Proc. Natl. Acad. Sci. USAVol. 81, pp. 5767-5771, September 1984Cell Biology

Localization of the gangliosides GD2 and GD3 in adhesion plaquesand on the surface of human melanoma cells

(melanoma ganglioside antigens/adhesion plaques)

DAVID A. CHERESH, JOHN R. HARPER, GREGOR SCHULZ, AND RALPH A. REISFELD

Department of Immunology, Scripps Clinic and Research Foundation, 10666 North Torrey Pines Road, La Jolla, CA 92037

Communicated by Nathan 0. Kaplan, June 7, 1984

ABSTRACT The predominant gangliosides produced bytwo cultured human melanoma cell lines are GD3 and/or GD2.These gangliosides were found to be cell associated and presentin substratum-attached material after cell removal by EDTA.Monoclonal antibodies directed to GD2 and GD3 specified thecell-surface distribution of these gangliosides and localizedthem in focal adhesion plaques at the interface of cells andtheir substratum. These attachment sites did not represent in-discriminant membrane fragments remaining after removal ofcells with EDTA, because neither melanoma-associated pro-teoglycan nor class I histocompatibility antigens were detectedby their respective antibodies. Our data suggest that the disial-ogangliosides GD2 and GD3 may be involved in the interactionbetween human melanoma cells and solid substrata.

The development of murine monoclonal antibodies (mAbs)that react specifically with antigens on human tumor cellshas led to a more complete understanding of the topographyand molecular profile of these tumor cell-surface markers.Such antibodies have also aided in defining functional prop-erties of various tumor-associated glycoproteins (1-3), pro-teoglycans (4, 5), as well as glycolipids (6-9).Recent technological advances have facilitated the charac-

terization of mAbs directed to the carbohydrate moieties oftumor-associated gangliosides (6-9). In this regard, mAbsthat react with such molecules associated with neuroblasto-ma (10), melanoma (7, 9), as well as carcinoma of the colon(8) have been described. The use ofmAbs specifically direct-ed to gangliosides may help to strengthen and extend obser-vations that implicated these molecules as putative cellularreceptors for hormones (11) and toxins (12) as well as to gainfurther evidence for their possible role in cell-substratuminteractions (13, 14).

Previous studies have demonstrated that the gangliosidesGD3* (7) and GD2 (16) are highly enriched in tumors of neu-roectodermal origin. Specifically, two murine mAbs pro-duced against cultured melanoma cells were reported to re-act with GD3 (7, 17), and a recently described human mAbwas shown to react with GD2 (16). In the present study, wedemonstrate that GD2 and/or GD3 are major gangliosidesproduced by two human melanoma cell lines and that thesemolecules are deposited in the substratum-attached materialof these tumor cells. We show that two murine mAbs (126and MB3.6) produced in our laboratory react with GD2 andGD3, respectively. Using these mAbs, we were able to de-fine the topographical distribution of GD2 and GD3 on thehuman melanoma cell surface and to localize these ganglio-sides in focal-adhesion plaques, thus further implicating gan-gliosides as important molecules in cell-substratum interac-tions.

MATERIALS AND METHODSCell Lines and Tissues. The human melanoma cell lines

M14 and M21 were provided by D. L. Morton (University ofCalifornia, Los Angeles), and the Melur cell line was a giftfrom Ursula Koldovsky (University of Dusseldorf, F.R.G.).Portions of freshly explanted malignant neuroblastoma tis-sues were obtained from A. Yu and F. Kung (Department ofPediatric Oncology, University of California, San Diego).The M14 cells were grown in serum-free chemically definedmedium (Centaurus, Santa Ana, CA); the M21 cells werepropagated in RPMI 1640 (GIBCO) medium supplementedwith 10% fetal calf serum. The Melur cells were grown inDulbecco's modified Eagle's medium supplemented with10% fetal calf serum.mAbs. mAb 126 was produced against human neuroblasto-

