Histochemical Journal 24, 805-810 (1992)

A metastasis-associated antigen is present on a 60 kDa glycoprotein in transitional cell carcinoma of the human urinary bladder TETSUSHI MATSUSAKO TAKASHI MURAMATSU

~, H I S A K O M U R A M A T S U z, T S U T O M U z and Y O S H I T A D A OHI 1

SHIRAHAMA

~,

Departments of 1Urology and eBiochemistry, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Japan 890 Received 4 February i992 and in revised form 27 April 1992

Summary We have previously shown that the degree of expression of LeX-related carbohydrate epitopes, namely, Lotus tetragonolobus agglutinin (LTA) receptors, SSEA-1 and FH6, correlates with the metastatic potential of transitional cell carcinoma of the human urinary bladder. In an effort to obtain a better reagent with which to detect a metastasis-associated epitope, monoclonal antibodies were produced against LTA receptors from BOY bladder carcinoma cells. One antigen defined by such a monoclonal antibody, MM4, indeed showed better correlation with the metastatic potential of the tumour than did other carbohydrate markers. In the LTA receptors, MM4 antigen was located only on a 60 kDa glycoprotein. In extracts from primary carcinomas and lymph node metastases, the 60 kDa glycoprotein was the principal carrier of MM4 antigen. LTA receptors from these sources were composed of arrays of glycoproteins, while the 60 kDa one was invariably present. Metastasis-associated carbohydrate epitopes on the 60 kDa glycoprotein may promote metastasis by interaction with carbohydrate-recognizing proteins such as selectins on host cells.

Introduction Understanding the mechanism of metastasis will greatly contribute to clinical control of cancer. 'A variety of cell-surface molecules are thought to be involved in metastatic processes. Indeed, cadherins (Vleminckx et al., 1991), CD-44 (Gfinthert et al., 1991) and cell-surface carbohydrates (Elizabeth et al., 1981; Dennis et al., 1987; Matsusako et al., 1991, Matsumoto et al., 1992; Shirahama et at., 1992) have been reported to enhance or suppress metastasis. Bladder carcinoma is a suitable system with which to analyse the metastatic process in human cancer since the histological variety is limited. We found that expression of carbohydrate epitopes related to Le~ or CD15 [Gal~l--+4(Fuc0~l--+3)GlcNAc] is correlated with increased metastatic potential of transitional cell carcinoma of the human urinary bladder. Thus, receptors for the fucose-binding protein of Lotus tetragonolobus (LTA) (Shirahama et al., 1992), SSEA-1 (Shirahama et at., submitted), which is a Lex antigen, and FH6 antigen (Matsusako et al., 1991), whose epitope is sialyl dimeric LeX, were more intensely expressed in cancer with lymph node metastasis than in that without. 0018-2214 9

1992 Chapman & Hall

The aim of the present investigation was to obtain a better reagent with which to detect a metastasisassociated epitope and to gain a comprehensive picture of glycoproteins whose expression is correlated with metastasis. Thus, a monoclonal antibody was prepared against LTA receptors from the bladder carcinoma, and the mode of antigen expression was compared with that of other carbohydrate epitopes. The glycoprotein with metastasis-associated epitopes was identified by Western blotting.

Materials and methods

Materials LTA was prepared as described by Ogata et al. (19.85) and was coupled to Affigel 10 (Kamada et al., 1987). LTA which bound to mucin-agarose (Ogata et al., 1985) was used throughout. SSEA-1 monoclonal antibody (Solter & Knowles, 1978) was kindly given by Professor D. Solter, and the Y001 monoclonal antibody was purchased from Biocarbo Chemical, Lund, Sweden.

