Ganglioside Gd1a Functions in the Adhesion of Metastatic Tumor Cells to Endothelial Cells of the Target Tissue'

[CANCER RESEARCH57. 1882-1888. May IS, 19971 Ganglioside GD1a Functions in the Adhesion of Metastatic Tumor Cells to Endothelial Cells of the Target Tissue'

Takao Taki,2 Dai Ishikawa,3 Misa Ogura, Motowo Nakajima, and Shizuo Handa Department of Biochemistry, Faculty of Medicine. Tokyo Medical and Dental University. 1-5-45 Yushima, Bunkyo-ku 113 Tokyo, fT. T., D. 1., M. 0., S. H.], and Bio-organic Research Department, International Research Laboratories, Ciba-Geigy Japan Limited, 10-66 Miyuki-C'yo. Takarazuka, Hyogo 665 [M. N.]. Japan

ABSTRACT metastatic tumor cells (9, 10). Takada et a!. (1 1, 12) found that sialyl Lex as well as sialyl Lea, epitopes and selectin molecules function in We studied the role of glycosphingolipids expressed on the cell surfaces the adhesion of colon tumor cells to human embryonic umbilical vein of a metastatic tumor cell line. Glycosphingolipid compositions of the endothelial cells. low-metastatic murine lymphosarcoma cell line RAW117-P and its sub line, RAW117-H1O,which shows higher metastatic potential for the liver We investigated the possible involvement of glycosphingolipids in than P cells, were compared. Both types of cells had LacCer, Gg3Cer,and the adhesion of metastatic tumor cells to the target tissue using the Gg@Ceras the major neutral glycosphingolipids and GM1b and GD1a as metastatic murine lymphosarcoma cell line RAW1 17-P and its sub the gangliosides. There are differences in glycosphingolipid contents, the line, RAWI l7-HlO, the latter showing high metastatic potential for neutral glycosphingolipid contents in the parental cells being 1.5-fold the liver. The variant Hl0 cells selected 10 times in vivo for liver higher than that in the variant ones. In contrast, the level of GD1a in H1O colonization form more than 200-fold more gross liver tumors than cells was twice as much as that in the P cells; however, the expression of the parent cells (13). Murine HSE cells were used as the target cell other gangliosides was down-regulated. On the basis of the results of (14). The adhesion of HlO cells to HSE cells was twice as high as that glycosphingolipid analysis, we investigated the functional role of GD1a in of P cells with a 60-mm incubation (15). In the previous and present H1Ocells in the adhesion of the tumor cells to the target tissue by using reports, we have compared the glycosphingolipid composition in both hepatic sinusoidal endothelial (HSE) cells. GDIa and GM1b inhibited the adhesion when HSE cells were incubated prior to coculture with the cell lines and found that GD1a is up-regulated during the mutation to tumor cells. This inhibitory effect by GD1a and GM1b was observed Hl0 cells from P cells.4 On the basis of these findings, we here report within 30 mm after addition of H1O cells to HSE cells and was dose the involvement of GD1a ganglioside in the adhesion of RAWI 17- dependent. GD1a showed a higher inhibitory effect on the adhesion than HlO cells to the target tissue. GM1b, whereas other glycosphingolipids showed no inhibitory effect. Anti-GD1a monoclonal antibody also inhibited the adhesion between the H10 and HSE cells. When cultured without fetal bovine serum for 30 mm MATERIALS AND METHODS in a various glycosphingolipids-coated dish for bacterial culture, HSE cells adhered to the area coated with GD1a but not to areas coated with other Glycosphingolipids. The neutral glycosphingolipids and gangliosides used glycosphingolipids. HSE cell adhesion depended on the amount of GD1a were prepared in our laboratory. GMlb (16) and GDla (5) were obtained from coated on the plate. These data indicate that GD1a functions as an rat ascites hepatoma AH7974F cells. Gg3Cer and Gg.@Cerwere prepared from adhesion molecule in the process of metastasis of H1Ocells. GM2 and GMla, respectively, by mild acid hydrolysis (17). GM3 was pre pared from human placenta (18). MAbs. MAbs directedto Gg3Cer,Gg.