4948.Full.Pdf

(CANCER RESEARCH 52. 4948-4953. September 15. 1992] Cell Surface Accessibility of Individual Gangliosides in Malignant Melanoma Cells to Antibodies Is Influenced by the Total Ganglioside Composition of the Cells1

Kenneth O. Lloyd,2 Claudia M. Gordon, Immac J. Thampoe, and Cheryl I)iBeni-detto

Memorial Sloan-Ketlering Cancer Center and Cornell University Graduate School of Medical Sciences, New York, New York 10021

ABSTRACT In this study, we have compared the total ganglioside com position of a panel of melanoma cell lines with the ability of a The reactivity of a panel of antiganglioside monoclonal antibodies number of antiganglioside mAbs (anti-GM3, -GM2, -GD3, and with a number of melanoma cell lines having different ganglioside com -GD2) to react with these cell lines. The results show that the position profiles was studied. One cell line synthesized only GM3, one produced both CM3 and GD2, 2 had GM3 and GD3 as their major reactivity of a given mAb with a cell line is dependent not only gangliosides, and 2 others synthesized approximately equal amounts of on the presence or absence of its cognate ganglioside antigen GM3, GM2, GD3, and GD2 gangliosides. Antibody reactivity with but also on the expression of other gangliosides by the target viable cells was analyzed by: (a) flow cytometry on suspension cells; and cell. (i) mixed hemagglutination assays or immune adherence assays on monolayer cells in culture. GM3 was efficiently detected only in the cell line having GM3 as its sole ganglioside. In the other cell lines, GM3 MATERIALS AND METHODS was difficult to detect even in cells in which it made up a high proportion (up to 50%) of the total ganglioside content. GM2 was easily detectable Cell Lines. Human melanoma cell lines SK-MEL-28, - 31, -37, and only in JB-RH melanoma cells (which contain only GM3 and GM2). MeWo have been described (9). B78 is a subclone of the mouse mela GD3 was the most reactive ganglioside in 2 cell lines and GD2 in 2 other noma cell line B16 (10) and was kindly provided by Dr. A. Albino, lines. In general, the most complex ganglioside present in a cell was the Sloan-Kettering Institute. JB-RH is also a mouse melanoma cell line, one most accessible to antibody. The differential exposure at the cell and it was derived by Dr. Jane Berkelhammer, University of Missouri, surface of specific gangliosides may have implications for antibody- Columbia, MO, and subsequently subcloned for high GM2 expression directed tumor detection and therapy and for cell-protein or cell-cell (11). The cell lines were cultured in minimal essential medium supple interactions that involve glycolipids. mented with fetal bovine serum (10%), L-glutamine (2 HIM),nonessen- tial amino acids (1%), and penicillin-streptomycin (100 Mg/ml) as de INTRODUCTION scribed previously (9). Antibodies. mAb M2590 |anti-GM3; IgM (6)], DH2 [anti-GM3; Melanoma cells, as well as other cell types of neuroectoder- IgG3 (12)], and GMR6 [anti-GM3; IgM (13)] were kindly provided by mal origin, are comparatively rich in gangliosides (1). In human Drs. T. Itoh and M. Taniguchi, by Dr. S. Hakomori, and by Dr. T. Tai, melanoma tissue and cell lines, the predominant gangliosides respectively. mAb 3F8 (anti-GD2; IgG3) was a gift from Dr. N-K. are GM33 and GD3, although in some instances GM2 and Cheung (5). mAb R24 (anti-GD3; IgG3) and 5-3 (anti-GM2; IgM) GD2 can also be expressed in substantial amounts (2). mAb,4 have been described previously (11, 14). both mouse and human, have been used extensively in the anal Flow Cytometry. For flow-cytometric experiments, confluent cell cultures were disrupted with 10 min EDTA-PBS, trypsin (0.1%)-EDTA ysis of ganglioside expression in melanoma (1, 3). Moreover, (0.02%), or Pronase (1 Mg/ml in PBS; Calbiochem, San Diego, CA) to mAbs to certain gangliosides have found use in the diagnosis obtain single cell suspensions. Cells (1.0 x 106/ml) were incubated with and therapy of tumors. Specifically, anti-GD3 mAbs have been studied for the therapy of malignant melanoma (4), and anti- saturating concentrations of antibody as follows: M2590 (20 Mg/ml), DH2 (culture supernatant), GMR6 (culture supernatant), 5-3 (ascites GD2 has been applied to both the therapy and diagnosis of fluid, 1:100), R24 (20 Mg/ml), and 3F8 (20 Mg/ml). After incubation at neuroblastoma (5). 0Â°Cfor 45 min, the cells were washed 3 times with PBS-1% bovine Although the reactivity of antiganglioside mAbs with specific serum albumin. They were then incubated for 30 min at 4Â°Cwith cells and tissue is, in general, related to ganglioside composition anti-mouse IgM-fluorescein conjugate (Sigma Chemical Co., St. Louis, of the cells, there are indications that other factors also play a MO) for mAbs M2590, GMR6, and 5-3 and anti-mouse Ig (H- and role in this process. For example, although GM3 ganglioside is L-chain-specific)-fluorescein conjugate (Sigma) for mAbs DH2, R24, widely distributed in a variety of cell types, antibodies to GM3 and 3F8. After washing as described above, the cells were suspended in react selectively with certain melanoma cell lines (6-8). This PBS-1 % bovine serum albumin at 1.0 x 106cells/ml and analyzed in an phenomenon has been partially explained by the high density of Epics Profile II fluorocytometer. GM3 found on melanoma cells (7). Another important param Mixed Hemagglutination and Immune Adherence Assays. MHA eter, which has not been as well studied, may be the influence of and IA assays for cell surface reactivities on adherent cells were carried other components of the cell surface, i.e., glycoproteins and out as described previously (9, 15). The MHA method with protein A-conjugated red blood cells was used for the IgG3 mAbs (DH2, R24, other glycolipids, on antibody reactivity. and 3F8), whereas the IA method was used for the IgM mAbs (M2590 and 5-3). Received 1/28/92; accepted 7/3/92. Glycolipids. Gangliosides were isolated from cells as described pre 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 accord viously (16). Briefly, cell pellets were extracted with chloroform-meth- ance with 18 U.S.C. Section 1734 solely to indicate this fact. anol (2:1, 1:1, and 1:2, sequentially) and the glycolipid fraction was 1Supported by grants from the National Cancer Institute (CA 21445 and CA isolated by Florisil chromatography of the acetylated derivatives (17). 08478). 2 To whom requests for reprints should be addressed, at Memorial Sloan- Neutral glycolipids were separated from gangliosides by DEAE- Kettering, 1275 York Avenue. New York, NY 10021. Sephadex ion exchange chromatography (18). The ganglioside fraction 3 Ganglioside nomenclature is according to Svennerholm (40). was finally isolated by chromatography on a Sep-Pak C|8 (Waters- 4 The abbreviations used are: mAb, monoclonal antibody; PBS, phosphate- buffered saline, 0.15 M NaCI-0.05 M sodium phosphate, pH 7.2; TLC, thin layer Millipore, Milford, MA) column (19). The sialic acid content of the ganglioside fractions was determined by chromatography; Gb, globoside series glycolipid; IA. immune adherence: MHA, hydrolysis in 0.1 N HC1 at 80Â°Cfor l h and separation of the released mixed hemagglutination. 4948

