Production of Monoclonal Antibody to Human Esophageal Cancer Cell Line Motoyasu NAITO,Masayuki IMAMURA,Reiji KANNAGI*and Takayoshi TOBE ABSTRACT: The immunization of Balb/C mice with esophageal cell line KYSE-50 established from poorly-differentiated esophageal squamous cell carcinoma, resulted in obtaining the monoclonal antibody KYMN-28-5. This monoclonal antibody is of the IgM class and recognizes a carbohydrate antigen contained in glycoproteins with molecular weights of 53 and 56K, and in neutral glycolipids extracted from teratomas. Tissue staining revealed that this monoclonal antibody reacts strongly with malignant tumors but only weakly, or not at all, with normal tissues, apart from squamous epithelial tissue. KYMN-28-5 is thus a useful tumor marker which will improved the accuracy of serological diagnosing squamous cell carcinoma when combined with the measurement of SCC antigen. KEY WORDS: monoclonal antibody, esophageal cancer cell line, SCC antigen, squamous cell carcinoma, carbohydrate antigens

INTRODUCTION

Since Koprowski et al.1 produced the first monoclonal antibody for human colorectal cancer by detecting that CA19-9 (carbohydrate antigen 19-9) was associated with such cancers, many lnonoclonal antibodies have been subsequently produced and used for the serological diagnosis of cancers of the digestive tract. However, most of these monoclonal antibodies are used for the diagnosis of adenocarcinoma and as yet, no monoclonal antibody which reacts with the squamous cell carcinoma (SCC) associated with esophageal cancer has been found,

The First Department of Surgery, Faculty of Medicine, Kyoto University, *the Department of Clinical Science and Laboratory Medicine, Faculty of Medicine, Kyoto University, Kyoto,Japan Reprint requests to: Motoyasu Naito, MD, The First Department of Surgery, Faculty of Medicine, Kyoto University, 54 Shogoinkawara-cho, Sakyo-ku, Kyoto 606,Japan

except for one which recognizes the SCC antigen?-4 In a recent experiment, we immunized Balb/C mice with KYSE-50, a cell line established from an esophageal cancer, and produced a monoclonal antibody that recognizes esophageal cancer by the cell fusion method described by Koehler and Milstein? The antigen recognized by our antibody was analyzed by the immunological detection method for a carbohydrate component, since many antigens detected by these monoclonal antibodies have recently been demonstrated as carbohydrate in nature, We thus produced a monoclonal antibody for esophageal cancer by the cell fusion method, and analyzed the antigen recognized by our antibody by the immunological detection method. METHODS

Production of the monoclonal antibody KYSE-506 cells (3)< 107), KYSE-50 being a

JAPANESEJOVIAL OVSURCERV,VOL.20, No. 2 pp. 170-179, 1990

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Monoclonal ab to human esophageal ca

cell line established from a poorly differentiated squamous cell carcinoma of the esophagus, were intraperitoneally administered to 8 week old Balb/c mice. After 3 weeks, the procedure was repeated using the same number of cells. The spleen was resected 3 days later, and splenic cells (1)~ 108) were fused to the mouse myeloma cell line X63.6.5.3 (2 X l0 t) at room temperature in the presence of 50 per cent (w/v) polyethyleneglycol 4000 (ART. 9727, Merck Co.)2 The hybridomas were then selected in HAT medium. Fourteen days after the cell fusion, the supernatant of the medium from the wells in which the hybridomas had formed colonies, was stained by the enzyme labeled antibody method (ABC method: VECTASTAIN ABC kit PK-4002) using 96 well plates (COSTAR catalog #3596) coated with the KYSE-50 cells in order to select the hybridomas producing the antibody to this cell line. The hybrid cells were cloned twice by the limiting dilution method, and those producing the monoclonal antibody KYMN-28-5 were obtained. The class of the antibody was IgM, as determined by the Ouchterlony method using a Monoclonal Typing Kit (Miles Scientific Co.). Western Blotting The KYSE-50 cells (2 X I0 ~) used for the immunization were suspended in 1 ml of TE containing 0.5 per cent triton X100. After the addition of para-aminophenylmethylsulphonyl fluoride (10 # tool), a strong proteinase inhibitor, the cells were disrupted by ultrasonication and centrifuged at 5000 G for 10 minutes. The supernatant was then subjected to electrophoresis using TEFCO-Gel SDS-PAGE mini (8 per cent) after which, the separated components were transferred to nitrocellulose membranes. The membranes were treated with 0.05 tool periodic acid or 12.5 /~m/m neuraminidase at 37~ for 1 hour, exposed to the supernatant from the cultures of hybridomas producing KYMN-285 as the primary antibody, and stained by the enzyme-labeled antibody method (ABC method).7 As the substrate for peroxidase, 4-