@Cer(19),GM1b, and GDla were INTRODUCTION used. MAbs against GM1b, and GDla (KA17) were kind gifts from Dr. Y. Hirabayashi (Riken, Saitama, Japan; Ref. 20). Tumor metastasis contains many biological processes, such as Cell Lines. Two tumorcell lines were used. The low-metastaticmurine dissociation of tumor cells from primary tissue, invasion to vessel, lymphosarcoma cell line, RAW117-P, had been established from spleen cells adhesion and invasion to the target tissue, and generation of secondary after Abelson leukemia virus infection (21). The othercell line, RAW! 17-H10, tumor. Tumor cell-endothelial cell adhesion probably is the first step is a subclone selected 10 times in vivo for liver colonization that has been in the process of organ preference of metastasis, but the adhesion established as a cell line with high metastatic potential for the liver (13, 22). process in metastasis has yet to be clarified. During oncogenic trans The biological properties of RAWI 17 cells were assessed by iv. injection of formation, dramatic changes in the glycosphingolipids on the cell 5 x l0@cellsinto BALB/c mice. Mice receiving RAW1 17-PandHlOcells had surface membrane have been reported (1â€”7).These changes suggest median numbers of liver tumor colonies of 0 (range, 0â€”5) and >200, respec that glycosphingolipids are involved in the adhesion of metastatic tively. Median lung colonies of both subline cells were 0 (range, 0â€”5). HSE tumor cells to the endothelial cells of the target tissue. We reported cells had been established by Belloni et a!. (14). We performed the adhesion experiments using HSE cells of passage numbers 6â€”8 after a large-scale @ elsewhere the accumulation of Lex. and sialyl glyco expansion. HSE cells of such lower passage numbers possessed the phenotypes sphingolipids in human colon cancer that metastasized to the liver (8). described by Belloni et a!. (14). Sialyl Lex epitopes are reported to be recognized by various MAbs3 in TLC. Glycosphingolipidswereseparatedona precoatedsilica gel 60 plate [(HPTLC plate) purchased from E. Merck, Darmstadt, Germany]. The devel Received I 1/5/96; accepted 3/24/97. aping system for neutral glycosphingolipids was chloroform/methanol/0.2% The costs of publication of this article were defrayed in part by the payment of page CaCI, (60/35/8 by volume). Gangliosides were separated with a solvent charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mixture of chloroform/methanol/0.2% CaCl2 (55/45/10 by volume). Glyco I This study was supported in part by the Special Coordination Fund from the Science sphingolipids were made visible by spraying the plate with orcinol-H2S04 and Technology Agency of the Japanese Government. reagent, and gangliosides by spraying the plate with resorcinol-HCI reagent. 2 To whom requests for reprints should be addressed. Phone: 8 1-886-65-2272; Fax: 81-886-65-5662; E-mail: [email protected]. 3 Present address: Cellular Technology Institute, Otsuka Pharmaceutical Co., Ltd., Galf3l-3GalNAcfJl-4(NeuAca2-3)Galf3l-4GlcCer;GMlb. NeuAca2-3Galj3l-3GalNAc@l- 463-10 Kagasuno. Kawauchi-cho, Tokushima 771-01, Japan. 4Gal@3l-4GlcCer, GDla, NeuAca2-3GalfJl-3Ga1NAc@1@(NeuAca2-3)Ga1@314G1cCer, 4 The abbreviations used are: MAb, monoclonal antibody; FBS, fetal bovine serum; GC, GD1 a, NeuAca2-3Galj3l-3(NeuAca2-6)GalNAcj3l-4Galf3l-4GIcCer; GTlb, NeuAca2- gas chromatography; HPTLC, high-performance TLC; HSE cell, hepatic sinusoidal endothe 3Gal@3l-3GalNAc@3l-4(NeuAca2-3NeuAca2-3)GaI@3l4GlcCer;GT1j3.NeuAca2-3GalfJl hal cell; Cer, ceramide; GIcCer, Glcj3l-l â€˜Cer;sulfatide,SO3-3Galj3l-l'Cer; LacCer, Gal@1- 3(NeuAca2-8NeuAca2-6)GalNAc@3l-4Galj3l -4GlcCer, GQlba; NeuAca2-3Gal@I- 4GlcCer; Gg3Cer,GalNAcj3l-4Galj3l-4GIcCer,Gg.@Cer,Galj31-3GalNAc@3l4Gal@3l-4Glc 3(NeuAca2-6)Ga1NAcf3l-4(NeuAca2-8NeuAca2-3)Gal@l-4GlcCer. Cer; Gb3Cer. Galal-4Galf3l-4GlcCer, Gb4Cer, GalNAc@3l-3Galal-4GaIj3l-4GIcCer; GM3, 5 D. Ishikawa, T. Kasama, M. Nakajima, S. Handa, and T. laid, Gangliosides in NeuAca2-3GalfJl -4GlcCer; GM2, GalNAcfJl -4(NeuAca2-3)Gal@l -4GlcCer; GMla, metastatic tumor cells, submitted for publication. 1882

