Int. J. Cancer (Pred. Oncol.): 74, 7–14 (1997) r 1997 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer METANESTIN, A GLYCOPROTEIN WITH METASTASIS-ASSOCIATED EXPRESSION IN TRANSITIONAL CELL CARCINOMA OF THE URINARY BLADDER Masakazu TAKEMOTO,1,2,3* Tsutomu SHIRAHAMA,1 Teruo MIYAUCHI,2,4 Tetsusi MATSUSAKO,1 Norio KANEDA,3 Hisako MURAMATSU,2,3 Masayuki OZAWA,2 Yoshitada OHI1 and Takashi MURAMATSU2,3 1Department of Urology, Faculty of Medicine, Kagoshima University, Kagoshima, Japan 2Department of Biochemistry, Faculty of Medicine, Kagoshima University, Kagoshima, Japan 3Department of Biochemistry, Nagoya University School of Medicine, Nagoya, Japan 4Japan Immunoresearch Laboratories, Takasaki, Gumma, Japan A 60-kDa glycoprotein named metanestin was identified by molecular cloning. The glycoprotein had a twice-repeated motif of Pro-Gly-Pro-Gly and carried metastasis-associated carbohydrate epitopes. The antibody to the protein portion of metanestin strongly reacted with lymph-node metastasis of transitional-cell carcinoma of the urinary bladder, but the reactivity to the primary tumor was generally weaker and the normal urothelium was scarcely reactive. Thus both metanestin and the carbohydrate on it showed metastasis-associated expression. In addition, we identified a 100-kDa glycoprotein with a 4-times-repeated motif of Pro-Ala-Pro-Ala. The antibody to the 100-kDa glycoprotein showed reactivity similar to that to metanestin. Int. J. Cancer 74:7–14. r 1997 Wiley-Liss, Inc. The most important issue in management of patients with urinary-bladder carcinoma is lymph-node metastasis. When invasive urinary-bladder carcinoma does not penetrate the muscle layer (pT3a), the 5-year survival rate of patients is 50 to 60%. In cases of more advanced carcinoma (pT3b, T4), this figure decreases markedly, to 15 to 20%, demonstrating the importance of lymph-node metastasis (Wishnow et al., 1987; Pagano et al., 1991; Kakehi et al., 1992.). Therefore, clarification of the mechanism of lymphnode metastasis is important for developing effective methods of treatment of urinary-bladder carcinoma. Various reports have described expression of carbohydrate antigens, especially blood group-related antigens, in malignant tumors. Some of the carbohydrate antigens are known to play important roles in metastasis of carcinomas (Hoff et al., 1989; Muramatsu, 1993.). In blood-borne metastases of colon carcinomas, sialyl Lex antigen and sialyl Lea antigen (blood-group-related carbohydrate antigens) are considered to function as ligands of E-selectin, and to enhance tumor metastasis (Hoff et al., 1989; Takada et al., 1993). Physiologically, these adhesion molecules is known to play an initial role in adhesion of leukocytes to endothelial cells of blood vessels (Lasky, 1992). We have studied the expression of blood-group-related carbohydrate markers, namely Lex, sialyl Lex and binding sites for Lotus tetragonolobus agglutinin (LTA) in urinary-bladder carcinomas and have reported that strong expression of these markers correlated with the occurrence of lymph-node metastasis and, consequently, poorer prognosis (Matsusako et al., 1991; Shirahama et al., 1992a,b, 1993). Furthermore, we prepared monoclonal antibodies (MAbs) to LTA-binding glycoproteins isolated from urinarybladder carcinoma cells, and one of these antibodies, MM4, reacted with an antigen (MM4 antigen) whose expression in primary tumors correlated with the presence of lymph-node metastasis. MM4 antigen was also strongly expressed in metastatic tumor nests in lymph nodes (Matsusako et al., 1992). The aim of the present investigation was to determine the antigenic epitope and to characterize the molecule carrying the epitope. MATERIAL AND METHODS Cells The BOY transitional-cell-carcinoma cell line, established in our laboratory (Kayajima et al., 1989), was cultured in Dulbecco’s modified Eagle’s minimum essential medium containing 10% FCS (ICN, Costa Mesa, CA), 50 IU/ml ampicillin/50 IU/ml streptomycin (Flow, Irvine, UK), and amino acids (1/100 V/V) (Flow) and fungison (GIBCO, Grand Island, NY) under 12% CO2 at 37°C. After washing twice with