Neuroectoderm-Associated Antigens on Ewing's Sarcoma Cell Lines1

[CANCER RESEARCH 47, 183-187, January 1, 1987] Neuroectoderm-associated Antigens on Ewing's Sarcoma Cell Lines1

Marc Lipinski,2 Karim Braham, IrÃ¨nePhilip,JoÃ«lleWiels,Thierry Philip, Christo Goridis, Gilbert M. Lenoir, and Thomas Tursz Laboratoire d'lmmunobiologie des Tumeurs, CNRS VA 1i56, Institut Gustave Roussy, 94805 Villejuif Cedex, France / M. L., K. B., T. TJ; Centre LÃ©onBÃ©rard,69373 Lyon Cedex, France [I. P., T. P.]; Biochemical Oncology and Membrane Research, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104 [J. WJ; Centre d'Immunologie de Marseille-Luminy, 13288 Marseille Cedex 9, France [C. G.]; and International Agency for Research on Cancer, 69372 Lyon Cedex, France [G. M. L.J

ABSTRACT MATERIALS AND METHODS

The histogenesis of Ewing's sarcoma, the second most frequent pri Cell Lines. With the exception of the SK-ES1 cell line that originated from the Sloan Kettering Cancer Center (New York), all ES lines were mary bone tumor in humans, remains controversial. Ten Ewing cell lines established by some of us (I. P., T. P., and G. M. L.) from metastatic were analyzed by immunological methods. Surface antigens recognized cells of different sites of origin in nine different patients. The karyotypic on Ewing cells were found to be related to the neuroectoderm lineage. analysis of these lines has been published in part (5, 6) as the first They included ganglioside GD2,a marker of neuroectodermal tissues and description of the t(l 1;22) (q24;ql2) translocation of ES. The HNK-1 tumors, and an acidic glycolipid detected by monoclonal antibody HNK- hybridoma was purchased from the American Type Culture Collection 1 in the nervous system. The P61 rat monoclonal antibody that reacts (Rockville, \ll)). All lines were grown as monolayers or in suspension with a peptide moiety of neural cell adhesion molecule (N-CAM) and a rabbit antiserum raised to purified mouse N-CAM also stained Ewing and were carried in RPMI 1640 supplemented with fetal calf or horse serum. Adherent cells were resuspended using trypsin-EDTA (0.05- cells. Flow cytometry analysis performed using these reagents allowed 0.02%) (Flow Laboratories, United Kingdom) with no effect on surface the definition of four distinct Ewing phenotypes: all reagents equally stained group 1 lines; group 2 lines were strongly reactive with anti-N- antigen expression CAM reagents, by contrast with a fainter staining with HNK-1 and anti- Staining Antibodies. Monoclonal antibodies used in this study origi nated from hybridomas grown in our laboratories or were kindly made Go2 antibodies; all reagents but P61 were strongly reactive with group 3 available to us. Mouse antibody HNK-1 was originally shown to detect lines; in group 4, Ewing lines were stained by P61 but only poorly by the anti-N-CAM antiserum. Several antibodies to melanoma and neuroblas a blood cell population with natural killer cell activity (10) but was later shown by us and others to also detect a neuroectoderm associated toma associated antigens including two monoclonal antibodies to the antigen (11-13). Rat antibody P61 has been raised to the purified nerve growth factor receptor were also found to react with Ewing cells. mouse N-CAM (14) and was shown to detect a peptide determined By contrast, all antibodies detecting antigens specifically expressed in epitope of this molecule (IS). This antibody has been shown to react hematopoietic cell lineages were totally unreactive. HLA class II antigens with N-CAM from all major species tested including humans. Mouse were never detected while the level of expression of class I antigens monoclonal antibodies 126.4 and MB3.6 directed to gangliosides (;,,.. varied to a large extent. Ewing cells are characterized by a specific t(ll;22) (q23-24;ql2) translocation also observed in neuroepithelioma, a and GD3[according to nomenclature of Svennerholm (16)], respectively, neuroectodermal tumor. Thus, Ewing's sarcoma cells share