow System-For lectins, fibronectin, laminin, and GSLs used in this study, 10 pl of solution having a concentration of 20-200 pg/ml was placed on a marked area (0.5-cm diameter) on a glass plate (38 X 75 mm, Corning Glassworks, Corning, VA) and dried in a refrigerator a t 4 "C. Dried plates were immersed in PBS at37 "C of PBS. For GSL for 1 h and washed extensively with several changes coating, GSL-liposomes were prepared from 200 pg of GSL, 200 pg of cholesterol, and 400 pg of PC in 1ml of PBS asdescribed previously (11).10 pl of GSL-liposome solution was placed on glass plate and dried at 4 "C, and plates were washed with PBS as described above. Quantity of adsorbed molecules was determined using1'21 labeling for lectins, fibronectin, or laminin, or [3H]cholesterol labeling for GSLliposomes. Under these conditions,almost theentirequantity of protein, regardless of whetherfibronectin,laminin, or lectin, was adsorbed on the glass plate. For example, when 100 pg/ml fibronectin was applied, 12.5 ? 1.8 ng/mm' was adsorbed. Likewise, almost all GSL-liposome dried on the glass plate was adsorbed; e.g. when 200 pg/ml GSL-liposome was applied, 31.3 k 5.2 ng GSL/mm2 was adsorbed. Endothelial cells were coated by placing 100 pl of a suspension containing 2 X lo5mouse or human endothelial cells on glass plates and culturing in a COZ incubator a t 37 "C until confluence. Plates coated with adhesion molecules or endothelial cells were affixed in a flow chamber, and a suspension of B16 melanoma cells was passed through the chamber as described in the preceding section. B16 cells were harvested from culture by 0.02% EDTA in PBS, and suspended in PBS ata concentration of 1 X 105/ml.
Log FIuorcsccIIce irtensily
B LocCsr-
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Gg3 Gb d
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3
4
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2
3
FIG. 1. GSL analyses of B16 melanoma cells and endothelial cells. Panel A, GM3 expression in B16 cells. Cells were treated with anti-GM3 mAb DH2 andanalyzed by flowcytometry. Line with short dashes, control. Long dashes with circles, WA4 cells. Solid line, F1 cells. Alternating long and short dashes, BL6 cells. Reactivity of F10 cells is almost identical to that of BL6 cells (data not shown). Panel B, GSLs from mouse endothelial cells were extracted and analyzed by TLC. Lane 1, neutral GSL standards LacCer, Gg3, Gb4, and Le". Lane 2, neutral GSLs from mouse endothelial cells. Lune 3, acidic GSLs (gangliosides) from mouse endothelial cells. Lane 4, ganglioside standards GM3, GM1, GDla, and GTlb.Panel C,GSL characterization of endothelial cells after cell surface labeling. Endothelial cells were treated withGal oxidase and then reduced with NaB'H4 as described in the text. GSLs were extracted from labeled cells and analyzed by TLC. Lane 1, human endothelial cells. Lane 2, mouse endothelial cells. Lune 3, mouse lung microvascular endothelial cells.