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1992 American Association for Cancer Research. ACCESSIBILITY OF (Â¡ANGLIOSIDES IN TUMOR CELLS sugars was done with high performance aniÃ³nexchange chromatography using a pulsed amperiometric detector (Dionex Corp., Sunnyvale, Resorcinol B Anti - GD2 CA) according to a modification (20) of the method of Hardy et al. (21). Fatty acid composition was determined after methanolysis in l N HC1 in methanol at 80Â°Cfor 6 h, and analysis of the released fatty acid GM3- esters by gas chromatography-mass spectometry using a Delsi-Nermag GM2- (Houston, TX) spectrometer. GD3- Thin Layer Chromatography. Thin layer chromatography of the iso GD2- lated gangliosides was carried out in chloroform-methanol-0.2% CaCl2 (60:40:9) on high performance silica gel G TLC plates (E. Merck), and the gangliosides were visualized with resorcinol-HCI. Plates were then 1 23456 1 2 3 scanned with a Shimadzu TLC scanner, and the percentage of individ Fig. 1. A, ganglioside patterns of 6 melanoma cells determined by TLC and ual gangliosides determined by comparison with known amounts of visualization with resorcinol-HCI reagent. Lane I, B78; Lane 2, JB-RH; Lane 3, gangliosides. TLC immunostaining was performed using aluminum- SK-MEL-28; Lane 4, MeWo; Â¡Mne5, SK-MEL-37; Lane 6, SK-MEL-31. Ap backed silica gel G plates (E. Merck) as described (16), except that the proximately 6 ng of NeuAc were applied per Lane except for Lane 2, which contained 2 un NeuAc. B, TLC immunostaining of gangliosides from melanoma reactivity was visualized by using rabbit anti-mouse IgG-horseradish cells with anti-GD2 antibody (3F8): Lane I. B78; Lane 2. JB-RH; Lane 3, SK- peroxidase as the second reagent. MEL-28; Lane 4, MeWo; Lane 5, SK-MEL-31; Lane S, standard mixture of Radiolabeling of Cell Surface Components Containing Sialic Acid. GM3, GM2, GD3, GD2, GDla. and GTlb gangliosides. Approximately 6 Mg Melanoma cells were specifically labeled in cell surface sialic acid res NeuAc were applied per Lane. idues using a slight modification of the method of Gahmberg and Andersson (22). A monolayer culture of melanoma (grown in a T-75 Table 1 Ganglioside composition of panel of melanoma cell lines" flask) was mixed with PBS and after cooling the flask to 4Â°C,3.0 ml of 2 mMsodium periodate in PBS were added. After incubating at 4Â°Cfor Ganglioside (%) 30 min, 3 ml of 100 IHMglycerol-PBS were added. After 5 min at room CelllineB78JB/RHSK-MEL-28MeWoSK-MEL-37SK-MEL-31GM380e4951461924GM2047WV2015GD3003236IS19GD200VV1936Other*2041718273 temperature, the cell layer was rinsed 4 times with PBS. Sodium [3H]- borohydride (0.4 mCi, 7.7 Ci/mmol; New England Nuclear-Dupont, Boston, MA) in PBS (3 ml) was then added and allowed to react for 30 min at room temperature. Unlabeled sodium borohydride (5 mg/ml, 1.0 ml) was then added. After 5 min, the cell layer was rinsed gently with 4 " Determined by TLC and detection of gangliosides by resorcinol-HCI spray as portions of PBS. The cells were then detached from the flask by scrap described in "Materials and Methods." ing, and acidic glycolipids were extracted from the cell pellet as de '' Unidentified gangliosides and nonspccifically stained components. scribed above and analyzed by TLC and fluorography (16). ' Percentage of total ganglioside in each cell type (average of 2 experiments). ''Small amounts of GD2 could be detected by TLC immunostaining with mAb 3F8. RESULTS Ganglioside Profiles of