ma cells in our laboratory and was shown to be of IgM iso-type and reactive with tumors of neuroectodermal origin.mAb MB3.6 was produced against the FM9 human melano-ma cell line established in our laboratory and was deter-mined to be of IgG3 isotype. The mAb 9.2.27 developed ear-lier in our laboratory specifically recognizes a melanoma-as-sociated chondroitin sulfate proteoglycan (4). mAb W6/32directed to common determinants of all HLA-A, -B, and -Cantigens was provided by P. Parham (Stanford University,Palo Alto, CA), and mAb 3E3 directed to human fibronectinwas a gift from M. Pierschbacher (La Jolla Cancer ResearchFoundation, La Jolla, CA).

Preparation and Purification of Gangliosides. Biopsy ma-terial or a known volume of cultured tumor cells was homog-enized in 20 vol of chloroform/methanol (2:1) for 5 min withmild sonication and Vortex mixing. The homogenate was fil-tered through a scintered glass filter, the residue was re-ex-tracted as described above with 10 vol of chloroform/meth-anol (1:1) refiltered, and the filtrates were combined, evapo-rated, and dissolved in 10 ml of chloroform/methanol (2:1).Gangliosides were partitioned into an aqueous phase as de-scribed by Ledeen and Yu (30). This material was dialyzedexhaustively against cold distilled water and lyophilized.The lyophilized crude ganglioside preparation was furtherpurified as follows: it was dissolved in 50 ml of methanol/chloroform/water (60:30:8) and applied slowly to a column(1 x 15 cm) of DEAE-Sepharose CL-6B (Pharmacia). Thecolumn was washed extensively with the above solvent andgangliosides were eluted with methanol/chloroform contain-ing 0.8 M aqueous sodium acetate (60:30:8). Fractions werecollected and assayed for the presence of gangliosides byTLC using a variety of ganglioside standards. Fractions con-taining gangliosides were evaporated and dissolved in dis-tilled H20 to be dialyzed and lyophilized. The freeze-driedmaterial was dissolved in chloroform/methanol (1:1) and ap-

Abbreviation: mAb, monoclonal antibody.*Gangliosides are termed according to the nomenclature of Svenner-holm (15).

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plied to a column of latrobeads as described by Ledeen andYu (30). The material eluted from this column was consid-ered relatively free from contaminants.Removal of Attached Cells from the Substrata. Monolayers

of melanoma cells grown on plastic tissue culture dishes(Falcon) or glass coverslips (S/P) were treated with phos-phate-buffered saline (pH 7.2) containing EDTA (1.4 mg/ml)and KCl (0.16 mg/ml) at room temperature for 5-10 min untilmicroscopic examination indicated that all cells had been re-moved. The surfaces were washed three times with phos-phate-buffered saline to remove any residual cells.

Biosynthetic Labeling of Melanoma Gangliosides. 3H-la-beled gangliosides were isolated from melanoma cells grownin their respective media, supplemented with [3H]glucosa-mine (New England Nuclear). Details of the growth condi-tions have been described (9). The substratum-associatedgangliosides were removed by scraping the tissue culturedishes with a rubber spatula in the presence of methanol.Ganglioside material was then extracted by the addition ofchloroform so that the ratio of chloroform to methanol was2:1. The ganglioside purification was carried out as de-scribed above.

Ganglioside Standards. The purified ganglioside standardsGD2, GDlb, GTlb, and GQ1b were kindly supplied by R. K.Yu (Yale University, New Haven, CT). Purified melanomaGD3 was a gift from S. Hakomori (Fred Hutchinson CancerResearch Institute, Seattle, WA). GM3 and GM2 were sup-plied by J. Sundsmo from our institution, and GM1 and GDiawere purchased from Supelco (Bellefonte, PA).ELISA Binding Assay. Reactivity of mAb was analyzed by

serological means using a solid-phase ELISA. Target cellswere dried onto 96-well flat-bottom polyvinyl microtiterplates (Dynatech, Alexandria, VA), and the ELISA bindingassay was carried out as described by Harper et al. (18).Spent culture supernatant containing mAb was diluted to -5,ug/ml in phosphate-buffered saline/0.1% bovine serumalbumin/0.02% Tween 20.For binding inhibition studies, total melanoma ganglio-