antigenic and both highly enriched in neuroectodermal tissues and tumors (17) were donated by Dr. David Cheresh (La Jolla, Ã‡A).A panel of anti-neuro- karyotypic features with derivatives of the neuroectoderm possibly indi blastoma reagents including antibodies UJ13A, UJ 127:11, UJ181, and cating a related histogenesis. UJ167 was obtained after immunization with human fetal brain (18) and provided by Dr. John Kemshead (ICRF, London, England). A panel of antimelanoma reagents was a kind gift from Drs. A. H. Ross INTRODUCTION and H. Koprowski (Wistar Institute, Philadelphia, PA). Included were monoclonal antibodies ME20.4 and ME82-11 that both precipitate the ES,3 a childhood tumor first described in 1921 (1), is the NGF receptor protein expressed on melanomas (19), ME492 to an M, second most frequent primary bone tumor in humans but it 30,000 to 60,000 melanoma associated protein (20), and ME31-3 to a melanoma associated proteoglycan (21). Monoclonal antibody 425-3- also occurs in extraskeletal localizations. Morphologically it 75-4 to the epidermal growth factor receptor4 was also provided and belongs to the group of small round cell tumors that because of included in the study. the absence of unequivocal features of differentiation poses A rabbit antisemiti was obtained as described (22) after immunization many problems to the pathologist (2). For the same reason, the with purified mouse N-CAM. This serum does not contain detectable histogenesis of ES is still debated; the postulated endothelial antibodies recognizing the HNK-1 determined epitope (23) and reacts origin proposed by Ewing has not received confirmation and with typical N-CAM proteins in immunoblot from human brain. most authors consider it to derive from a mesenchymal cell (3, Indirect Immunofluorescence Assay. Indirect immunostaining was 4). performed as described (11) by incubating cells with hybridoma super We have used cell lines with the characteristic ES transloca natant, ascites fluid, purified antibody, or antiserum at the appropriate tion (5, 6) also described in fresh tumors (7) to investigate their dilutions. Reactions were revealed with fluoresceinated goat antisera to immunological phenotype. Within the large panel of antibodies mouse immunoglobulin or IgM, rat immunoglobulin or rabbit Â¡mum used, all found positive detected antigens whose expression is noglobulin, as necessary. Fluorescence of live cells was quantitatively analyzed by flow cytometry (Epics C, Coulter, France), as described by related to the neuroectoderm lineage, thus supporting our pre Herzenberg and Herzenberg (24), or in some cases examined under a vious hypothesis (8, 9) that ES and other neuroectodermal fluorescence microscope. Negative controls were included in every tumors derive from developmentally related precursors. experiment and consisted of cells incubated in the presence of irrelevant antibodies or in the absence of staining antibody. Received 6/23/86; accepted 9/22/86. Thin Layer Chromatograph}' Immunostaining. Glycolipids were ex 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 tracted from cell pellets and partitioned as described (25). The most accordance with 18 U.S.C. Section 1734 solely to indicate this fact. polar fraction was eluted from a DEAE-Sephadex column with 0.45 M 1Supported in part by Grants 83D7 from the Institut Gustave Roussy and ammonium acetate in methanol and then applied after extensive dialysis 2098 from the Association pour la Recherche sur le Cancer. 2To whom requests for reprints should be addressed. against water onto a thin layer chromatography plate. After migration 3The abbreviations used are: ES, Ewing's sarcoma; (.,, , disialoganglioside; for 45 min in chloroform:methanol:water (50:40:10), the plate was GDJ, trisialoganglioside; N-CAM, neural cell adhesion molecule; NGF, nerve growth factor. ' Unpublished observations. 183 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1987 American Association for Cancer Research. EWING CELL ANTIGENIC PHENOTYPES