Dulbecco’s PBS without Ca21, Mg21 [PBS(—)], cells were harvested mechanically and stored at 270°C until use. Construction of cDNA library Total RNA was extracted from tumor tissues by the guanidiumisothiocyanate/cesium-chloride method (Maniatis et al., 1982). Poly (A)1 RNA was isolated on an oligo (dT) cellulose column. First-strand cDNA was synthesized using oligo (dT) and random primers. Second-strand cDNA was synthesized using DNA polymerase, and the double-stranded cDNA was blunt-ended. After the endogenous EcoRI sites were protected by EcoRI methylase, EcoRI adapters were ligated to the synthesized cDNA. After digestion with EcoRI, cDNA was separated from adapters by chromatography on Sepharose 4B (Pharmacia, Milwaukee, WI). The cDNA was ligated to lgt 11 arms and packaged. Antibodies and screening of the cDNA library The MAb MM4, established and characterized in our laboratory (Matsusako et al., 1992), was used for screening. The library was screened by the procedure of Young and Davis as modified by Schuh et al. (1986). Phage clones with extended cDNA sequence were screened by the plaque hybridization method (Maniatis et al., 1982). Sub-cloning and DNA sequence analysis Phage DNA was prepared by the plate lysate method (Maniatis et al., 1982), and the cDNA inserts were cloned into Bluescript SK (—). DNA sequencing was performed by the dideoxy-chaintermination method (Sanger et al., 1977). Homology was searched using the data bases of EMBL-GDB (European Molecular Biology Laboratory/Gene Data Bank). Preparation of BNN 103 lysogen The fusion protein produced by the recombinant lgt 11 clones, ST-1 and ST-2, was prepared as described (Schuh et al., 1986). BNN 103 lysogen was cultured in LB (Luria-Bertani) medium at 30°C until optimal density was reached; phage production was induced at 42°C for 20 min and the cells were cultured at 37°C for 2 hr. Preparation of antibody against a synthetic peptide A synthetic peptide, GPAPASAGGDHSPAPAS (amino acids 51–67 in Fig. 1b), was prepared and conjugated to branched *Correspondence to: Masakazu Takemoto, Department of Biochemistry, Nagoya University School of Medicine, 65 Tsurumai-cho Showa-ku, Nagoya 466, Japan. Fax: 81-52-744-2065. Received 26 April 1996; Revised 23 July 1996 8 TAKEMOTO ET AL. FIGURE 1 – Nucleotide and deduced protein sequence. (a) Partial sequence of cDNA of metanestin (ST-1-derived protein). The sequence of the initially isolated clone (ST-1) is shown underlined and the repeating motif by dashed lines. (b) Partial sequence of cDNA of a 100-kDa protein (ST-2-derived protein). The sequence of initially isolated clone (ST-2) is underlined. Double underline shows Poly A signal. The characteristic repeating motif is shown by dashed lines. A METASTASIS-ASSOCIATED GLYCOPROTEIN polylysine oligomer (Peptide Institute, Suita, Osaka, Japan). New Zealand White rabbits were immunized with 1 mg of the conjugated peptide mixed with Freund’s complete adjuvant. Three booster immunizations were performed at 2-week intervals, and anti-serum was collected 10 days after the last immunization. Five milligrams of the synthetic peptide were coupled to 1 ml of CNBr-activated Sepharose (Pharmacia) according to the manufacturer’s protocol. The antiserum (2 ml) was dialysed against 20 mM Tris-HCl buffer (pH 7.0) and applied to an affinity column with the peptide-Sepharose mentioned above, which was equilibrated with 20 mM Tris-HCl buffer (pH 7.0). After washing with 10 to 20 volumes of Tris-HCl buffer, antibody was eluted with 0.1 M glycine-HCl buffer (pH 2.5) and immediately neutralized with 1.5 M Tris-HCl buffer (pH 8.8). The antibody, anti-PAPA antibody, was mixed with BSA (final concentration, 1 mg/ml) and stored at 280°C until use. 9 Glutathione-S-transferase(GST) fusion protein was prepared for purification of antibody against MBP fusion protein. The cDNA (nucleotides 611-1255 in Fig. 1a) was sub-cloned into a GST-fusionprotein vector pGEX-5X-3 (Pharmacia). E. coli JM 109 cells were Preparation of fusion protein and antibody The cDNA of ST-1 (nucleotides 462-813 in Fig. 1a) was sub-cloned into a maltose-binding-protein(MBP) fusion