TLC Immunostaining. Immunological detection of Gg3Cer, Gg,@Cer, 1640 without serum in 24-well trays. The monolayers were washed GM1b, and GDlct on a HPTLC plate was done as described elsewhere (23). with PBS, and the adhesion of RAWI 17 cells were observed under a Horseradish peroxidase-conjugated antimouse 1gM antibody (Cappel Labora light microscope. Fig. 1 shows that the adhesion of HlO cells (2 X iO@ tories, West Chester, PA) was the second antibody. cells) is higher than that of P cells (6 X l0@ cells), indicating that Methylation Analysis. Glycosphingolipids were methylated by the method metastatic potential is correlated with the adhesiveness of the tumor of MAnsson et a!. (24). Methylated glycosphingolipids were derivatized to cells to the endothelial cells of the target tissue. alditol acetates as described in a previous paper (25) and analyzed by GC-mass Glycosphingolipid Compositions of the RAW117-P and spectrometry using a Varian 3400 gas chromatograph interfaced to a TSQ 70 triple-stage quadruple mass spectrometer(Finnegan MAT, Inc., San Jose, CA). RAW117-H1O Cells. In the present study, neutral glycosphingolipids The GC column was a 30 m DB-5 (J & W Scientific) chemically bonded, fused were identified, and the compositions of glycosphingolipids in P cells silica capillary column. The temperature was programmed to change from 140 and Hl0 cells were compared. The TLC profiles of neutral glyco to 250Â°Catthe rate of 4Â°C/mm.Electhcimpact mass spectra were acquired sphingolipids in the RAW1I7-P and HlO cells are shown in Fig. 2. from m/z 50 to 500 at 1 s/scan. Structural characterization was done by immunostaining with anti Effect of Glycosphingolipids on the Adhesion of RAW117-H1O Cells to Gg3Cer and anti-Gg4Cer MAbs (Fig. 2B). TLC blotting/secondary ion HSE Cells. RAWIll-HlO cells (1 X l0@cells) were labeled metabolically mass spectrometry analysis, gas chromatographic sugar composition with an addition of [35S}methionine (370 kBq) in RPMI 1640 containing 5% analysis, and linkage analysis by methylation study with GC-mass FBS for 12 h. The cells were centrifuged, washed with PBS several times, and spectrometry, together with the immunostaining, confirmed that both suspended in RPM! 1640. This cell suspension was designated the 35S-labeled RAW117-HlOcells. HSE cells (1 X 10â€•cells)were seeded in each well of a cell lines contained GlcCer, LacCer, Gg3Cer, and Gg@Cer as neutral 96-microwell plate or a 24-well tray. The cells were cultured in RPM! 1640 glycosphingolipids (data not shown). Mobility on a HPTLC plate is containing 10% FBS for 2 days. The monolayers were washed with PBS. shown in Fig. 2A. The glycosphingolipid content in RAW1 17-P cells Individual glycosphingolipid solutions (0â€”200jig/miin 100 jil of RPM! 1640 (11.6 mg/l0'Â° cells) was 1.5-fold that in the metastatic subline HlO without serum) were added to the HSE culture wells, and the plate was left in cells (7.5 mg/l0'Â° cells). The ganglioside fractions separated by the a CO2 incubator at 37Â°Cfor30 mm, after which 35S-labeledRAW! 