Melanoma Cell Panel. The total gan- The fatty acid composition of the gangliosides (GM3 and glioside profile of 6 melanoma cell lines was determined by GD3) isolated from SK-MEL-28 cells was also determined. TLC and visualization with resorcinol-HCI (Fig. \A). The TLC The major fatty acids in GM3 were C16:0 (11.9%), C18:l plates were also scanned and the percentage of individual gan (14.1%), C 18:0 (8.3%), C22:l (62.5%), C24:l (1.3%), and gliosides determined (Table 1). The identity of the individual C24:0 (1.9%), and in GD3 were C 16:0 (17.6%), C 18:1 (7.9%), gangliosides was confirmed by antibody immunostaining5 C18:0 (15.9%), C22:l (33.7%), C24:l (15.7%), and C24:0 (Fig. \B). The cell lines were chosen for their range of gangli- (9.7%). The data on the fatty acid composition of GD3 differ oside composition. Thus, mouse B78 cell line has GM3 as its from those presented by Nudelman et al. (23), who found a only detectable ganglioside; mouse JB-RH has both GM3 and predominance of C24 fatty acids. The reason for this discrep GM2 in approximately equal amounts. SK-MEL-28 and ancy is unclear, but it could be due to differences in culture MeWo have GM3 and GD3 as their major ganglioside compo conditions or isolation procedures. nents [this pattern is typical of the majority of human mela Reactivity of Antiganglioside Antibodies with Melanoma noma cell lines and tumor tissue samples (1-3)]. Even though Cells as Analyzed on Suspension Cells by Flow Cytometry. The GD2 could not be detected in these 2 cell lines on TLC by reactivity of the panel of antiganglioside mAbs with cell sus resorcinol-HCI reagent, small amounts of GD2 could be de pensions of the 6 melanoma cell lines described in the previous tected by immunostaining with anti-GD2 mAb (Fig. 1Ã„);the section was analyzed by flow cytometry. Typical patterns are level of GD2 detected was estimated to represent less than 1% shown in Fig. 2, and quantitative data are given in Table 2. of the total cell ganglioside. SK-MEL-31 and -37 are human Several interesting aspects of the reactivity are evident. For melanoma cell lines showing more complex ganglioside pat most of the cell lines, only one mAb reacted with the majority terns with GM3, GM2, GD3, and GD2 all being expressed in of the cells in the culture with strong uniform staining. For substantial amounts. SK-MEL-28 and MeWo, this was mAb R24, reacting with The total ganglioside content of the different cell lines was GD3; for SK-MEL-31, mAb 3F8 (identifying GD2) reacted determined by assaying the amount of sialic acid present in the maximally; and for JB-RH only mAb 5-3 (identifying GM2) extracted ganglioside fractions. The lipid-bound sialic acid con reacted strongly. Only SK-ME-37 was an exception, showing tents of the cell lines were: 0.42 tig/107 cells in B78 cells, 5.07 quite strong immunoreactivity with both mAb R24 (anti-GD3) Mg/107 cells in JB-RH; 4.20 Â¿ig/107cells in SK-MEL-28; 2.72 and 3F8 (anti-GD2). Mouse melanoma cell line B78 reacted Mg/107 cells in MeWo; 5.00 Mg/107 cells in SK-MEL-37; and only with anti-GM3 mAbs (mAbs M2590, GMR6, and DH2). 1.08 Mg/107 cells in SK-MEL-31. The degree of reactivity depended on the antibody used: with mAb DH2, 58.2% of the cells were positive and the intensity of 5 K. O. Lloyd, C. M. Gordon, 1.J. Thampoe, and C. DiBenedetto. unpublished fluorescence was 7.3; with mAb M2590, 55% of the cells were observations. positive with a mean fluorescence intensity of 5.4; and mAb 4949