RESULTS
Patterns of GSL Surface Expression on Mouse Melanoma B16 Variants,Human and Mouse Endothelial Cells, and Mouse Lung Microvascular Endothelial Cells-GSL compositions of BL6, F10, and F1 cells were essentially the same, consisting of GlcCer, GM3, LacCer, and a minor quantity of SPG. WA4 was characterized by a much lower chemical quantity of GM3, but higher quantities of SPG, PG, and IV3FucnLc4.These B16 variants differed markedly in surface reactivity with anti-GM3 mAb DH2 (BL6 = F10 > F1 >> WA4) (Fig. IA). Mouse endothelial cells contained GlcCer, LacCer, and Gb4 or PG asneutral GSLs, a major ganglioside with the same mobility as GDla, and relatively small quantities of GM3 (Fig. 1B). GD3 was absent from both mouse and human endothelialcells, as indicated by lack of immunostaining with anti-GD3 mAb (data not shown). Based on labeling experiments, LacCer was the major GSL exposed a t the cell surface in both mouse and human endothelial cells. In the Gal oxidase/NaB3H4 experiment with mouse lung microvascular endothelial cells, three GSL bands were observed LacCer, Gb3 (or Gg3), and Gb4 (Fig. IC). Human endothelial cells contained GlcCer, LacCer, Gb4, and PG as neutral GSLs, and GM3 and SPG asmajor gangliosides (data not shown), in agreement with a previous report (34). Adhesion of Mouse Melanoma B16 Variants to Non-activated Human and Mouse Endothelial Cells Is Based on GM3/ LacCer Interaction, and Occurs Prior to Integrin-mediated Adhesion"B16 melanoma variants BL6, F10, F1, and WA4, which show declining metastatic potential and reactivity with anti-GM3 mAb DH2 in that order (see "Experimental Procedures"), also showed relative adhesion to LacCer- or Gg3coated plates in the same order (i.e. BL6 > F10 > F1 >>
i mc
m,n,
01y2.0 dynes/cm2). Under low shear stress, 37 "C, and washed with PBS. Labeled BL6 cells (3 X 105/well) were WA4 cells adhered more strongly than F1 cells. In the static added to endothelial cells and incubated as described above. Human system, WA4 (compared to F1) also showed a greater number endothelial cells were treated with IL-lj3 for 4 h (Fj.mAbs used were: 1-4, , seeabove; 5 , mAb MIA-15-5 (anti-H); 6, mAb 3B7 (antiof cells adhering to mouse endothelial cells with a 5-min incubation period. In the dynamic system, BL6 adhesion to ELAM-1); 7, mAb 5D7 (anti-Gal-binding lectin).
mouse endothelial cells was inhibited in the presence of 0.1 M lactose, 50 mM ethyl-p-lactoside, or by pretreatment of BL6 cells with anti-GM3 mAb DH2 (Fig. 8B).
on B16 cells, and the order of adhesion of the four B16 variants on endothelial cells (both human and mouse) is the same as their order of GM3 expression (BL6 > F10 > F1> WA4). (ii) LacCer is the major cell surface-exposed GSL DISCUSSION strongly labeled by Gal oxidase/NaB3H4in human endothelial Cell adhesion mediated by various mechanisms (see Intro- cells (34), mouse endothelial cells, and mouse lung microvasduction) has been repeatedly studied under static conditions. cular endothelial cells. (iii) Order of adhesion of the B16 Physiologically, however, adhesion of blood cells (or tumor variants on LacCer- or Gg3-coated plates is the same as their cells) among themselves (aggregation) or to endothelial cells takes place under dynamic flow conditions, i.e. in a moving order of GM3 expression. (iv) Adhesion of B16/BL6 (the bloodstream. While the role of CHOs in cell adhesion based highest GM3 expressor) to human and mouse endothelial on lectins (5-7), selectins (8,9), or GSL/GSL interaction (10) cells is inhibited by pretreatment of BL6 cells with anti-GM3 has been clearly documented, there have been no comparative mAb DH2 or with sialidase (which eliminates cell surface studies of adhesion under static versus dynamic flow condi- GM3). (v) Adhesion of BL6 to human or mouse endothelial tions. In this study, we have (i) demonstrated that adhesion cells is inhibited by pretreatment of the endothelial cell monoof melanoma cells to non-activated endothelial cells is based layer with anti-LacCer mAb T5A7, but not with other mAbs on GMB/LacCer interaction, and (ii)compared this adhesion including anti-ELAM-13B7. Onthe otherhand, pretreatment system to lectin- and fibronectin-dependent adhesion under of BL6 cells with anti-Gal-binding lectinmAb 5D7 did inhibit adhesion (on mouse endothelial cells only), supporting the dynamic flow conditions. There are several lines of evidence that adhesion of B16 previous suggestion that Gal-binding lectin associated with melanoma cells to non-activated endothelialcells depends on B16 melanoma plays some role in BlG/endothelial cell adheGM3/LacCer interaction: (i) GM3 is the major GSL expressed sion (25). Bindingof mAb 5D7 to B16 variants, measured by