sides containing 3 nmol of each ganglioside standard weredried in tubes and incubated for 1 hr at 25°C with 200 ,ul ofhybridoma supernatant (10 ug per ml of antibody). After abrief centrifugation of the supernatant, the antibody was ap-propriately diluted and added to microtiter plates containingappropriate target cells. Antibody reactivity was measuredby ELISA and compared with control antibody that waspreincubated without gangliosides.TLC. Silica gel plates (plastic backed; Merck) were acti-

vated by heating at 110°C for 1 hr. Chloroform/methanol/0.2% aqueous CaCl2 (60:45:10) was used for the develop-ment of chromatograms. Samples were spotted 1.5 cm fromthe bottom of the TLC plates, which were then placed in adeveloping tank presaturated with 100 ml of the solvent de-scribed above. Chromatographs were developed for 1.5 hr atroom temperature, and then the plates were allowed to dry.Appropriate lanes of chromatograms were cut and sprayedwith resorcinol reagent to visualize gangliosides (19).Immunostaining of Gangliosides Separated by TLC. The re-

activity of mAbs 126 and MB3.6 with gangliosides separatedby TLC was determined directly by using a modification ofan immunostaining method originally described by Magnaniet al. (20). As described (9), we modified this procedure byusing an ELISA detection system.

Detection of GD2 and GD3 on Cultured Melanoma Cells byIndirect Immunofluorescence. Approximately 2 x 104 mela-noma cells (Melur and M21) were suspended in 2 ml of ap-propriate growth medium and allowed to attach to glass cov-erslips for 48 hr at 37°C in a humidified incubator containing5% CO2. The coverslips were rinsed with Hanks' balancedsalt solution and treated with 3% paraformaldehyde inHanks' balanced salt solution for 30 min at 25TC. After fixa-

tion, the cells were washed in Hanks' balanced salt solutioncontaining 0.1 M glycine followed by two additional washesin Hanks' balanced salt solution. The coverslips containingthe fixed melanoma cells were overlayed with 0.2 ml of (hy-bridoma supernatant) mAb containing spent culture mediumfor 1 hr at 25TC, followed by three washes in Hanks' bal-anced salt solution. Fluorescein-conjugated goat anti-mouseIgG and IgM (Bio-Rad) diluted 1:50 in Hanks' balanced saltsolution (0.2 ml) was then allowed to react with cells for 1 hrat 25TC. After three additional washes in Hanks' balancedsalt solution, the coverslips were mounted in 90% (vol/vol)glycerol buffered to pH 8.0 with 0.1 M Tris-HCl, and thecells were observed through a Zeiss epifluorescent micro-scope and photographed. To examine focal-adhesionplaques, cell-coated coverslips were removed with EDTAand stained as described above.

RESULTSGanglioside Profile of Melanoma Cells and Substrate-At-

tached Material. Gangliosides labeled intrinsically with[3H]glucosamine were isolated from M21 or Melur melano-ma cells and separated by TLC. The autoradiogram depict-ing the ganglioside profile of each cell line appears in Fig. 1.The M21 cells and their substrate-attached material containabundant amounts of GD3 and GD2, as represented by theprominent doublets (lanes 1 and 2). The Melur cell line pro-duces large amounts of GD3; however, GD2 appears to be arelatively minor component (Fig. 1A, lanes 1 and 2). Thus,the gangliosides deposited onto the substrate compare readi-ly to those associated with the cells themselves.mAbs 126 and MB3.6 React Directly with Specific Ganglio-

side on Thin-Layer Chromatograms. To demonstrate anti-body-binding specificity, we attempted to directly visualize

A; 13

*.