806

MATSUSAKO, MURAMATSU, SHIRAHAMA, MURAMATSU and OHI

Preparation of LTA receptors

Preparation of monoclona] antibody

The transitional carcinoma cell line BOY, established in our laboratory from a patient with a urinary bladder carcinoma, was cultured in Eagle's minimum essential medium containing 10% fetal calf serum. Cells were harvested mechanically. The following procedures were carried out at 4~ Frozen cells (4 g) were homogenized with 16ml of Dulbecco's phosphate-buffered saline without Ca2+, Mg 2+ (PBS). After centrifugation at 105 000 g for I h, the precipitate was homogenized with 16 ml of 0.01 M Tris-HC1 buffer, pH 7.6, containing 2% Triton X-100, 0.15 M NaC1, I mM phenylmethyIsulphonyl fluoride and 3 mM MgC12. The homogenate was centrifuged at 105 000 g for I h, and the supernatant was applied to a column of LTA-agarose (0.8 x 4 cm) equilibrated with 0.1 M Tris-HC1 buffer, pH 7.6, containing 0.1% Triton X-100 and 0.15 M NaC1 (buffer A). The column was washed with 80 ml of buffer A and eluted with 20 ml of buffer A containing 0.1 M fucose, The LTA receptors thus obtained contained 500 ~tg protein, as determined by the method of Lowry et al. (1951) using bovine serum albumin as the standard. The same procedure was applied to surgically removed tumour specimens.

LTA receptors (50 ~tg total protein) with complete Freund adjuvant were injected subcutaneously into male BALB/c mice; the procedure was repeated three times at 4-week intervals, using the same amount of antigen with incomplete Freunds adjuvant. The last injection was performed intraperitoneally. Three days after the last immunization, 8.3 x 1 0 7 spleen cells from the mouse were fused with 1.7 x 1 0 7 NS-1 cells using polyethylene glycol 4000 (Wako Pure Chemical, Japan). Hybridomas obtained by the procedure of K6hler and Milstein (1976) were cultured in RPMI 1640 medium containing 10% fetal calf serum. If necessary, the culture supernatant containing monoclonal antibodies was concentrated 5-10-fold by precipitation with ammonium sulphate.

Western blotting SDS polyacrylamide gel electrophoresis was performed using 7,5% gel as described by Laemmli (1970). After transfer to a nitrocellulose membrane (Towbin et at., 1979), the membrane was pre-incubated in PBS containing 3% (w/v) bovine serum albumin at 4~ overnight, reacted with 5Xconcentrated monoclonal antibody

Fig. 1. Immunohistochemical staining of invasive transitional cell carcinoma of the human urinary bladder by monoclonal antibody MM4. A. Primary carcinoma. Bar = 100/am. B. Metastatic lymph node of the carcinoma. Bar = 25 l/re.

Metastasis-associated antigen for 2 h and washed with PBS containing 0.1% Triton X-100. The membrane was then reacted with biotinylated horse anti-mouse IgG antibody (Vector Laboratories) in PBS for I h, washed, and incubated with avidinbiotin-peroxidase complex (Vector Laboratories) in PBS for I h and washed. Colour was developed using 0.05% 4-chloro-naphthol in 0.1 M Tris-HC1 buffer, pH 7.6, with 0.05% H202.

807 Table 1. Expression of MM4 antigen in primary tumours correlates with metastatic potential Lymph node metastasis a Antigen expression

+

-

Strongly reactive Weakly reactive Non-reactive

14 0 0

I0 12 11

~The figures show the number of cases. Immunohistochemistry

Specimens were surgically removed from 72 patients with transitional cell carcinoma of the bladder at Kagoshima University Hospital. Of those, 47 had invasive carcinomas at, or more advanced than, stage pT2, and 25 were at stage pT1, or less. The turnout stages were classified according to General Rules of Clinical and Pathological Studies on Bladder Cancer. In addition to primary cancers, metastasized lymph nodes from 15 patients were analysed. The tumour specimen, as well as adjacent normal tissues, was fixed in buffered formalin and embedded in paraffin. Sections of 4 l.tm were deparaffinized, and endogeneous peroxidase was blocked with 0.3% hydrogen peroxide in methanol for 30 min. Sections were first incubated with normal horse serum diluted 25-fold with PBS for 20 rain, then reacted with monoclonal antibodies for I h, washed with PBS, and reacted with biotinylated horse anti-mouse IgG for 30min. The specimen was then incubated with avidin-biotin peroxidase, and colour was developed by reacting with 0.005% 3,3'-diaminobenzidine (Nacalai Tesque, Japan) and 0.008% hydrogen peroxide in PBS for 8 min. Normal mouse serum diluted 20-fold with PBS was used as the control. Although many of the monoclonal antibodies used were IgM, the horse anti-mouse IgG which we used cross-reacted with IgM.