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chloro-l-naphtol was used. TLC (thin-layer chromatography) immunostaining Glycolipids from tumors and red blood cells were extracted by methods previously reported. 8 The glycolipids were separated by high performance TLC (HPTLC) and reacted with supernatant from the cultures of hybridomas producing KYMN-28-5 as the primary antibody. Rabbit anti-mouse IgM antibody (ICN Immunobiologicals, Cat #64365) was used as the secondary antibody, followed by incubation with II=5-protein A (Dupon, NEX-146L) and radioautography on X-ray filmsY Simultaneously, the separated glycolipids were reacted with orcinol sulfuric acid, and the TLC patterns compared with those of the plates after TLC immunostaining. ELISA To evaluate the reactivity of KYMN 28-5 to the cell lines of malignant tumors, 96-well flat-bottom culture dishes (COSTAR catalog #3596) were coated with each culture cell line and the ceils fixed with glutaraldehyde. Of the cell lines used, KYSE-30, 50, 110, 150, 170, 180, and 220 were established by Shimada at our laboratory (unpublished), and TE-1, 2, 3, and 8 were established and kindly provided by the Second Department of Surgery, Tohoku University. MIA-PaCa-2 was obtained from JCRB, A-431 from Dr. Ri of the Institute for Virus Research, Kyoto University, and the other cell lines from Dr. Miyake of the Chest Disease Research Institute, Kyoto University. Cells from each line were reacted with serial dilutions of the culture supernatant from the cultures of hybridomas producing KYMN-28-5 as the primary antibody and stained by the enzymelabeled antibody method (ABC method:indirect method: Cappel cat #3211-0201). Absorbance was measured at 500 nm. To examine the propemes of the antigen recognized by KYMN-28-5, 96-well flat-bottom culture dishes (COSTAR catalog #3296) were coated with cells from the esophageal cancer cell line KYSE-50. The cells were

1990 Jn.prchJ.l Surg. a

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fixed with glutaraldehyde, and one of the three following compounds was added to each experimental well: 50/tl o f 0.2 per cent pronase, 0.1 tool o f periodic acid, or 25 miliunit/ml neuraminidase. After incubation at 30~ for 30 minutes, the cells were treated with serial dilutions o f the supernatant from the cultures of hybridomas producing KYMN-28-5 a n d stained by the e n z y m e labeled a n t i b o d y m e t h o d (ABC m e t h o d : VECTASTAIN ABC KIT PK-4010). T o evaluate the reactivity of the KYMN-285 antibody to glycolipids, 10-20 ng/well 96 well flat-bottom culture dishes ( L i n b r o / Titertek cat. #76-381-04) were coated with various glycolipids using an ethanol solution of phosphatidylcholine and cholesterol T h e cells were reacted with serial dilutions o f the culture supernatant o f hybridomas producing KYMN-28-5 as the primary antibody and stained by the e n z y m e - l a b e l e d a n t i b o d y m e t h o d (indirect method). Absorbance was measured at 500 rim, 1~ using o-phenylenediamine as the substrate for peroxidase in all cases.

1"

0.90.8-

~0.70.6. 0.50.4"~ 0.3 ~ .~ 0.2~0.1 2-1 2-z

2-3 2-4 2-5 ,)-6 2-7 2-s 2-9 Dilution of Antibody

Fig. 1. Effect of periodic acid and protease treatment on the reactivity of KYMN-28-5 antibody to the esophageal cancer cell line KYSE-50 as measured by ELISA. Reactivity was markedly decreased in the wells treated with periodic acid. proteinase, - . . . . periodic acid, ..... neuraminidase, - - " - - control -

-

53k *-- 56k

Tissue staining Surgical specimens were fixed in 10 per cent formalin, e m b e d d e d in paraffin, and cut into sections. After the culture supernatant of h y b r i d o m a s p r o d u c i n g KYMN-28-5 was added as the primary antibody, the tissue sections were stained by the enzyme-labeled antibody method (ABC method: VECTASTAIN ABC KIT PK-4010). Diaminobenzidine tetrachloride was used as the substrate for peroxidase. RESULTS

Studies of antigens recognized by KYMN-28-5 in cell membrane glycoproteins ELISA using plates coated with the esophageal cancer cell line KYSE-50 showed a marked decrease in the reactivity to KYMN28-5 after treatment with periodic acid and only a slight decrease after treatment with proteinase (Fig. 1). According to the staining of nitrocellulose

Fig. 2. Reactivity of KYMN-28-5 antibody to membrane proteins of esophageal cancer cell line KYSE-50 cells. Western blotting showed that KYMN-28-5 recognizes antigens with molecular weights of 53 and 56K from neuraminidase-treated nitrocellulose membranes. Left: Gel stained with silver. Right: A nitrocellulose membrane after blotting stained by the enzyme-labeled antibody method (ABC method).