GD; 3

1 2

FIG. 1. Ganglioside profile of melanoma cells and their substra-tum-attached material. 3H-labeled gangliosides were isolated fromMelur and M21 melanoma cells and their substrate-attached materi-al. Labeled gangliosides (8 x 103 cpm) were spotted onto TLCplates, separated, and sprayed with EN3HANCE (New EnglandNuclear) before exposing to x-ray film for 72 hr. (A) Lane 1, Melurcells; lane 2, Melur substrate-attached material; (B) lane 1, M21cells; lane 2, M21 substrate-attached material. The braces corre-spond to the migration of GD2 and GD3 standards.

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GM,- 0

GD,,,- O 0

GTi- 9

_ -

A B C D E F G

FIG. 2. Reactivity of mAbs 126 and MB3.6 with gangliosides onthin-layer chromatograms. Gangliosides purified from melanoma orneuroblastoma cells from a 1-,ul packed cell equivalent were separat-ed by TLC along with 0.1 pg of authentic GD2 and GD3. Visualiza-tion was achieved either by resorcinol spray or by immunostainingwith mAbs 126 and MB3.6. Lane A, ganglioside standards; lane B,gangliosides from melanoma cells (M14) (lanes A and B were stainedwith resorcinol); lane C, authentic GD2; lane D, gangliosides frommelanoma cells (Melur); lanes E and F, gangliosides from a humanneuroblastoma tumor; lane G, authentic GD3. Lanes C-E and F andG were immunostained with mAbs 126 and MB3.6, respectively.

the reactivity of mAbs 126 and MB3.6 on TLC plates, whichcontained total ganglioside extracts from either melanoma orneuroblastoma cells, as well as authentic GD2 and GD3, sep-arated by TLC and then subjected to immunostaining. Asshown in Fig. 2, mAb 126 specifically reacted with authenticGD2 (lane C), which comigrated with a minor component in aganglioside extract from melanoma cells (lane D). A ganglio-side extract from a neuroblastoma tumor biopsy showed arelatively strong immunostaining pattern, also correspond-ing to the migration of GD2 (lane E). mAb MB3.6, however,reacted with both authentic GD3 (lane F) and a prominentcomponent of total melanoma gangliosides whose migrationwas comparable to that of the GD3 standard.mAbs 126 and MB3.6 React Directly with Purified Ganglio-

side Standards. To further determine the specificity of mAbs

126 and MB3.6, ganglioside standards were tested for theirability to inhibit binding of these mAbs to tumor-cell targets,as measured by a competitive ELISA. As shown in Fig. 3A,the ganglioside GD2 Ga1NAc/31-4(NeuAca2--8SNeuAca2-+3) Galp1-+4Glc-ceramide specifically inhibited binding ofmAb 126 to melanoma cell targets. We could observe a mi-nor cross-reactivity of mAb 126 with other ganglioside com-ponents with a similar core structure; however, the bindingof mAb MB3.6 was essentially inhibited in this assay only bythe ganglioside GD3 NeuAca2--8NeuAca2-*3Gal31-4Glc-ceramide (Fig. 3B). The sole structural difference betweenthese two gangliosides is the terminal GalNac present onGD2. Thus, it is apparent that the terminal sugars most likelyplay a major role in the antigenic determinants defined bythese antibodies.

Localization of GD2 and GD3 on the Melanoma Cell Sur-face. Melanoma cells (M21 and Melur) were grown on glasscoverslips, and the topographical distribution of the antigensGD2 and GD3 was determined by indirect immunofluores-cence. As shown in Fig. 4A, the immunofluorescent stainingpattern of mAb 126 on Melur cells indicates that GD2 ap-pears to be mainly associated with the plasma membrane. Inaddition, GD2 as defined by mAb 126 also seems to be local-ized in microspike projections that emanate from the cellsurface and extend toward other cells as well as the glasssubstratum. Only a small proportion of cells of the Melur linecontained GD2 < 50% as demonstrated by this procedure(data not shown). The topographical pattern of GD3 on Me-lur cells as visualized by immunofluorescent staining withMB3.6 is represented in Fig. 4C and indicates that GD3 ap-pears in clusters on the surface of >90% of these cells. M21melanoma cells were also stained with each antibody andexhibited prominent surface staining of GD2 (t1000 posi-tive) and >80% positive for GD3 (Fig. 4 E and G, respective-ly).