B 78 SK-MEL-28 " These data were derived from cell suspensions prepared using either an EDTA-containing or trypsin-EDTA buffer. Essen tially the same results were obtained using either procedure. â€¢¿/' The effect of more vigorous protease treatment was examined V by treating the cell suspensions with Pronase (using a maximum of 1 Mgenzyme/ml enzyme, inasmuch as higher concentrations resulted in cell lysis). Under these conditions, the reactivity of the 2 cell lines tested (B78 and SK-MEL-28) with anti-GM3 -*â€¢â€¢¿-.._v.._. was slightly enhanced, but reactivity with anti-GD3 was un changed (data not shown). Reactivity of Antiganglioside Antibodies as Analyzed on Mel anoma Motiola) IT Cells by Mixed Hemagglutination and Im

\ mune Adherence Assays. In order to determine whether the accessibility of cell surface gangliosides to antibodies was the FLUORESCENCE same on monolayer cells as with cell suspensions, the reactivity Fig. 2. HoÂ«c>torneine analysis of cell linci with antiganglioside antibodies using indirect immunofluorescence method. Cell lines: A. D, and G, B78; B. E. with mAbs was studied on melanoma cells growing in the wells and H. JB-RH: C. F, and I. SK-MEL-28. Antibodies: A. B, and C, GMR6 of microtiter plates using two red blood cell rosetting assays: (anti-GM3); D. E, and F. 5-3 (anti-GM2); G, H. and I. R24 (anti-GD3). The either the MHA or the 1A. The former was used for the IgG3 fainter line in each panel is a control representing staining with fluoresceinated- anti-lgG alone. antibodies using protein A-coupled erythrocytes and the latter assay was used for IgM antibodies. In general, the results (Table GMR6 was more strongly reactive, with 90% of the cells being 3) were very similar to those obtained with flow cytometry. Thus, anti-GM3 mAbs were strongly reactive only with B78 positive with an intensity of 10.4. It should be noted that mAb GMR6 shows some cross-reactivity with GDla and GMlb, but cells; JB-RH cells reacted only with anti-GM2 and not with anti-GM3 antibodies, and SK-MEL-31 was reactive only with since these gangliosides are not present in melanoma cells, this anti-GD2 mAb despite having substantial amounts of the other is not a complication of the analysis. Anti-GM3 mAbs DH2. M2590, and GMR6 did not react 3 gangliosides. Accessibility of Gangliosides to Cell Surface Labeling by Per- significantly with any of the other cell lines tested. mAb M2590 iodate-[3H]Borohydride. Since GM3 was poorly recognized reacted with 35% of SK-MEL-37 and GMR6 reacted with 76% of MeWo cells, but the mean intensities were only 2.4 and 2.9, by antibodies in a number of cell lines, the accessibility of this respectively. The low reactivity of anti-GM3 with all the cell ganglioside to chemical reagents was analyzed. Specifically, the lines, except B78, was quite suprising, considering that the radiolabeling of gangliosides by treatment with sodium perio date and sodium [3H]borohydride was analyzed in 3 cell lines GM3 ganglioside content was quite high, ranging from about 50% of total ganglioside content for SK-MEL-28 and JB-RH to (B78, JB-RH, and SK-MEL-28). This procedure has been dem 19% for SK-MEL-37. A similar phenomenon was noted with onstrated to specifically label cell surface components contain some of the other antibodies. Thus, even though SK-MEL-31 ing sialic acid, at least in erythrocytes (22). The results showed had substantial amounts of GD3 (20% of total gangliosides), that GM3 was efficiently labeled in all 3 cell lines (Fig. 3). This anti-GD3 mAb (R24) reacted only weakly with this cell lineâ€” was despite the fact that GM3 was poorly accessible to anti 71% of the cells were positive but the average intensity was only body in JB-RH and SK-MEL-28 cells. GM2 was efficiently 4.1. Likewise, SK-MEL-37 had 19% of its ganglioside content labeled in JB-RH cells and GD3 was radiolabeled, although as GM2, but anti-GM2 (mAb 5-3) reacted only weakly (19.7% not strongly, in SK-MEL-28 cells. These data confirm that positive cells with a mean intensity of 6.8). As expected, anti- these gangliosides are cell surface components in the cell lines GD2 reacted very strongly with SK-MEL-31 and SK-MEL-37, examined. which contain large amounts of GD2. More suprisingly, a high proportion of SK-MEL-28 and MeWo cells, which have ex DISCUSSION tremely low GD2 levels, reacted with anti-GD2, although the intensity of fluorescence was rather low. From these data, it The phenomenon of the "crypticity" of some glycolipids in appears that the reactivity of an antigangliosidc mAb with a the plasma membrane of mammalian cells has been known melanoma cell is strongly influenced by the nature of the other for some time. The classic example is the inaccessibility of gangliosides expressed by the cell. There was a tendency for a globoside, the major neutral glycolipid of adult human eryth mAb to react most strongly with the most complex ganglioside rocytes, to antigloboside antibodies (24). In this instance, treat in the cell when more than one ganglioside was present. ment of the cells with trypsin or sialidase enhances globoside

Table 2 Reactivity of antiganglioside antibodies with melanoma cells as assayed by fluorescence staining and flow cytometry Anti-GM3DH2Cell

lineB78 Pos"58.3* Pos0.558.7 Pos2.6

JB/RH 6.4 2.92.5 2.4 2.2 7.7 12.5 19.8 2.7 6.0 4.4 SK-MEL-28 0.88 2.1 1.3 24.7 1.9 4.4 4.0 97.7 36.7 64.2 2.53.2 MeWo 11.9 3.7 5.8 1.8 76.0 2.9 2.5 4.1 97.6 64.8 61.7 SK-MEL37 3.8 5.5 35.0 2.4 1.2 1.1 19.7 6.8 90.0 21.1 91.1 23.8 SK-MEL-31% 0.4Int.6.3* I.IM2590%Pos55.10.6Int.5.4 1.1GMR6%Pos90.010.41.1Int.10.4 2.0Anti-GM25-3%70.5Anti-GD3R24Int.8.44.1%Pos5.074.1Int.3.4 3.6Anti-GD23F8%97.8Int.3.6 35.5 % Pos. percentage of cell showing positive fluorescence in comparison to staining with lluoresceinated anti-Ig alone; Int. mean fluorescence intensity. h Average of 2 separate experiments. 4950