protein vector pMAL-c2 (Bio-Labs, Beverly, MA). E. coli TB-1 cells were transformed with recombinant plasmid. The cells were cultured and induced to produce the fusion protein, which was collected by centrifugation. The pellets were suspended in column buffer (20 mM Tris-HCl, pH 7.4, 0.2 M NaCl, 1 mM EDTA, 10 mM b-mercaptoethanol, 1 mM NaN3 ), and frozen and thawed. The suspended cells from 1 liter of culture were disrupted by sonication and centrifuged at 14,000 g for 20 min. The supernatant was applied to an amylose-resin column (30 ml) (Bio-Labs), which was then washed with 10 volumes of column buffer. The fusion protein was eluted with 10 mM maltose in the column buffer. One hundred micrograms of this protein was mixed with Freund’s complete adjuvant and used for immunization. FIGURE 3 – Northern-blot analysis of mRNA corresponding to ST-1 and ST-2. (a) ST-1 mRNA; 5 µg of Poly A1 RNA from BOY cells were analyzed. (b) ST-2 mRNA; 20 µg of total RNA from BOY cells were analyzed. FIGURE 2 – Western-blot analysis to detect MM4 antigenic epitope in fusion proteins. Bacterial pellet was lysed in SDS-PAGE sample buffer and applied to each lane. SDS-PAGE was performed using 8% polyacrylamide gels. Lane 1, extract of BNN 103 lysogen which had b-galactosidase gene fused with the initially isolated insert of ST-2 (underlined in Fig. 2b) and expressed the 118-kDa fusion protein. Lane 2, extract of BNN 103 lysogen which had b-galactosidase gene fused with the initially isolated insert of ST-1 (underlined in Fig. 2a) and expressed the 128-kDa fusion protein. Lane 3, extract of BNN 93 expressing 116-kDa b-galactosidase. (a) Staining with Coomassie brilliant blue; (b) staining with MM4 MAb. FIGURE 4 – Western-blot analysis to detect antigenic proteins in extracts from BOY cells; 400 µg of protein was applied to each well of 8% polyacrylamide gels. After SDS-PAGE, the gel was stained by specific antibodies. (a) Anti-metanestin (ST-1 protein) revealed a 60-kDa band. (b) anti-PAPA antibody reacted with a 100-kDa band. TAKEMOTO ET AL. 10 FIGURE 5 – Expression of metanestin in clinical samples as revealed by immunohistochemical analysis using anti-metanestin antibody. (a) Normal epithelium (bar, 100 µm); (b) primary tumor (bar, 50 µm); (c) metastasized lymph node at low magnification (bar, 200 µm). Almost all metastatic tumor cells were stained. (d) Metastasized lymph node at high magnification (bar, 50 µm). All samples were taken from one patient. Concentration of the antibody was 7 µg/ml. TABLE I – STAINING OF PRIMARY TUMOR AND METASTATIC LYMPH NODES BY ANTI-METANESTIN AND ANTI-100-kDa-PROTEIN ANTIBODIES Number of cases Strong staining1 Anti-metanestin Primary tumor Lymph-node metastasis Anti-100-kDa protein Primary tumor Lymph-node metastasis No or weak staining2 9 6 15 1 12 8 6 2 1Strong staining, more than 20% of tumor stained.–2Weak or no staining, less than 20% of tumor stained. transformed with the recombinant plasmid. The cells were cultured and induced to produce fusion protein, which was then collected by centrifugation. The pellets from 1 liter of culture were suspended in PBS (—) and disrupted using sonicator. After centrifugation at 14,000 g for 20 min, the supernatants were applied to glutathioneSepharose column (10 ml) (Pharmacia) equilibrated with 10 bed volumes of PBS (—). The column was drained and washed with 30 bed volumes of PBS (—) and eluted with elution buffer (10 mM glutathione, 50 mM Tris-HCl, pH 8.0). The protein solution was dialysed against coupling buffer (0.5 M NaCl, 0.1 M NaHCO3, pH 8.3) and used for coupling to CNBr-activated Sepharose. The column was used to purify the antibody against the MBP fusion protein, as described for purification of the antibody to synthetic peptide. Western-blot analysis BOY cells were lysed in Laemmli’s sample buffer (Laemmli, 1970). Proteins were separated by SDS-PAGE on 8% gels and were electrophoretically transferred to nitrocellulose membranes. The membrane was reacted with mouse anti-MM4 MAb or affinitypurified rabbit antibodies, followed by HRP-labeled goat antimouse or rabbit IgG as described (Matsusako et