Localization of GD2 and GD3 on Melanoma-Associated Ad-hesion Plaques. To visualize GD2 and GD3 in melanoma-as-sociated focal adhesion plaques, either M21 or Melur cellswere grown on glass coverslips, removed with EDTA, andthe remaining substrate-attached material was stained by in-direct immunofluorescence with mAbs directed against therespective gangliosides. Typical adhesion plaques of Melurcells (Fig. 4 B and D) or M21 cells (Fig. 4 F and H) can bedetected with either of'these antibodies (mAb 126, Fig. 4 Band F; mAb MB3.6, Fig. 4 D and H). In contrast, mAbW6/32 (Fig. 5C), known to react with a determinant com-mon to essentially all class I histocompatibility antigens on

GM3GM2GM,GD3lGDTaG02

GT~brGQlbl

^ ^ n o~~~~~~~~nanU 1 0 20 JU

% Inhibition

FIG. 3. Reactivity of mAb 126 and MB3.6 with individual ganglioside standards. mAb 126 (A) or mAb MB3.6 (B) was preincubated withindividual ganglioside standards and then tested for its ability to bind to melanoma cell (Melur) targets by ELISA. Reactivity of each antibodywith individual gangliosides is expressed as the percentage of inhibition of antibody binding compared to that obtained with controls that werepreincubated in the absence of standard gangliosides.

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-w

FIG. 4. Detection of GD2 and GD3 on human melanoma cells byindirect immunofluorescence. Melur (A-D) or M21 (E-H) melano-ma cells (2 x 104) were grown on coverslips, fixed, incubated witheither mAb 126 or MB3.6, and then stained by indirect immunofluo-rescence. Melur cells or M21 cells were stained with mAb 126 (A, B,E, and F) or MB3.6 (C, D, G, and H). Adhesion plaques of respec-tive cell types appear in B, D, F, and H.

the plasma membranes of melanoma cells, failed to reactwith these adhesion plaques (Fig. 5D). In addition, mAb9.2.27 (Fig. 5 A and B) directed to an epitope on a melanoma-associated chondroitin sulfate proteoglycan and mAb 3E3 di-rected to fibronectin (data not shown) also failed to localizein these membrane attachment sites.

DISCUSSIONEvidence is presented in this report indicating that the disial-ogangliosides GD2 and GD3 are produced by human melano-ma cells. We also show with two mAbs that GD2 and GD3are not only components of the plasma membrane, but arealso localized in adhesion plaques at the interface betweenthese cells and the substratum to which they are attached.The contents of substratum-attached material have beenchemically analyzed after the removal of cells by chelatingagents (21, 22) and/or detergents (23). Culp and Black (22)reported that removal of normal or simian virus 40-trans-

FIG. 5. Detection of proteoglycan and histocompatibility anti-gens on human melanoma cells by indirect immunofluorescence.Melur melanoma cells were grown on glass coverslips and stained asdescribed in Fig. 4, using mAbs 9.2.27 (A) or W6/32 (C) as primaryantibodies. The coverslips were stained with these same antibodiesafter the cells were removed with EDTA as described in Fig. 4. (B)9.2.27; (D) W6/32. mAb 9.2.27 was applied as spent culture medium(10 ,ug/ml), whereas mAb W6/32 was used as ascites fluid diluted inHanks' balanced salt solution to -10 4g/ml.