17269
Cell Adhesion through Glycosphingolipids in Dynamic Flow System
%
A
3 t
Shear Stress (dynes/cm2)
FIG. 7. Inhibition by various reagents of BL6 cell adhesion to LacCer-coatedplates in a dynamic system. The glass plate in the dynamic system was coated with 200 pg/ml LacCer-liposomes. BL6 cells were harvested, washed, and suspended in PBS (1 X lo6 cells/ml). The cell suspension was mixed with 1 ml of mAb DH2 (10 pg/ml) or 1 ml of sialidase (0.1 unit/ml) at 4 "C for 30 min, washed, resuspended in PBS at 1 X IO5 cells/ml, and injected into the flow chamber. In another experiment, 1 X lo6 BL6 cells were suspended in 9 ml of PBS plus 1 ml of 0.5 M ethyl-p-lactoside, and the cell suspension (1 X lo6 cells/ml) was incubated at 37 "C for 30 min and injected into the flow chamber. Cell adhesion during 3 min at various shear stresses was quantified. 0, untreated BL6 cells (control); A, cells treated with 0.1 M ethyl-p-lactoside; A, sialidase; 0, anti-GM3 mAb DH2. FIG. 6. Adhesion of BL6 cells to glass plates coated with various GSLs or adhesive proteins in dynamic flow and static systems. Panel A , freshly harvested BL6 cells were suspended in PBS (1 X 106/ml) and injected into a dynamic flow system (see text) under various shear stressesas shown on the abscissa. GSL-liposomes (200 pg of GSL/ml) were coated on a marked area (0.5-cm diameter) of the glass plate. Quantity of GSL adsorbed on the marked area was calculated as 31.3 5.2 ng/mm2. After 3 min of flow, the number of adherent cells on the marked area was counted. 0, Gg3; A, LacCer; 0,GM3; V, PG; A, control liposome without GSL. Panel B, procedure as in panel A, except that glass plates were coated with various adhesive proteinsinstead of GSL-liposomes. Whena 100 pg/ml solution of fibronectin was applied, a quantityof fibronectin adsorbed on a marked area of 0.5-cm diameter was found to be 12.5 ng/mm2; values for otherproteins were similar. V, ConA (200 pglml); A, Erythrina lectin (200 pg/ml); 0, fibronectin (100 pg/ml); V, laminin (100 pg/ml); A,ConA (20 pg/ml); 0,Gg3 (200 pg/ml) (for comparison with panel A ) . Panel C, glass plates were coated with ConA, fibronectin, and Gg3 at the same concentrations as in panels A and B and tested under static conditions. Labeled BL6 cells (1 X lo6; 180,000 cpm) were added to each plate and incubated a t 37 "C for various durations as shown on the abscissa. V, ConA (200 rg/ml); A, ConA (20 pglml); A, fibronectin (100 pglml); 0, Gg3 (200 pg/ml).
*
cytofluorometry, was minimal. Therefore, the role of Galbinding lectin in adhesion of melanoma cells to endothelial cells appears to be minor compared to the role of GM3/ LacCer interaction. While BL6 adhesion to LacCer- or Gg3-coated plates was evident at an earlier time than adhesion to fibronectin- or laminin-coated plates under static conditions, maximal strength of fibronectin-, laminin-, or ConA-mediated adhesion was greater than that of adhesion based on GSL/GSL interaction. Adhesion under dynamic flow conditions differed in many important respects. In the dynamic system, BL6 adhesion to LacCer- or Gg3-coated surfaces was stronger than integrin-dependent adhesion to fibronectin- or laminincoated surfaces, and adhesion to ConA or Erythrina lectincoated surfaces was negligible at low concentration (20 pg/ ml) even at low shear stress, but became evident at high concentration ( ~ 2 0 pg/ml). 0 In the staticsystem, as little as 10-20 pg/ml of these lectin8 was sufficient to induce strong cell binding or spreading. Similarly, in the staticsystem, BL6
Wall S n s u Stress
(dymlcd)
FIG. 8. Inhibition by various reagents of B16 cell adhesion to mouseendothelial cells in a dynamic system. Mouse endothelial cells were cultured on glass plates which were then placed in the flow chamber (see text). B16 cells (1X 105/ml) in PBS were passed through the chamber, and thenumber of adherent cells was counted. Panel A, adhesion of untreated B16 variants. 0, BL6; A, F10; 0, F1; A, WA4. Panel B, adhesion of BL6 cells treated with: A, mAb DH2; A, lactose (0.1 M); 0, ethyl-S-lactoside (50 mM); 0, control (no treatment).