Table 3 Reactivity of antiganglioside monoclonal antibodies with melanoma cell lines assayed by red blood cell rosetting methods (MHA and IA) Anti-GM3

Cell line M2590" DH2* Anti-GM2, 5-3Â° Anti-GD3, R24* Anti-GD2, 3F8* B78JB/RHSK-MEL-28MeWoSK-MEL-37SK-MEL-3164'(2+f0032

<+)8 (+)16 (+)32 (+)> (+)0128(3+)04 <+)2 1024(3+)1024(3+) (Â±)0128(2+)0004 (+)001024(3+)128(3+)>1024(3+)2 (Â±)00512(3+)32 " Assayed on monolayer cells with IA method. * Assayed on monolayer cells with protein A-red blood cell MHA method. ' Last dilution showing positive cells. d Estimate of strength of reaction: 3+, very strong; 2+, strong; +, weak; Â±,very weak.

I04"-B(3H)4 position), this ganglioside was essentially unreactive with anti- GM3 antibodies. A similar finding was the relatively poor re activity of anti-GM2 antibody with SK-MEL-37 cells and of anti-GM2 and anti-GD3 with SK-MEL-31, even though these cell lines contained substantial amounts of the target antigens. In all cell lines examined, it appeared that the more complex GM3- ganglioside present was the most readily available for reactivity ^^ with antibody. A striking example of this was the ready detec GM2- tion of GD2 in SK-MEL-28 and MeWo cells by anti-GD2 GD3- antibody (albeit with low fluorescence intensity), even though this ganglioside was undetectable in the ganglioside fraction extracted from these cells by chemical means (resorcinol-HCI) and could only be detected by immunostaining of TLC plates with anti-GD2 antibody (Fig. 15). These results show that the organization of gangliosides in 1 2 3 the plasma membrane influences the exposure of a specific ganglioside at the cell surface. Since the effect is found on cell Fig. 3. Fluorogram of TLC plate showing cell surface radiolabeling of gangliosides with sodium periodate-['H]borohydride method. Lane l, B78; Lane 2, suspensions, as well as with monolayer cells, it appears unlikely JB-RH; Lane 3, SK MEL-28. that some gross change in membrane architecture or interfer ence by membrane proteins is responsible for this phenomenon. Also the possibility that the undetected gangliosides are not expression, showing that membrane proteins, glycoproteins, or found in the plasma membrane, but are located intracellularly, gangliosides may be shielding the globoside molecules (24). was discounted by showing, in 3 cell lines, that GM3 was avail Similarly, Stein et al. (25) showed that GM1 expression in able at the cell surface for radiolabeling by small chemical re murine lymphocytes is enhanced after treatment of the cells agents that are thought not to penetrate the cell (22). with trypsin, and Urdal and Hakomori (26) found that the Although this phenomenon has not been studied systemati expression of Cer in murine lymphoma cells Gg3 is greatly cally, previously published studies indicate that it has been ob influenced by existing glycoconjugates. Cell surface glycolipids served in other systems also. For instance, Kannagi et al. also vary in their degree of exposure to enzymes such as (29) studied the reactivity of an antigangliotriosylceramide neuraminidase (27) and galactose oxidase (28). In the studies (GgjOse) antibody with sublines of a mouse lymphoma cell line presented here on melanoma cells (summarized in Fig. 4), the and showed that its reactivity was reduced in sublines also ex effect is more specific in that we show that the accessibility of a pressing gangliotetraosylceramide and GM1. Other examples, given ganglioside to antibody is influenced by the coexpression although not commented on by the authors, can be noted in the of other gangliosides in the cell. The phenomenon is unlikely to study of Kawashima et al. (30) on the characteristics of anti- be due to the influence of other cell surface glycoproteins, in GD2 antibodies reacting with melanoma and neuroblastoma asmuch as protease treatments had little effect on the results. cell lines. They showed quite strong reactivity of an anti-GD2 The possibility that the carbohydrate structures detected by the antibody (A 1-425) with MeWo and G361 cells even though various antibodies are present on glycoproteins, as well as on GD2 was very low or undetectable on TLC plates by resorcinol- glycolipids, is also unlikely since Pronase treatment enhances, HCI reagent. rather than diminishes, antibody reactivity. Moreover, our stud Although the presence of other gangliosides in the cell mem ies have failed to demonstrate reactivity of antiganglioside an brane seems to affect the reactivity of a specific ganglioside with tibodies with cell surface glycoproteins on melanoma cells using a variety of approaches.6 its cognate antibody, the mechanism underlying this phenom enon is still uncertain. An obvious explanation would be that The observed phenomenon was most marked with GM3. the longer gangliosides physically block accessibility to the Thus, although all the melanoma cell lines examined contained shorter gangliosides, particularly when the glycolipids are clus GM3, only in B78 cells (in which GM3 is the sole ganglioside present) was GM3 readily available to antibody. In JB-RH and tered together in the membrane. In fact, Kannagi et al. (29) SK-MEL-28 cells, in which GM3 is one of 2 major gangliosides proposed such a mechanism to explain the lower reactivity with present (representing about 50% of the total ganglioside com- antibody of gangliotriosyl ceramide in some sublines of a mouse lymphoma cell line as discussed above. It seems unlikely, how ever, that a glycolipid longer than the inaccessible glycolipid by 6'K. O. Lloyd, unpublished observations. one sugar unit (e.g., GM2 versus GM3, or GD3 versus GM3) 4951