dried, incubated for 2 h with 5% bovine serum albumin in phosphate matography of the most polar fraction of glycolipid material buffered saline, and then incubated overnight at 10Â°Cwith HNK-1 extracted from ES cells has revealed the presence of an acidic supernatant. After a washing with phosphate buffered saline supple glycolipid with migration properties identical to those already mented with 0.5% bovine serum albumin, the plate was incubated for detected by HNK-1 in the peripheral nervous system (27) (Fig. 2 h with the appropriate dilution of a rabbit anti-mouse IgM antiserum, washed, and further incubated for 45 min with ' "I labeled protein A. 2). After the plate was washed and dryed, it was submitted for autoradiog- We then investigated the presence on ES cells of a variety of raphy. surface antigens with an association with neuroectoderm lin eage that has been demonstrated. Antibodies raised to human melanoma cell lines, raised to human fetal brain, or obtained RESULTS after immunization with purified mouse N-CAM were used. Immunofluorescence studies using monoclonal and poly- The majority of these reagents proved to be reactive with most clonal antibodies were carried on cell suspensions of ES lines ES lines (Table 2). IARC-EW1, IARC-EW3, IARC-EW7, IARC-EW11, IARC- Among the best characterized antigens defined by this collec EW12, IARC-EW14, IARC-EW15, IARC-EW16, IARC- tion of antibodies, the N-CAM and the nerve growth factor EW17, and SK-ES1 and analyzed by flow cytometry. All ex receptor are of paramount importance. The role of N-CAM in pressed class I but not class II HLA antigens. None was stained by a series of 23 antibodies taken from the panels of the Second International Workshop on Leukocyte Antigens and chosen for their specificity to 12 different clusters of differentiation of the T, B and myelomonocytic hematopoietic lineages (Table 1). By ABCD contrast, several structures associated with the neuroectoderm lineage were demonstrated at the surface of ES lines. The first example was the reactivity observed on all lines but one with the HNK-1 antibody (Fig. 1). According to its original description as a reagent for a leukocytic subset with natural killer cell activity (10), we first included it in our panel of hematopoietic markers. An extensive study of human tumors and normal tissues made us soon realize, however, that HNK- 1 in fact was an excellent marker for neuroectodermal organs and malignancies (13). Three glycoconjugates of the nervous system have been implicated as recognition structures for HNK- 1, two glycoproteins, myelin associated glycoprotein (26) and N-CAM (23), and an acidic glycolipid (27). Immunoprecipita- tion studies have not provided evidence of an HNK-1 recog nized glycoprotein on ES cells. By contrast, thin layer chro-

Table 1 Absence of Hematopoietic specific antigens on Ewing cell lines sarcoma cell lines AntigenCD2CD3CD4CDSCD6CDSCD10CDUCDwl4CDwlSBIB2DistributionT-cellsT-cellsTâ€ž-cellsÂ°T-cellsT-cellsT.negative/tested4/44/44/44/44/44/44/44/44/44/44/44/4

Fig. 2. HNK-1 immunostaining of thin layer chromatography of polar glycolipids extracted from Ewing's sarcoma lines IARC-EW1 (A), IARC-EW3 (B), and cellsGranulocytesMonocytesMonocytes-plateletsGranulocytesB-cellsB-cellsEwing'sIARC-EW7 (Q and neuroblastoma line IMR32 (fl). The most acidic glycoiipids were separated on a DEAE-Sephadex column and eluted with 0.45 M ammonium acetate in methanol. Migration of GDI,(arrow) and GMI (arrowhead) standards is indicated. Top, origin.

Table 2 Expression of neuroectoderm associated antigens on Ewing cell lines sarcoma Â°TH-cells,helper T-cells; Ts-cells, suppressor T-cells. cell lines AntibodyME20-4ME82-11ME31-3ME492126.4MB3.6UJ13AUJ127:11UJ181UJ167P61AntiserumImmunogenMelanomaMelanomaMelanomaMelanomaMelanomaMelanomaFetalAntigenpositive/testedNGF receptorNGF receptorProteoglycanM, 30,000-60,000 A pro teinGo,GD3ND"M, ID

brainFetal brainFetal 220,000-240,000proteinNDNDN-CAMN-CAM9/99/93/33/37/100/102/42/41/40/48/108/10