al., 1992). Color was developed using 0.05% 4-chloro-naphthol in 0.1 M Tris-HCl buffer, pH 7.6, with 0.05% H2O2. Northern-blot analysis Total cellular RNA was extracted by the guanidium-isothiocyanate/cesium-chloride method (Maniatis et al., 1982). RNA was denatured with glyoxal and dimethyl sulfoxide, separated on 1% agarose gels and transferred to nylon membranes in 20 3 SSC. DNA fragments were labeled with 32P and used as probes (metanestin: nucleotide 611-1255 in Fig. 1a; a 100-kDa protein, nucleotide 63-645 in Fig. 1b). Hybridization was performed under conditions of low stringency (5 3 SSC, 50% formamide, 42°C) for 20 hr, and washes were performed with 0.1 3 SSC, 0.1% SDS, at 55°C. Immunohistochemical staining Transitional-cell-carcinoma tissues of the bladder were resected by radical cystectomy in Kagoshima University Hospital from 1994 to 1995; in all, 24 primary tumors of the bladder and 10 metastasized lymph nodes were analyzed, while some specimens were available only for analysis of expression of one antigen. The specimens were fixed with PBS(—)-buffered folmaldehyde and then embedded in paraffin. They were cut into sections 4 µm thick, A METASTASIS-ASSOCIATED GLYCOPROTEIN 11 FIGURE 6 – Expression of antigenic epitopes of a 100-kDa (ST-2) protein in clinical samples as revealed by immunohistochemical analysis using anti-PAPA antibody. (a) Normal epithelium (bar, 50 µm); (b) primary tumor (bar, 50 µm); (c) metastasized lymph node (bar, 50 µm). Only umbrella cells were faintly stained in normal epithelium, while primary tumors and the metastatic lesions were strongly stained. All samples were taken from one patient. Concentration of the antibody was 10 µg/ml. TAKEMOTO ET AL. 12 and endogenous peroxidase was blocked in methanol for 30 min. Specimens were then rinsed with PBS(—), incubated with normal goat serum (Nichirei, Tokyo, Japan) for 15 min at room temperature, then reacted with affinity-purified antibodies for 1 hr. After washing with PBS (—), sections were reacted with biotinylated goat anti-rabbit IgG (Nichirei) for 10 min at room temperature. The specimens were incubated with avidin-biotin-peroxidase (Nichirei) for 5 min, and color was developed with 0.05% 3,38-diaminobenzidine and 0.008% hydogen peroxide in 0.01 M Tris-HCl buffer, pH 7.4 for 5 min. The sections were washed with distilled water, counterstained with hematoxylin, dehydrated, washed with xylene, and mounted with permount. Negative control sections were incubated with normal rabbit serum instead of antibody. The positive control in each experiment was BOY transitional-cellcarcinoma cells, which were collected, fixed, embedded and sectioned as described above. RESULTS Predicted partial sequences of 2 proteins with MM4 antigen We screened an expression library constructed from BOY bladder-carcinoma cells with MM4 antibody, and obtained 2 clones, ST-1 and ST-2. Repeated screening failed to detect additional clones, and we tentatively concluded that protein sequences specified by the 2 clones included the MM4 antigenic epitope. Indeed, b-galactosidase fusion proteins of the inserts of the 2 clones reacted with the antibody, but b-galactosidase itself did not (Fig. 2). Thus, we decided to characterize the proteins encoded by these clones. Using the inserts as probes, we screened the library and obtained extended cDNAs. DNA sequences derived from the two clones (Fig. 1) did not overlap and were thus considered to encode different proteins. The length of the extended insert of ST-1 was about 1.2 kb, and that of ST-2 was 1.0 kb. Northern-blot analysis of Poly(A)1 RNA from BOY cells revealed that mRNA corresponding to ST-1 was 5 kb, while that to ST-2 was 3.5 kb (Fig. 3). Thus, these were partial clones. Both ST-1 and ST-2 had extended open reading frames. By analyzing the predicted protein sequences, we found that ST-1derived protein (ST-1 protein) contained 2 repeats of a Pro-Gly-ProGly motif (Fig. 1a) and ST-2-derived protein (ST-2 protein) had 4 repeats of a Pro-Ala-Pro-Ala motif. The initial clones, which reacted with MM4 MAb, each had 2 of Pro-Gly-Pro-Gly or Pro-Ala-Pro-Ala sequences (Fig. 1a,b, underlined sequences). Glycine and alanine are homologous amino acids; addition of a methyl group to glycine yields alanine. Since there are no other identical or homologous sequences between the predicted protein sequences, we conclude that the repeated Pro-Gly(or Ala)-ProGly(Ala) motif is the antigenic epitope of MM4 antigen. The predicted partial sequences of ST-1 protein and ST-2 protein were not related to any other protein sequences so far reported. That of ST-1 is rich in proline, serine and threonine. Since mucin is also known to be rich in these amino acids, ST-1 appears to be a new mucin-type glycoprotein. The partial sequence of ST-2 has a 4 times-repeated sequence of SXXXNHGPAPA. Occurence of repeated structure is a characteristic of mucin, and ST-2 protein is also considered to be a mucin-type glycoprotein. ST-1 protein, re-named metanestin, was preferentially expressed in metastatic nests in lymph nodes Since the predicted open reading frame of ST-1 encodes a 42-kDa polypeptide chain, we prepared a rabbit antibody against ST-1-maltose-binding protein fusion protein. The affinity-purified antibody reacted strongly with a 60-kDa protein from BOY cell extract (Fig. 4a). MM4 MAb also reacted principally with a 60-kDa band among LTA-binding glycoproteins isolated from BOY cells (Matsusako et al., 1992). Thus, we concluded that ST-1 encodes a partial sequence of the 60-kDa protein. Although a band of 80 kDa faintly reacted with the anti-ST-1-fusion-protein antibody, this was considered to be due to cross reactivity. The antibody to ST-1 fusion protein scarcely reacted with normal urothelium (Fig. 5a), and reactivity to primary tumors of transitionalcell carcinoma of the urinary bladder was generally weak (Fig. 5b, Table I). However, in 6 out of 7 cases of lymph-node metastasis, the staining was generally strong (Fig. 5c,d, Table I). Among the 6 strongly positive cases in lymph-node metastasis, in 4 cases the primary cancers did not react with the antibody, or reacted with it weakly. These results indicated that ST-1 protein was expressed in metastatic nests of lymph nodes, but was generally expressed only weakly or not at all in primary tumors. From the data of Table I, we conclude that the stronger expression of metanestin in lymph-node metastasis than in primary tumors is statistically significant ( p 5 0.0329, Fisher’s exact probability test). Thus, ST-1 protein is a novel protein which is preferentially expressed in metastatic nests in lymph nodes as compared with the primary tumor or with normal urothelium. We re-named ST-1 metanestin, because of its preferential expression in metastatic nests in lymph nodes. TABLE III – EXAMINATION OF POSSIBLE CORRELATION BETWEEN ANTIGEN EXPRESSION AND TUMOR STAGE Antigen Metanestin TABLE II – EXAMINATION OF POSSIBLE CORRELATION BETWEEN ANTIGEN EXPRESSION AND TUMOR GRADES Antigen Metanestin 100-kDa protein Tumor grades Primary tumor G1 G2 G3 Lymph-node metastases G1 G2 G3 Primary tumor G1 G2 G3 Lymph-node metastases G1 G2 G3 Number of cases Total Strongly positive 2 6 16 1 3 5 0 0 7 0 0 6 1 8 9 0 5 7 0 2 8 0 1 7 100-kDa protein Tumor stages Primary tumor T1 T2 T3a T3b T4 Lymph-node metastases T1 T2 T3a T3b T4 Primary tumor T1 T2 T3a T3b T4 Lymph-node metastases T1 T2 T3a T3b T4 Number of cases Total Strongly positive 10 2 5 6 1 2 2 1 4 0 1 0 1 4 1 1 0 1 3 1 6 1 4 6 1 2 1 3 5 1 1 0 3 5 1 1 0 1 5 1 A METASTASIS-ASSOCIATED GLYCOPROTEIN A protein with PAPA motif also showed tumor-associated expression Since insert of ST-2 was small, we prepared an antibody against a synthetic peptide with the repeating motif, PAPA. The antibody reacted with a 100-kDa protein on Western blots of BOY cell extract (Fig. 4b). The antibody faintly reacted with umbrella cells (Fig. 6a), but not with basal cells of the urothelium. Both primary tumors and metastatic lymph nodes reacted with the antibody (Fig. 6b,c). However, strong staining was also more frequently observed in lymph-node metastasis than the primary tumor (Table I). Thus, the 100-kDa protein with the PAPA motif also showed tumorassociated and metastasis-associated