B78 JB/RH SK-MEL-28 MeWo SK-MEL-37 SK-MEL-31 Fig. 4. Summary of ganglioside content and immunoreactivity (flow cytometry) of mela noma cells. A, ganglioside composition (per CD cent of total ganglioside in cell line), l, GM3; O) 2, GM2; 3, GD3; 4, GD2. fi. flow-cytometric m analysis with antibodies (percent of cells posi Â£ tive compared with negative control). 1. DH2 o (anti-GM3); 2, 5-3 (anti-GM2); 3, R24 (anti- B B78 JB/RH SK-MEL-28 MeWo SK-MEL-37 SK-MEL-31 o; GD3); 4, 3F8 (anti-GD2). *. intensity of reac O. ' II tivity with antibodies is low (2-3 intensity units). J could be physically blocking the smaller by virtue of its size the cell surface will be an important parameter in both cell-cell alone. Indeed, molecular modeling studies have indicated that and cell-antibody interactions. Specifically, the presence of a the oligosaccharide moiety of GD3 is not appreciably longer certain glycolipid in the cell membrane does not necessarily than that of GM3, inasmuch as the conformation of GD3 oli mean that the glycolipid is able to participate in these interac gosaccharide was calculated to be hook-shaped with the termi tions. A specific example of this could be the selectively shown nal Neu Ac being folded back onto the G M 3 sequence.7 A pos by anti-GM3 monoclonal antibodies for a few melanoma cell sible explanation of this phenomenon lies in an understanding lines even though GM3 is widely distributed in many cell types of the nature of glycolipid-glycolipid interactions in the cell (6, 8, 12). Ã‘ores et al. (7) have explained this phenomenon on membrane and of interactions of the oligosaccharide chain with the need to exceed a certain antigen density before an antibody the lipid surface of the membrane. These are both very poorly can react effectively with a target cell. While this could be one understood processes. Pertinent to this topic are the recent important parameter, the data presented here indicate that the studies of StrÃ¶mberg et al. (31) who, using molecular modeling total ganglioside composition of the cells is another contribut techniques, showed that the orientation of the saccharide chain ing factor. The question of accessibility to antibody could also of globo-series glycolipids plays an important role in determin be important in selecting antibodies for use in tumor diagnosis ing their binding to Escherichia coli. Specifically, they sug and therapy. Thus, when a cell type has more than one major gested that the sugar chain of globotriosyl-ceramide (GbOse3) is glycolipid, then the use of an antibody to the more complex oriented parallel to the membrane surface, whereas the sugar glycolipid in the target cell could be most effective. To some moieties of longer glycolipids (GbOse4 and GbOse5) have more extent this has already happened in that anti-GD3 is the anti vertical orientations. In the context of the present study, it body of choice for studying malignant melanoma cells (4, 14), could be suggested that the presence of longer glycolipids in a even though GM3 is found in approximately equal amounts in cell causes the shorter glycolipids to assume a more horizontal, the melanoma cells (14, 37). Also anti-GD2 is widely used in and therefore less accessible, orientation at the cell surface. studies on neuroblastoma (5, 38), even though GM2 is an Biophysical studies, such as those that have been carried out on equally prominent ganglioside in this cell type (39). Neverthe model membrane systems, may help in understanding this phe less, attention to the question of antigen accessibility could help nomenon (32-34). The tendency of certain glycolipids to coa in the choice of antibodies or combination of antibodies for use lesce in clusters or patches, as demonstrated in these studies, in tumor diagnosis and therapy in the future. could influence their reactivity with antibodies. A number of studies have suggested that subtle differences in ACKNOWLEDGMENTS ceramide structure, such as fatty acyl chain length, can influ ence the antigenicity of glycolipids (29). In the present study, we We thank Marvin Olsen (Sloan-Kettering Institute) for assistance compared the fatty acid composition of GM3 and GD3 isolated with the mass spectrometric analysis of fatty acids and Ken Class for the from SK-MEL-28 melanoma cells. The overall fatty acid com flow cytometric data. position of the 2 gangliosides was found to be quite similar. Although the GD3 sample had a slightly greater proportion of REFERENCES C24 fatty acids, this difference does not seem to be large enough 1. Furukawa, K., and Lloyd, K. O. Gangliosides in melanoma. In: S. Ferrane to result in greater exposure of the majority of the GD3 mole (ed.). Human Melanoma. Immunology, Diagnostics, and Therapy, pp. 15- 30. Heidelberg: Springer, 1989. cules to antibody. 2. Tsuchida. T., Ravindrananth, M. H., Saxton. R. E., and Irie, R. F. Gangli Although the function of glycolipids is still poorly under osides of human melanoma: altered expression in vivo and in vitro. Cancer stood, recent work has shown that they can play important roles Res.. 47: 1278-1281, 1987. in cell-cell interactions by virtue of glycolipid-glycolipid (35) 3. Lloyd, K. O.. and Old, L. J. Human monoclonal antibodies to glycolipids and other carbohydrate antigens: dissection of the humoral immune response in and glycolipid-protein (selectin) recognition (36). They also cancer patients. Cancer Res., 49: 3445-3451, 1989. serve as important targets for the reactions of cells with anti 4. Houghton, A. N., Mintzer, D., Cordon Cardo, C., Welt, S., Fliegel, B., Vadhan, S., Carswell, E.. Melamed, M. R., Oettgen, H. F., and Old, L. J. bodies. Specifically, since tumors often express altered gly- Mouse monoclonal lgG3 antibody detecting GD3 ganglioside: a phase I trial colipid patterns, antibodies to glycolipids provide important in patients with malignant melanoma. Proc. Nati. Acad. Sci. USA, H2: 1242- 1246, 1985. reagents for the analysis, diagnosis, and therapy of tumors. 5. Cheung. N. K., Neely, J. E., Landmeier, B., Nelson, D., and Miraldi, F. Obviously, the degree of exposure of individual glycolipids at Targeting of ganglioside GD2 monoclonal antibody to neuroblastoma. J. NucÃ.Med., 28: 1577-1583, 1987. 6. Wakabayashi, S., Saito. T., Shinohara, N., Okamoto, S., Tomioka, H., and 'S. L. Laban. H. M. Dunniway, K. O. Lloyd, and T. A. W. Koerner, Confor- Taniguchi, M. Syngeneic monoclonal antibodies against melanoma antigens mational analysis of ganglioside oligosaccharides using a molecular mechanics with species specificity and interspecies cross-reactivity. J. Invest. Dermatol., force field, manuscript in preparation. 83: 128-133. 1984. 4952