17-H10 DEAE-Sephadex A-25 column were subjected to TLC (Fig. 3A) cells (5 X l0@ cel!s/100 jil, 6 x l0@ cpm) were added to each well, and the followed by immunological staining on a HPTLC plate (Fig. 3B). whole plate was left for 30 mm at 37Â°Cinthe CO2incubator. After incubation, The major ganglioside of RAW1 17-P cells was determined to be the culture medium was removed by suction, and the wells were washed with GMlb from the following observations. (a) The mobility of the PBS threetimes.TheadheringRAW117-HlOcells wereobservedunderalight ganglioside on the HPTLC plate was identical with that of GM lb microscope and then harvested with 0.05% trypsin/0.53 mist EDTA. The collected cells were made soluble with 100 jal of lysis solution (10% Triton obtained from rat ascites hepatoma AH7974F cells. (b) Treatment X-l00 and 30% methanol in 2 N NaOH) at 37Â°C for 10 mm. This solution was with Clostridium perfringens sialidase without detergent converted neutralized by an addition of 100 jal of 2N HC1, and its radioactivity was the ganglioside to a glycosphingolipid that had the same mobility on counted with a liquid scintillation counter (Beckman Instruments). the HPTLC plate as Gg@Cer. (c) This glycosphingolipid was stained Detection ofGDla on RAW117 Cells by Anti-GD1a MAb KA17. P and by the MAb against Gg4Cer. Hl0 cells (1 X i0@cells) were incubated with anti-GD1a MAb KAI7 in 100 Two major ganglioside bands were found in RAWI l7-HlO cells. pi of PBS for 30 rain at 4Â°C.After incubation, both cell lines were washed One was identified as GM lb from the observations described above. with PBS three times and then incubated with FITC-conjugated antimouse 1gM The other was identified as GDla from the following results: The antibody (Cappel Laboratories) for 30 mm at 4Â°C.Both cell lines were washed ganglioside showed the same mobility on a HPTLC plate as the GD1a with PBS, and then the presence of GD1a on the cell surfaces was observed from AH7974F cells. The ganglioside was stained with the MAb with fluorescence microscopy. Inhibition by Anti-GD1a MAb (KA17) of the Adhesion of H1OCells to against GDla, KA17. After sialidase treatment, the ganglioside was HSE Cells. Hl0 cells (1 X 10' cells) suspended in 100 ji! of serum-free converted to a glycosphingolipid that had the same mobility on the medium were incubated with or without the anti-GDla MAb KAI7 (400 jag) HPTLC plate as Gg@Cer. This glycosphingolipid was immunostained for 30 mm. The mixture was then added to the HSE cell culture wells (24-well with MAt, against Gg.@Cer(Fig. 2). The compositional study analyzed tray), and the tray was incubated for 60 mm. After incubation, the medium was removed by suction, and the well was washed three times with PBS. The adhering Hl0 cells were observed under a light microscope. Adhesion of ElSE Cells to the Glycosphingolipid Area Coated on a Petri HSE + P HSE+H1O Dish. One pAof glycosphingolipid solution (10 mg/mI methanol) was placed on a Petri dish (10 cm in diameter, Eiken Co. Ltd., Tokyo, Japan), and the @ . .. methanol was evaporated. HSE cell (3 X 106 cells) suspension in RPM! 1640 without serum was added to the glycosphingolipid-coated dish. Incubation took place at 37Â°Cina CO2 incubator. After 30 mm, the culture medium was changed with RPM! 1640 containing 10% FBS, and then the cells were , cultured at 37Â°Covernight.The culture medium was removed by suction, and the dish was washed with PBS three times. The cells adhering to the dish were observed under a light microscope. 35S-labeled HSE cells (5 x 106 cells, @ approximately 1 X 106 cpm) were used for the experiment on the dose ,: i: -* dependent HSE adhesion to the glycosphingolipid-coated areas. After incuba