formed BALB/c 3T3 cells with EGTA left behind substrate-attached material, enriched in such extracellular matrix com-ponents as glycosaminoglycans and fibronectin, as well asthe cytoskeletal proteins actin and myosin. Moreover, adhe-sion plaques of Rous sarcoma virus-transformed cells wereshown by indirect immunofluorescence to contain the srcgene product (23).The presence ofGD2 and GD3 in melanoma-associated ad-

hesion plaques is not surprising, because the immunofluo-rescent staining patterns indicate that these disialoganglio-sides represent some of the major constituents of the cellsurface. However, other melanoma membrane antigens alsoknown to be associated with the plasma membrane were notdetectable in these adhesion plaques. Specifically, compo-nents produced by human melanoma cells (i.e., class I histo-compatibility antigens as defined by mAb W6/32, chondroi-tin sulfate proteoglycans determined by mAb 9.2.27, and fi-bronectin detected by mAb 3E3) could not be detected onthe substratum after removal of the cells from the glass cov-erslips, suggesting that these adhesion plaques do not repre-sent indiscriminant pieces of plasma membrane. In fact, nei-ther of these two antigens were detectable by polyacrylam-ide gel electrophoresis of [3H]leucine-labeled extracts ofmelanoma-associated substrate-attached material (data notshown). The apparent absence of melanoma-associated pro-teoglycans and fibronectin in adhesion plaques suggests thatthe attachment of cultured human melanoma cells may notrequire the direct involvement of extracellular matrix com-ponents produced by the cell, as has been suggested for oth-er cell-attachment models (24, 25).

Previous studies from our laboratory have shown that, al-though human melanoma cells synthesize and secrete pro-teoglycan and fibronectin, they lack the ability to organizethese molecules into an extracellular matrix (25). The findingthat gangliosides are specifically deposited in the adhesionplaques of melanoma cells implicates these molecules in analternative adhesion mechanism independent of extracellularmatrix components. In this regard, we have shown that mAb

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9.2.27 has little or no effect on adhesion, while significantlydecreasing cytoplasmic spreading after attachment (26).Blackburn and Schnaar recently reported that specific celladhesion was mediated by immobilized glycolipids (27). Tak-en together, these observations suggest that gangliosidesmay participate in the early events of adhesion and that mol-ecules such as proteoglycans may become involved in eventsimmediately after cell attachment (i.e., spreading and migra-tion). This contention is supported by a recent report thatdescribed the addition of various mAbs to substrate-attachedmelanoma cells, each of which was directed to different mel-anoma-associated antigens. In this case, only anti-GD3 wascapable of causing loss of cell attachment accompanied by achange in morphology, suggesting that GD3 was playing anactive role in the adhesion process of these cells (28). A rolefor gangliosides in cellular adhesion has also been suggestedby other studies that demonstrated increased adhesive prop-erties of cells whose membranes were enriched with exoge-nous gangliosides (14). In addition, Bremer et al. showedthat detergent removal of hamster fibroblasts left an adhe-sioh matrix rich in ganglioside GM3 (29).The data presented in this report indicate that GD2 and/or

GD3 are predominant gangliosides synthesized by two hu-man melanoma cell lines. Furthermore, these gangliosidesare present in the membrane and in focal-adhesion plaques,implicating them as molecules that may play an importantrole in the adhesion ot melanoma cells to substrata.

Note Added in Proof. Recent findings reported by Okada et al. (31)demonstrated that hamster fibroblasts, particularly viral-trans-formed hamster fibroblasts, deposited a "detergent-insoluble sub-strate attachment matrix" relatively rich in the ganglioside GM3.Their study indicated that this ganglioside played a functional role inthe regulation of attachment of these cells. These data are in accordwith our results presented in this report implicating gangliosidesGD2 and GD3 as potentially important molecules in the attachmentof human melanoma cells to solid substratum.

The authors wish to thank Ms. Laura E. Wolff and Susan K. Per-ry for their expert technical assistance and Ms. Bonnie Pratt Filiaultfor her excellent secretarial assistance. This work was in part sup-ported by U.S. Public Health Service Grant CA 28420 from the Na-tional Institutes of Health. D.A.C. and J.R.H. are recipients of Na-tional Institutes of Health Fellowship Awards CA 07544 and CA07222, respectively. G.S. is supported by Deutsche Forshungsge-nIeinschaft Grant I-3-Schu 512/1-1. This is Scripps Publication no.Imm-3385.

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