(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). 0°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 4°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 80°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 (¡ANGLIOSIDES IN TUMOR CELLS sugars was done with high performance aniónexchange chromatogra- phy 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 80°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; ¡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 4°C,3.0 ml of 2 mMsodium periodate in PBS were added.