@@@ tion, radioactivity was counted with a liquid scintillation counter. .5'. .,@ â€˜

$; @ RESULTS â€˜:j@.:. @@@ Adhesionof RAW117-Pand H1Oto HSE Cells. We firstcom :. ..@:::@; pared the adhesion of low-metastatic RAWI 17-P cells and high metastatic Hl0 cells to HSE cells. Each RAW! 17 cell line (approx Fig. 1. Adhesion of RAWI 17-P and HlO cells to the HSE cells. RAWI 17-P and HlO cells were added to HSE cells, and their adhesion to the HSE monolayers was observed imately 5 X l0@ cells) was added to confluent monolayers of HSE under a light microscope. Bright, round cells on the HSE monolayers are RAWI 17-P (left) cells (approximately 5 X l0@cells) and cultured for 60 mm in RPMI and H10 (right) cells. 1883

GDIa AS ADHESION MOLECULE IN MURINE METASTATIC TUMORS

A B

mI@C& @& â€˜3@@ Fig. 2. Chromatographic profile and immuno staining of neutral glycosphingolipids in RAWII7-P and HlO cells on a HPTLC plate. A. TLC of the neutral glycosphingolipids in @@ RAWI 17-P and Hl0 cells. Glycosphingolipids LacCer-.@ â€”, were made visible by spraying the plate with or cinol reagent. B. immunostaining with anti-Gg3Cer antibody (left) and anti-Gg.@Cer antibody (right). Gg3Cer and Gg.,Cer were the standard glycosphin golipids. Lanes P and HIO indicate the neutral Gg.Cer @,.@ glycosphingolipids obtained from P and Hl0 cells, respectively.

with a TLC densitometer indicated that GDics content in the Hl0 cells GMlb and GDla are involved in the adhesion between HSE cells and was 2-fold greater than that of low-metastatic P cells. GDla seemed HlO cells. to be highly expressed in HlO cell surfaces during the mutation to Inhibition by the Anti-GD1a MAb KA17 on the Adhesion of high metastatic tendency for the liver. H10 Cells. To detect the expressionof GD1a on the H10 cell Inhibition by GM1b and GD1a of the Adhesion of RAW117 surfaces with anti-GD1a MAb KA17, we used a FITC-conjugated Cells to HSE Cells. On the basis of the results in shown in Fig. 1 and antimouse 1gM antibody as second antibody. The HlO cell surfaces the glycosphingolipid analysis, we examined the effect of glycosphin were stained with KA17 as shown in Fig. 5A, whereas the staining of golipids on the adhesion of RAW1 l7-HlO cells to HSE cells. When P cells was very weak (data not shown). Next, we examined the effect HSE cells were incubated for 30 mm with GDlcs at the concentration by KAI7 on the adhesion of H10 cells to HSE cells. The adhesion of of 40 jig/mI or with GMlb at the concentration of 200 jag/ml, Hl0 cells was inhibited by the addition of KA17 (Fig. SB). The adhesion of RAW1 17-Hl0 cells to the HSE cells was inhibited (Fig. addition of anti-GMlb MAb did not show a significant effect on the 4Ac and â€˜@.Theinhibition by GDlct or GMIb was observed within a adhesion, suggesting that GD1a is involved more predominantly in 30-mm incubation after addition of HlO cells to HSE cells (Fig. 4B). the adhesion of HlO cells than is GMlb. GD1a was more effective than GMlb and the inhibition was in a Adhesion of HSE Cells to a Petri Dish Coated with Glycosphin dose-dependent manner (Fig. 4D). No effect on inhibition was found golipids. To confirm whether the H10 cells adhere to HSE cells via when HSE cells were incubated with LacCer, Gg3Cer, Gg@Cer, GDla, we examined the attachment of HSE cells to glycosphingo Gb4Cer, or other gangliosides, or with GM3. The results suggest that lipids coated on a Petri dish. When HSE cells were seeded in a Petri

A B

antiGM1b anli-GOla

GM3@Ã¸a Fig. 3. Chromatographic profile and immuno @@ staining of gangliosides in RAWI 17-P and HlO t@..: . cells on a HP'FLC plate. A. TLC of the acidic glycosphingolipids in RAW1 17-P and H10 cells. @ Gangliosides were made visible by spraying the GMIaâ€”@ o plate with resorcinol-HCI reagent. B. immuno staining of gangliosides GM lb (left) and GD 1a (right) on the HVI'LC plate. GM3, mixture (bovine brain ganglioside fraction), GMIb, and GD/a are GD1aâ€”. . the standard gangliosides (Std.) used. Lanes P and HlO indicate the acidic glycosphingolipids of P and H10 cells, respectively. GD1b-.@ â€¢.@. GT1bâ€”@ @2 0 @D iQ -U â€˜0 -&@ @ a. 0 -& 0 _ 0â€¢ c@)