expression, although these tendencies were not as clear as those of metanestin, and no statistically significant conclusion was reached. We also examined the possible correlation between expression of metanestin or the 100-kDa protein and tumor grades (Table II), but at the present stage of analysis no clear-cut correlation was found. Similarly, antigen expression and tumor stages were not clearly correlated (Table III). DISCUSSION In transitional-cell carcinoma of the urinary bladder, we have found metanestin, a 60-kDa protein with tumor-associated and metastasis-associated expression, and elucidated its partial protein structure. Previous studies from our laboratories showed that the 60-kDa glycoprotein is the predominant carrier of carbohydrate epitopes, Lex and sialyl Lex, whose strong expression correlates with metastatic potential of the cancer cells (Matsusako et al., 1991, 1992). The 60-kDa glycoprotein corresponds to metanestin, identified herein. A 100-kDa protein with a repeated PAPA motif was also found to show tumor- and metastasis-associated expression, although these tendencies were less clear than those of metanestin. Comparison of protein sequences of metanestin and the 100-kDa protein revealed that the metastasis-associated antigenic epitope MM4 is a peptide, with twice-repeated PG (or A) PG (or A) motif. In other words, the MM4 antigenic epitope was concluded to be present both on metanestin and on the 100-kDa protein. Our results indicate that both the carbohydrate antigens and core proteins with the antigens show metastasis-associated expression. It is possible that expression of core protein in the tumor is a factor regulating the expression of an immunochemically detectable 13 carbohydrate antigen: the core protein may provide multiglycosylation sites, permitting production of multivalent carbohydrate antigens, which will bind to an antibody, especially Ig M with high affinity. Indeed, increased expression of Lex-related carbohydrate antigen in tumor cells may not be simply explained by the level of glycosyltransferase activities (Holmes and Macher, 1993). Both metanestin and the 100-kDa glycoprotein with the PAPA sequence were considered to be mucin-type glycoproteins, either from their amino-acid composition or from the presence of the repeating motif. Mucin-type glycoproteins have many glycosylation sites, and are suitable for carriers of carbohydrate antigens with high affinity (Shimizu and Shaw, 1993). Ligands of L-selectin are also carried by mucin-type glycoproteins (Lasky et al., 1992). Urinary-bladder carcinoma belongs to a group of cancers in which lymph-node metastasis is prevalent. Blood-borne metastasis occurs only in terminal stages or in a few cases of early cancer (Kyun et al., 1987). We have found that lymph-node metastasis occurs in 4.2% of cases in pL 0, 10% in pL 1 and 42.9% in pL 2 (data not shown). Thus, lymph-node metastasis is clearly correlated with lymphatic-vessel invasion: cancer cells are considered to enter into lymphatic vessels, and, through the lymphatic fluid, to metastasize to lymph nodes. Although E-selectin in blood vessels has been implicated in blood-borne metastasis, we do not know whether a selectin is present in lymphatic vessels. Thus, the metastasis-associated carbohydrate epitopes and their carrier, especially the 60-kDa glycoprotein metanestin, may function either as a ligand of hitherto undetected selectin in lymphatic vessels or by an entirely unrelated mechanism. In any event, demonstration of proteins with metastasis-associated expression will contribute significantly to understanding of the molecular mechanisms underlying lymph-node metastasis of urinary-bladder carcinoma. Metanestin may become clinically useful in detecting small lymph-node metastases of urinary-bladder carcinomas and in designing immunotherapy of the metastatic tumors. ACKNOWLEDGEMENTS We thank Ms. K. Yoshioka, K. Saito, C. Mashima, A. Horisawa and A. Miyata for secretarial assistance. This work was supported by grants from the Ministry of Education, Science and Culture of Japan. 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