7. Ã‘ores, G. A., Dohi. T., Taniguchi, M., and Hakomori, S. Density-dependent associated ganglioside antigen defined by a monoclonal antibody, 4.2. J. Biol. recognition of cell surface GM3 by a certain anti-melanoma antibody, and Chem., 257: 12752-12756. 1982. GM3 lactone as a possible immunogen: requirements for tumor-associated 24. Hakomori, S-L, Teather, C., and Andrews, H. Organizational difference of antigen and immunogen. J. Immunol., 139: 3171-3176, 1987. cell surface "hematoside" in normal and virally transformed cells. Biochem. 8. Yamaguchi, H., Furukawa, K., Fortunato. S. R., Livingston, P. O.. Lloyd, K. Biophys. Res. Commun.. 33: 563-568. 1968. O., Oettgen, H. F., and Old, L. J. Cell-surface antigens of melanoma recog 25. Stein, K. E., Schwarting. G. A., and Marcus, D. M. Glycolipid markers of nized by human monoclonal antibodies. Proc. Nati. Acad. Sci. USA, 84: murine lymphocyte subpopulations. J. Immunol., 120: 676-679, 1978. 2416-2420, 1987. 26. Urdal, D. L., and Hakomori, S-l. Characterization of tumor-associated gan- 9. Carey, T. E., Lloyd, K. O., Takahashi. T., Travassos, L. R., and Old, L. J. AU glio-n-triaosylccramide in mouse lymphoma and the dependency of its expo cell-surface antigen of human malignant melanoma: solubilization and par sure and antigcnicity on the sialosyl residues of a second glycoconjugate. J. tial characterization. Proc. Nati. Acad. Sci. USA, 76: 2898-2902, 1979. Biol. Chem., 258: 6869-6874, 1983. 10. Graf, L., Kaplan, P.. and Silagi, S. Efficient DNA-mediated transfer of se 27. Barton, N. W., and Rosenberg, A. Action of Vibrio cholerae neuraminidase lectable genes and unselected sequences into differentiated and undifferenti- (sialidase) upon the surface of intact cells and their isolated sialolipid com ated mouse melanoma clones. Somat. Cell Mol. Genet., 10: 139-151, 1984. ponents. J. Biol. Chem., 248: 7353-7358, 1973. 11. Natoli, E. J., Jr., Livingston. P. O., Pukel. C. S.. Lloyd, K. O.. Wiegandt. H.. 28. Gahmberg. C. G., and Hakomori. S. External labeling of cell surface galac Szalay, J., Oettgen. H. F.. and Old, L. J. A murine monoclonal antibody tose and galactosamine in glycolipid and glycoprotein of human erythrocytes. detecting /V-acetyl- and /V-glycolyl-GM2: characterization of cell surface re J. Biol. Chem., 248: 4311-4317, 1973. activity. Cancer Res.. 46: 4116-4120. 1986. 29. Kannagi, R.. Stroup. R., Cochran, N. A., Urdal, D. L., Young, W. W., Jr., 12. Dohi. T., Ã‘ores, G., and Hakomori. S. An IgG3 monoclonal antibody estab and Hakomori. S. Factors affecting expression of glycolipid tumor antigens: lished after immunization with GM3 lactone: immunochemical specificity influence of ceramide composition and coexisting glycolipid on the antige- and inhibition of melanoma cell growth in vitro and in vivo. Cancer Res., 48: nicity of gangliotriosyl-ceramide in murine lymphoma cells. Cancer Res., 43: 5680-5685, 1988. 4997-5005, 1983. 13. Tai, T., Kawashima, !.. Ozawa, H., and Kotani, M. Generation of murine 30. Kawashima, L, Tada. N.. Fujimori, T., and Tai, T. Monoclonal antibodies to monoclonal antibodies to ganglio-series gangliosides. Glycoconjugate J.. 8: disialogangliosides: characterization of antibody-mediated cytotoxicity 176, 1991. against human melanoma and neuroblastoma cells in vitro. J. Biochem., 108: 14. Pukel, C. S.. Lloyd, K. O., Travassos, L. R., Dippold, W. G., Oettgen, H. F., 109-115, 1990. and Old, L. J. GD3. a prominent ganglioside of human melanoma. Detection 31. StrÃ¶mberg, N., Nyholm. P-G., Pascher, J., and Normark, S. Saccharide and characterization by a mouse monoclonal antibody. J. Exp. Med.. 755: orientation at the cell surface affects glycolipid receptor function. Proc. Nati. 1133-1147, 1982. Acad. Sci. USA, 88: 9340-9344, 1991. 