1884

GDIa AS ADHESION MOLECULE IN MURINE METASTATIC TUMORS

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Fig. 4. Inhibition ofGMIb and GDla ofthe adhesion of RAWI 17-Hl0 cells to HSE cells. A. effect ofGMlb and GDIa on the adhesion of RAWI 17-HIO cells to HSE cells. HSE cells were incubated for 30 mm without glycosphingolipid (a), or with GM3 (200 p.g/ml; b), GMIb (200 p.g/ml; c). or GDla (40 p.g/ml; d). RAWI l7-Hl0 cells then were added and cultured for 30 mm. After the nonadhering cells were removed by suction, the monolayer was observed under a light microscope. B. effect of the incubation time of Hl0 cells. HSE cells were incubated with or without individual ganglioside (25 p.g/ml) for 30 mm, and then the adhesion of 35S-labeled HlO cells to HSE cells with time were counted. Adhesion is expressed as a percentage against control (without ganglioside) in each incubation time. â€¢,GM3;A, GM1b; & GDIa. C, effect of various glycosphingolipids on the adhesion of 35S-labeledHlO cells. D, dose-dependent inhibition effect ofGMlb and GDla on the adhesion of 35S-labeledHl0 cells to HSE cells. 0, GMla; â€¢,GM3;A, GM1b; A, GDIa. Bâ€”D, experiments were done in triplicate. Data are means; bars. SE.

1885

GD1a AS ADHESION MOLECULE IN MURINE METASTATIC TUMORS

anchoring was detected not only on the area coated with GD! a but on A the sulfatide-coated area (data not shown). This binding to the sul fatide-coated area seemed to depend on a serum component-mediated mechanism. Because laminin has an affinity for sulfatide (26), the laminin-mediated attachment of the HSE cells seemed to occur when FBS was present in the incubation medium. This finding indicates that

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Fig. 5. Immunofluorescence and effect of anti-GDIa MAb KA 17 on the adhesion of HlO cells to HSE cells. A, detection of GDIa on H10 cells by KAI7. H10 cells were S incubated first with KAI7 and then with FITC-conjugated antimouse 1gMantibody, after a. which HlO cells were observed with fluorescence microscope. B. H10 cells and KA17 were incubated together prior to their addition to the HSE monolayers. The extent of inhibition of adhesion by KAI7 was observed under a light microscope. C

â€˜a dish, which is used for bacterial culture, the cells could not adhere to 51 the dish. Taking advantage of this property, different areas (approx U â€˜a imately 6 mm in diameter) on the dish were first coated with various 51 U glycosphingolipids: GlcCer, LacCer, Gb3Cer, Gb4Cer, Gg3Cer, Gg.@Cer,GM3, GMla, GDla, GMlb, and GD1a; then, HSE cells were seeded. The HSE cells were incubated in RPM! 1640 without 0

serum for 30 mm, after which the medium was changed to 10% 51 E FBS-containing medium. Cell morphology was observed 1 day after .0 U incubation (Fig. 6A). Within 30 mm, HSE cells settled only on the area coated with GD1a and adhered (Fig. 6Ab). Under the present ,.@ experimental conditions, approximately 6 X l0@ cells of HSE cells 0.1 1 10 100 1 10 100 adhered to the dish; however, no HSE cells were present on the areas \_____ ng â€”â€˜ \@ @ig coated with the other glycosphingolipids used (Fig. 6B). Dose-depen Glycosphingolipids dent attachment of 35S-labeled HSE cells to areas of the Petri dish coated with the various glycosphingolipids is shown in Fig. 6C. HSE Fig. 6. Adhesion of HSE cells to a Petri dish coated with GDIa. A, HSE cells were cell attachment required more than 10 jag/cm2 GD1a, which suggests incubated in serum-free RPM! 1640 in Petri dishes coated with various glycosphingolipids (10 sg/cm2). After 30 mm, the medium was removed by suction. The HSE cells adhering that the clustering of the GDla epitope is important for cell attach to the dish were cultured overnight in 10% FBS containing RPM! 1640. HSE adhesion to ment. the glycosphingolipids was observed under a microscope. B. adhesion of 35S-HSE cells to various glycosphingolipids. Five X 106 cells (approximately I X 106 cpm) were used. C, When HSE cells were incubated for 30 mm in a Petri dish in dose-dependent adhesion of the 35S-labeled HSE cells to GDIa. B and C. experiments medium containing 10% FBS instead of in serum free-medium, cell were done in triplicate. Data are means; bars, SE. I886