cells adhered strongly to plates coated with 10 pg/ml fibronectin or laminin; however, an incubation period of >30 min was required for this adhesion process, in contrast to therapid adhesion based on GSL/GSL interaction. In the dynamic system, B16 cell adhesion to fibronectin- or laminin-coated glass surfaces was negligible evenat anfibronectin or laminin concentration of 100 pg/ml, and even at low shear stress (c1.0 dynes/cm2). B16 cells did not adhere to glass plates coated with GM3-liposomes, PG-liposomes, and control PC-cholesterol liposomes (lacking GSL). In these experiments, liposomes were applied on glass and air-dried in a refrigerator (see "Experimental Procedures"). The lack of adhesion to GM3 and other liposomes indicates that B16 cell adhesion to LacCer-liposome-coatedplates is highly specific. Blood-borne tumor metastasis is generally believed to be initiated by adhesion of tumor cells to microvascular endothelial cells (36,37) andto activate platelets, leading to tumor
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Cell Adhesion throughGlycosphingolipids in DynamicFlow System
cell-platelet or tumorcell-neutrophil aggregation, a major cause of microembolism and metastatic deposition. Expression of selectin GMP-140, which recognizes the tumor-associated antigens sialosyl-Le” and sialosyl-Le’,may play an important role in tumor cell adhesion and aggregation (38, 39). Results of the present study make clear the pathobiological significance of GM3/LacCer interaction as the basis for specific adhesion of melanoma cells to non-activated endothelial cells under dynamic flow conditions. The possibility that this represents the initial event in tumor cell metastasis is suggested by the apparent correlation of strength of adhesion (under high shear stress) with relative metastatic potential of the melanoma cells. Recent interest has been focused on the possible mediation of tumor cell adhesion by ICAMs or selectins which are expressed on activated endothelialcells (16, 17). For this process, a sufficient local concentration of factors secreted from tumor cells (e.g. TGFP or TNFa) is required to activateendothelial cells in situ. In dynamic microvascular flow, adhesion of circulating tumorcells to nonactivated endothelial cells is a prerequisite for the process of endothelial cell activation by tumor cells, and therefore an essential step in initiation of metastasis. Tumor-associated lectins which recognize the Gal residue of N-acetyllactosamine have been assumed to play a role in such adhesion (25, 40), and Gal-binding lectin onendothelial cells has been claimed to mediate adhesion of tumor cells to endothelial cells (41). It is highly plausible that initial adhesion of tumor cells, under physiological dynamic flow conditions, to non-activated endothelial cells is based on GSL/GSL interaction, which induces activation of endothelial cells, and is subsequently reinforced by induction of selectin or ICAM expression on activatedendothelial cells. This trend is particularlypronounced in a dynamic adhesion system, where cell adhesion mediated by GSL/GSL interaction is greater than that mediated by glycoprotein CHO/lectin interaction, and integrindependent adhesion is relatively minor. It should be noted that BL6 adhesion to LacCer strongly stimulated cell migration, in agreement with our previous report (35), thereby promotingtransendothelial migration. Adhesion of tumor cells to endothelial cells could induce activation of endothelial cells through TGFP or TNFa. Thus, a series of “cascade” reactions could be triggered by GSL/GSL interactionbetween tumor cells and non-activated endothelial cells. In vivo metastasis based on GSL/GSL interaction can be blocked by oligosaccharides, GSL derivatives, or GSL-liposomes. Studies
based on this approach are inprogress. Acknowledgments-Drs. Michael Lawrence and Timothy Springer (Dana Farber Cancer Research Institute, Boston, MA) provided two of theauthors (N. K. and K. H.) with “hands-on” training for construction and assembly of the dynamic flow adhesion system used at TheBiomembrane Institute. We also thank Dr. Stephen Anderson for scientific editing and preparation of the manuscript. 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41.
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