reactive' (more than 20% of the tumour area stained), 'weakly reactive' (less than 20% of the turnout area stained) and 'non-reactive' (no appreciable staining detected). Lymph node metastasis was found at the time of operation in the majority of the 'strongly reactive' carcinomas, but in none of the 'weakly reactive' or 'non-reactive' carcinomas (Table 1). The correlation between antigenic expression and metastatic potential is better than that found when LTA, SSEA-1 and FH6 expression was examined in this respect. In the case of FH6, a primary tumour without FH6 expression metastasized (Matsusako et aI., 1991), and slightly less correlation was found by using LTA and SSEA-I. For LTA, 15 cases metastasized among 34 strongly positive cases (Shirahama et al., 1992), and I4 cases metastasized among 28 SSEA-1 strongly positive cases (Shirahama et aI.,

1

2

3

KD 97 '

Other methods

Monoclonal antibody subclasses were determined using an isotyping kit for mouse monoclonal antibodies (Serotec). Results

45J"

MIMI4 as a metastasis-associated antigen

Monoclonal antibodies were prepared against LTA-binding glycoproteins isolated from BOY bladder carcinoma cells. The antibodies were screened using tissue sections of primary and metastasized transitional cell carcinomas of the human bladder. An IgM antibody MM4 was selected since it reacted strongly with the metastasized carcinoma, but variably with the primary carcinomas (Fig. I). In all 15 lymph node metastases, the MM4positive region occupied over 20% of the tumour area. The reactivity to the primary tumours of the invasive bladder carcinoma could be classified into 'strongly

Fig. 2. Identification of MM4 antigen by Western blotting. The samples analysed are as follows. LTA receptors from BOY bladder carcinoma cells (about 30 l,tg as protein) (lane I); membrane proteins from primary tumour of the invasive carcinoma (about 2 rag) (lane 2); membrane proteins (about 2 rag) from the lymph node metastasis (lane 3). The membrane proteins refer to the fractions just before LTA-agarose affinity chromatography (see Materials and methods). Arrows indicate a 60 kDa glycoprotein.

MATSUSAKO, MURAMATSU, SHIRAHAMA, MURAMATSU and OHI

808

Table 2. Correlation between expression of MM4 antigen, LTA, SSEA-1 or FH6 in primary tumours of transient cell carcinoma' L TA MM4

++

++ +

23 8

-

Total

SSEA-1 +

-

++

6 6

3 4

3

9

10

43

21

17

26 14

FH 6

+

-

++

25 6

+

4 3

2 I

5 3

2

4

16

0

2

42

11

19

3I

10

-

Total

2 9 20 31

32 18 22 72

~The figures show the number of cases. ~-+ = strongly reactive,more than 20% of the tumour area stained; + = weakly reactive, less than 20% of the tumour area stained; - = non-reactive. submitted). MM4 antigen was negative in normal uroepithelium, while it was present in umbrella cells, which also expressed LTA, SSEA-1 and FH6. The nature of MM4 antigen was examined by Western blotting. Only a 60 kDa band was detected when LTA-binding glycoproteins from BOY bladder carcinoma cells were examined (Fig. 2, lane I). Although other bands were detected in an extract from a primary tumour, the 60 kDa band was predominant (Fig. 2, lane 2). While the bands in a metastatic lymph node were quite heterogeneous, the 60 kDa band remained predominant (Fig. 2, lane 3). Thus, we concluded that the 60 kDa glycoprotein is the major carrier of MM4 antigen.

Correlation between metastasis -associated markers and M M 4 antigen

carbohydrate

Although MM4 antigen showed metastasis-associated expression most clearly, carbohydrate markers, LTA (Shirahama et al., 199i), SSEA-I (Shirahama et al,, submitted) and FH6 (Matsusako et aI., I99i) also did so. Thus, we investigated whether MM4 antigen expression correlated with that of LTA, SSEA-I and FH6 in invasive and superficial transitional cell carcinoma of the human bladder, and we found that it did (Table 2). The closest correlation was found between FH6 and MM4 expression. Ley antigen was not detected in the primary carcinomas by staining with monoclonal antibody Y001.