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1997 American Association for Cancer Research. GD1a AS ADHESION MOLECULE IN MURINE METASTATIC TUMORS a serum-free incubation condition is important to clarify the glyco sialyl La@and sialyl LaX are involved in the metastatic adhesion of sphingolipid-mediated adhesion system. GD1a is required for the human tumor cells. Taken together with these reports, GD1 a is a attachment of HSE cells to the substratum, and growth factor-like possible adhesion molecule in murine inflammatory response. components in FBS are required for proliferation. The attachment of Yang et al. (37) reported that GQlba, GTlb, GD1a, GM lb, and 35S-labeled HSE cells to GM1b was about 8% of the attachment to GTlf3 supported myelin-associated glycoprotein-mediated adhesion. GD1a, suggesting that the binding affinity of HSE cells for GD1a is Because GQlba and GT1f3 have the same terminal tetrasaccharide much stronger than that for GM lb. structure (NeuAca2-3Gal@l-3(NeuAca2-6)GalNAc) as GD1 a, mye lin-associated glycoprotein-like ligand(s) for GDIa may exist on the cell membrane of the HSE cells. DISCUSSION Because the sialyl Lea and sialyl Le5 as adhesion ligand epitopes To clarify the functional roles of the glycosphingolipids expressed have been reported to be expressed both on glycosphingolipids and on metastatic tumor cells, we compared the glycosphingolipid corn glycoproteins (30, 31), a experiment to detect glycoproteins bearing positions of murine lymphosarcoma cell lines RAW1 17-P (low met the GD1a oligosaccharide on the surfaces of HlO cells was done. A astatic potential) and RAW117-HlO (high metastatic potential). Total preliminary experiment showed that two distinct bands (Mr 33,000 glycosphingolipid level in H10 cells was decreased to about 65% of and 38,000) that were immunostained by KAI7 are present, and the that in P cells. In contrast, the amount of GD! a, a major component bands corresponded to the bands recognized with HSE cell surface in the high-metastatic H10 cells, was twice as much as that of P cells, protein(s) (data not shown). These experimental data suggest strongly which had GM1b as the major ganglioside. In both cell lines, Gg3Cer that GD1a epitopes of these glycoproteins were expressed on Hl0 cell and Gg@Ceras well as GlcCer and LacCer were found in the neutral surfaces and involved in the adhesion of H10 to HSE cells. glycosphingolipid fraction. Ganglioside synthesis is assumed to take Here, we propose that ganglioside GD1 a functions as an adhesion place through asialogangliosides. Joshi et a!. reported that expression molecule in adhesion of murine metastatic lymphosarcoma RAWI 17- of Gg.@Cerwas down-regulated to 50% during the metastatic mutation HlO cells to the target tissue. The isolation of the other adhesion of P cells to H10 cells (27) and that growth of RAW! 17-P cells in the molecule(s) that has affinity to GDla is on going in our laboratory. presence of tunicamaycin caused decreased surface glycosylation and increased their tumorigenic potential to levels approaching that ex ACKNOWLEDGMENTS hibited by H10 cells (28). The present results together with their observations indicate that the change of cell surface glycosylation We thank Dr. Y. Hirabayashi for supplying us with MAbs against GDla affects the metastatic potential of the murine lymphosarcoma cells. and GM1b. 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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1997 American Association for Cancer Research. Ganglioside GD1α Functions in the Adhesion of Metastatic Tumor Cells to Endothelial Cells of the Target Tissue

Takao Taki, Dai Ishikawa, Misa Ogura, et al.

Cancer Res 1997;57:1882-1888.

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