Fig. I. Flow cytometry analysis of ES lines IARC-EW1 (/), -EW3 (2), -EW7 (3), -EW11 (4), -EW12 (5), -EW14 (fi), -EW15 (7), -EW16 (Â«),and -EW17 (9) brainFetal and SK-ES1 (10) stained with monoclonal antibody HNK-1 developed with brainMouse N-CAMMouse fluoresceinated goat anti-mouse IgM antiserum as compared with control curves N-CAMEwing's obtained with irrelevant mouse IgM used as first antibody. Histograms appear with fluorescence plotted on logarithmic scale on jr-axis. ' ND, not determined. 184 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1987 American Association for Cancer Research. EWING CELL ANTIGENIC PHENOTYPES the mechanism of adhesion observed between neural cells has been analyzed (28). Flow fluorescence profiles obtained with the rat monoclonal antibody P61 and a rabbit antiserum, both raised to mouse N-CAM, are reported in Figs. 3 and 4, respec tively. The peptidic epitope defined by P61 and those, probably mostly carbohydrates, recognized by the rabbit serum were not expressed to the same extent on all lines. Six of the 10 ES lines were stained by both reagents while the remaining 4 (EW3, EW11, EW12, and EW16) were reactive with only 1. None was found to be negative with both antibodies. Even more striking was the positivity observed on all ES lines when tested for expression of the receptor for the nerve growth factor (Table 2). Because of the lack of differentiation of ES cells, the specific presence at the cell surface of this growth factor receptor, contrasting with the total absence of that for Fig. 5. Flow cytometry analysis of ES lines IARC-EW14 (A), -EW15 (fi), and the epidermal growth factor (Fig. 5), will possibly allow the -EW17 (Q stained with monoclonal antibodies ME20.4 (2) and ME82-11 (j), definition of culture conditions for induction of differentiation both to the nerve growth factor receptor, and 425-3-75-4 (4) to the epidermal growth factor receptor. The reaction was developed with fluoresceinated goat in vitro. anti-mouse immunoglobulin antiserum. Controls obtained with irrelevant mouse Ganglioside <;,,..was present on most ES lines while ( Â¡,,,was monoclonal immunoglobulin as tirsi antibody are shown in 1. Histograms plotted not detected. Extensive studies previously reported have dem as in Fig. 1. onstrated the strong association of GD2 with neuroectodermal tissues and tumors (29), whereas the expression of its precursor, GD3,seems to be principally restricted to melanomas (17). On the basis of the various reactivities of ES lines with HNK- 1, anti-GD25and anti-N-CAM antibodies, four phenotypes could be defined (Fig. 6). Group 1 included lines IARC-EW1, IARC- iAtsiM<*s^*S*nANSuTX>.H.Â«Â«Ã‰^_"**WÂ»lÂ«Â«*lk^(\ALkLV/VAv_. EW17, and SK-ES1 that were stained by all four reagents. Group 2 included lines IARC-EW7 and IARC-EW15 that were faintly stained by HNK-1 and anti-GD2 antibodies but strongly by both anti-N-CAM reagents. Lines IARC-EW3, IARC- EW11, and IARC-EW14 fell into group 3 that showed a poor or no staining with P61, contrasting with the other three reagents. Finally group 4 was defined by lines IARC-EW12 and

i\ivjuIIVC

'A8its.910kFig. 6. Definition of 4 types of antigenic flow cytometry profiles obtained with ES lines stained with monoclonal antibodies HNK-1 (.I). 126.4 (fi) and P61 (O and rabbit anti-N-CAM antiserum (D) developed by fluoresceinated goat anti- mouse IgM, rat immunoglobulin, or rabbit immunoglobulin antiserum, respec tively. Profile 1 included lines IARC-EW1 (shown) and -EW17 and SK-ES1; Fig. 3. Flow cytometry analysis of ES lines IARC-EW1 (/), -EW3 (2), -EW7 Profile 2 included lines IARC-EW7 (shown) and -EW15; Profile 3 included lines (3), -EW11 (4), -EW12 (5), -EW14 (6), -EW15 (7), -EW16 (Â«),and -EW17 (9) IARC-EW3, -EW11 (shown), and -EW14; Profile 4 included lines IARC-EW12 and SK-ES1 (10) stained with monoclonal antibody P61 developed by fluores (shown) and -EW16. Histograms are plotted as in Fig. 1. ceinated goat anti-rat immunoglobulin antiserum. Curves obtained with irrelevant rat antibody are presented as controls. Histograms plotted as in Fig. 1. IARC-EW16 that expressed GD2and the P61 epitope but were not, or only faintly, stained by the anti-N-CAM serum. 1a^23knA5L.