Fig. 3. SDS polyacrylamide gel electrophoresis of LTA receptors. A. The receptors (20 ~tg total protein) isolated from BOY carcinoma cells (lane 1), and membrane proteins (2 mg total protein) before LTA-agarose affinity chromatography (lane 2). B. LTA receptors (20 btg total protein) from the metastatic lymph node (lanes I and 4) and from primary tumours of the invasive carcinoma (lanes 2 and 3). After electrophoresis, the gels were stained by Coomassie Brilliant Blue. Arrows indicate 150 kDa and 60 kDa glycoproteins. Positions where standard proteins migrated are shown on the left as kD (molecular weights x 10-3).

Metastasis-associated antigen The next issue was whether other carbohydrate markers were expressed on the 60 kDa glycoprotein. We previously reported that FH6 was present on 60 and 44 kDa glycoproteins (Matsusako et aL, 1991). When LTA receptors from BOY cells, which were used as the antigen to produce monoclonal antibodies were analysed, the 60 kDa glycoprotein was present only as a minor component (Fig. 3A). However, in extracts from primary cancers and lymph node metastasis, the 60 kDa band was invariably present as a significant band together with many other bands (Fig. 3B). Thus, the 60 kDa band was present in all cases of LTA-binding glycoproteins so far examined.

809 i--

Sialyl LeX

' 60 KDa

Selectin

Discussion The MM4 antigen described in this report showed the closest correlation between the intensity of antigen expression and the metastatic potential of transitional cell carcinoma of the human bladder. Although FH6 antigen also correlated well, there was one exception, namely, a primary carcinoma without antigenic expression which metastasized to lymph node (Matsusako et aI., 1991). In contrast, no metastases were found in patients with primary tumours which did not express MM4 or expressed it only weakly. Practical application of MM4 antibody should be considered so that the treatment of patients can be designed according to MM4 antigenicity of the primary tumour. The antigenic epitope of MM4 antigen is not yet known. MM4 antibody did not react with a plastic plate with covalently bound Lex antigen (Plate X, Chemomed Ltd, Edmonton, Canada), and MM4 antigen was resistant to neuraminidase (Matsusako, T. et al., unpublished results). However, because of the close correlation between the antigenic expression of Le~ or sialyl Lex and MM4 antigen, we believe that MM4 antigen is a Lex- or sialyl Le• antigen. The 60 kDa glycoprotein was identified as the predominant carrier of MM4 antigen. FH6 antigen was also located on the 60 kDa glycoprotein and on a 44 kDa glycoprotein. Although LTA-binding glycoproteins were quite heterogeneous, the 60 kDa one was present in all samples examined. Thus, we regard the 60kDa glycoprotein as the most important carrier of metastasisassociated antigenic epitopes. We reported previously that the intense expression of Lex and related carbohydrate epitopes, especially FH6 (sialyl dimeric LeX), in the primary tumour is associated with metastatic potential of the bladder carcinomas. On the basis of these results, and on the present findings, we propose the following. A sialyl Lex or Lex epitope on a 60 kDa glycoprotein is recognized by a selectin on the surface of host cells, and this recognition promotes metastasis of the tumour (Fig. 4). Selectins are a group of cell adhesion proteins with a carbohydrate-recognizing domain, an EGF-like domain, multiple copies of comp-

Tumor cell

Lymph node cell Fig. 4. A model showing possible interaction between a selectin and a sialyl LeX-related carbohydrate epitope on a 60 kDa glycoprotein.

lement-binding domains and a transmembrane domain (Brandley et al., 1990; Butcher, 1991); one of them, E-selectin (ELAM-1), recognizes sialyl Lex and related structures (Lowe et at., 1990; Philips et al., 1990). So far, three selectins, namely L-, E- and P-selectins, are known. They play key roles in initial steps of leukocyteendothelial cell adhesion (Butcher, 1991). L-selectin expressed on lymphocytes serves as a homing receptor to peripheral lymph nodes. On the other hand, E-selectin expressed on cytokine-activated endothelial cells, and P-selectin expressed on activated platelet and endothelial cells, are important in leukocyte mobilization to inflammatory sites. Thus, it is quite likely that erroneous recognition of tumour carbohydrates by selectins on endothelial cells promotes tumour metastasis. Molecular identification of the 60 kDa glycoprotein, and fine structural analysis of the carbohydrate moiety, will be required for understanding the proposed selectin-tumour carbohydrate interactions.

Acknowledgements We thank Miss Kumiko Sato for her expert secretarial assistance. This work was supported by grants-in-aid from the Japanese Ministry of Education, Science and Culture. References

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