15. Real, F. X., Oettgen, H. F., and Old, L. J. Detection and analysis of anti 32. Maggio, B., Ariga, T., and Yu. R. K. Ganglioside GD3 lactones: polar head bodies against cancer cell surface antigens in the sera of cancer patients. ///. group mediated control of the inlermolccular organization. Biochemistry, 29: N. R. Rose, H. Friedman, and J. L. Fahey (eds.). Manual of Clinical Immu 8729-8734, 1990. nology, pp. 798-802. Washington, DC: American Society of Microbiology, 33. Ali, S., Brockman, H. L., and Brown, R. E. Structural determinants of 1986. miscibility in surface films of galactosylceramide and phosphatidylcholine: 16. Furukawa, K., Clausen, H., Hakomori, S., Sakamoto, J., Look, K., Lundblad. effect of unsaturation in the galactosylceramide acyl chain. Biochemistry, 30: A., Mattes. M. J.. and Lloyd, K. O. Analysis of the specificity of five murine 11198-11205, 1991. anti-blood group A monoclonal antibodies, including one that identifies type 34. Rock, P., Allietta, M., Young, W. W.. Jr., Thompson, T. E.. and Tillack. T. 3 and type 4 A determinants. Biochemistry'. 24: 7820-7826, 1985. W. Organization of glycosphingolipids in phosphatidylcholine bilayers: use 17. Saito, T.. and Hakomori, S-l. Quantitative isolation of total glycosphingolip- of antibody molecules and Fab fragments as morphologic markers. Biochem ids from animal cells. J. Lipid Res., 12: 257-259, 1971. istry, 29: 8484-8490. 1990. 18. Ledeen, R. W., and Yu, R. K Gangliosides: structure, isolation, and analysis. 35. Kojima, N., and Hakomori, S. Cell adhesion, spreading, and motility of Methods Enzymol.. 83: 139-191, 1982. GM3-expressing cells based on glycolipid-glycolipid interaction. J. Biol. 19. k muÃu,S. K.. and Suzuki, A. Simple micro-method for the isolation of Chem., 266: 17552-17558. 1991. gangliosides by reverse-phase chromatography. J. Chromatogr., 224: 249- 36. Brandley, B. K., Swiedler, S. J., and Robbins, P. W. Carbohydrate ligands of 256. 1981. the LEC cell adhesion molecules. Cell, 63: 861-863, 1990. 20. Lloyd, K. O., and Savage, A. High performance aniÃ³n exchange (HPAE) 37. INni. nikalian. J., Zwingelstein. G., and Dore. J. Lipid composition of human chromatography of reduced oligosaccharides from sialomucins. Glycoconju malignant melanoma tumors at various levels of maligant growth. Eur. J. gate J., 8: 493-498. 1992. Biochem.. 94: 19-23, 1979. 21. Hardy. M. R., Townsend, R. R.. and Lee, Y. C. Monosaccharide analysis of 38. Schulz, G., Cheresh, D. A., Varki, N. M., Yu, A.. Staffileno, L. K., and glycoconjugates by aniÃ³nexchange chromatography with pulsed amperomet- Reisfeld, R. A. Detection of ganglioside GD2 in tumor tissues and sera of ric detection. Anal. Biochem.. 170: 54-62, 1988. neuroblastoma patients. Cancer Res., 44: 5914-5920, 1984. 22. Gahmberg, C. G., and Andersson, L. C. Selective labeling of cell surface 39. Wu, Z-L., Schwartz, E., Seeger. R., and Ladisch, S. Expression of GD2 sialoglycoprotcins by periodate-tritiatcd borohydride. J. Biol. Chem., 252: ganglioside by untreated primary human neuroblastomas. Cancer Res., 46: 5888-5894. 1977. 440-443, 1986. 23. Nudelman, E., Hakomori. S-L. Kannagi. R., Levery, S., Yeh. M-Y.. Hell 40. Svennerholm, L. Chromatographie separation of human brain gangliosides. strÃ¶m, K. E., and HellstrÃ¶m, I. Characterization of a human melanoma- J. Neurochem.. 10: 613-623, 1963.

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1992 American Association for Cancer Research. Cell Surface Accessibility of Individual Gangliosides in Malignant Melanoma Cells to Antibodies Is Influenced by the Total Ganglioside Composition of the Cells

Kenneth O. Lloyd, Claudia M. Gordon, Immac J. Thampoe, et al.

Cancer Res 1992;52:4948-4953.

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