DISCUSSION

We have used an immunological approach in an attempt to clarify the question of the histogenesis of ES. A preliminary study had led us to postulate a neuroectodermal origin for Ewing cells (8, 9). We have presented here additional evidence A that the antigens expressed at the surface of 10 different ES Fig. 4. Flow cytometry analysis of ES lines IARC-EW1 (/), -EW3 (2), -EW7 lines are indeed related to this lineage. (3), -EW11 (4), -EW12 (5), -EW14 (6), -EW15 (7), -EW16 (Â«),and -EW17 (9) Two neuroectoderm associated glycolipids have been detected and SK I'M (ID) stained with rabbit anti-N-CAM antiserum developed by flu oresceinated goat anti-rabbit immunoglobulin antiserum. Curves obtained with a on Ewing cells. Ganglioside GD2 was present on the majority nonimmune rabbit serum are presented as controls. Histograms plotted as in Fig. of the lines. Its expression and distribution have been previously 1. reported in detail and were shown to be restricted to normal 185 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1987 American Association for Cancer Research. EWING CELL ANTIGENIC PHENOTYPES

tissues and tumors of neuroectodermal origin (29). The epitope Finally it should be noted that besides an identical chromo recognized by anti-GD2 antibody 126.4 probably includes the somal rearrangement, neuroepitheliomas and ES share further sialic acid residues of the ganglioside since the reactivity was characteristics, including expression of class I HLA antigens lost after neuraminidase treatment of the cells (data not shown). and absence of amplification of the N-wyc oncogene, by con The second neuroectoderm associated glycolipid was detected trast with neuroblastoma and as recently outlined (38). in ES cells by staining with the HNK-1 antibody and was also In addition to its interest for determining the histogenesis of isolated in the ganglioside containing fraction. On the basis of ES, the immunological phenotype here described could be its migration properties on thin layer chromatography, this found useful for the pathological diagnosis of Ewing's tumor. glycolipid is identical to the acidic compound detected by HNK- As described previously (13), the HNK-1 antibody stains par 1 in the peripheral and central nervous systems, the composi affin sections of ES, thus providing a first means for discrimi tion of which has been reported (27). nating between ES and other small round cell tumors not Several other antigens present on neuroectodermal tumors related to the neuroectoderm such as rhabdomyosarcoma. such as melanomas and neuroblastomas were further detected We have also observed some antigenic variability in the 10 on ES lines using antibodies raised to melanomas or human Ewing cell lines studied. Four distinct antigenic profiles have fetal brain. The most significant finding obviously was the been presented. Whether this tentative distinction will prove of reactivity obtained with antibodies to the NGF receptor. Two value for a subclassification of ES will be the subject of further distinct antibodies precipitating the same molecule (19) stained investigations. all nine ES lines tested while the 425-3-75-4 antibody that detects the epidermal growth factor receptor remained con stantly negative. Expression on Ewing's sarcoma cells of a ACKNOWLEDGMENTS receptor for the NGF makes it now possible to envision culture conditions that will allow /// vitro differentiation of this undif- The authors are grateful to David Cheresh, John Kemshead, Hilary Koprowski, and Alonzo Ross for providing monoclonal antibodies. The ferentiated tumor and thus provide further evidence of its expert assistance of CÃ©cileTÃ©taudandGilbert Hue is recognized. neuroectodermal potential. 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187

Marc Lipinski, Karim Braham, Irène Philip, et al.

Cancer Res 1987;47:183-187.

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