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membranes after Western blotting by the enzyme-labeled antibody method, KYMN-285 r e a c t e d with antigens with m o l e c u l a r weights o f 53K and 56K from the non-treated acid neuraminidase-treated membranes (Fig. 2). No positive reaction was observed with the membranes treated with periodic acid. Studies of glycolipid antigens recognized by KYMN-28-5 ELISA o f glycolipids extracted from lung tumor which had metastasized from esophageal cancer, lung squamous cell carcinoma or teratoma, as well as glycolipids from Otype red cells, CMH (ceramide monohexoside), CDH (ceramide dihexoside), CTH (ceramide trihexoside), nLc4 (neolactotetraosylceramide), and nLc6 (neolactohexosylceramide) was performed. T h e results showed that KYMN-28-5 reacted with some of the lung squamous cell carcinoma and the neutral glycolipid fraction o f teratoma but not with the other neutral glycolipid fractions or any of the acidic glycolipid fractions (Table

1). Furthermore, TLC i m m u n o s t a i n i n g revealed that this monoclonal antibody reacted

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with some o f the lung squamous cell carcinoma and the neutral glycolipid fraction from teratoma but not with acidic glycolipids containing sialic acid. Therefore, the epitope recognized by KYMN-28-5 is present in neutral glycolipids only. Tile TLC mobilities o f the positive glycolipids c o r r e s p o n d e d to those having 3 to 4 sugar residues but the reaction t ~ the standard C T H and nLc4 was negative. KYMN-28-5 did not react with the glycolipids extracted from A, B, or O type red blood cells, showing that the antigen recognized by this antibody is not contained in blood group glycolipids either (Fig. 3). Prevalence of the antigen recognized by KYMN28-5 in cultured human cancer cell lines ELISA using 96 well culture dishes coated with the cells o f cell lines established from malignant tumors such as esophageal cancer, showed moderate to strong reactions to KYMN-28-5. T h o u g h this monoclonal antibody was established from poorly differentiated esophageal squamous cell carcinoma, no difference was observed in its reactivity a m o n g cell lines established from esophageal cancers at various stages of differen-

Table 1. Reactivity of KYMN-28-5 to Glycolipids as Determined by ELISA Lung metastasis from esophageal cancer Neutral glycolipids Acidic glycolipids Lung squamous cell carcinoma Neutral glycolipids Acidic glycolipids Lung squamous cell carcinoma Neutral glycolipids Acidic glycolipids Teratoma Nentral glycolipids Acidic glycolipids

O type red blood cells ---

Neutral glycolipids Acidic glycolipids

m

CMH + --

CDH CTH

--

h

nLc4 nLc6

~+

--: absorbance (500 nm) ~0.2, +: 0.2-0.5, q+: 0.5-0.9, and ~: 0.9~ CMH: ceramide monohexoside, CDH: ceramide dihexoside, CTH: ceramide trihexoside, nLc4: Neolactotetraosylceramide, nLc6: Neolactohexosylceramide

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a

Fig. 3. Reactivity of KYMN-28-5 in TLCimmunostaining. The long chain group (upper A) and short chain group (lower) of the neutral glycolipids of O type red cell membrane antigens and the neutral glycolipids of teratoma (upper A) were applied to TLC. Immunostaining revealed a reaction of KYMN-28-5 only With the carbohydrate chains in the neutral glycolipids of teratoma but not with the bloodgroup substances. Left: HPTLC treated with orcinol sulfuric acid. Right: HPTLC treated with KYMN-28-5. tiation (Table 2). Distribution of the antigen recognized by KYMN-28-5 in normal and cancer tissues KYMN-28-5 reacted strongly to all specimens o f esophageal cancer (Fig. 4) and other malignant tumors (Table 3), however the antigen was not present in tissue samples of colorectal cancer. T h e reaction to normal tissues, apart from squamous epithelial tissue was w e a k a n d b o t h malignant and normal tissues o f the g l a n d u l a r e p i t h e l i u m a n d squamous epithelium were stained. Observation u n d e r high magnification of the strongly stained areas of the esophageal cancer tissue specimens from which KYSE-50 was established, revealed staining in a granular pattern, not only o f the cell m e m b r a n e but also o f the cytoplasm (Fig. 5). Stronglystained areas were also frequently observed in the cytoplasm o f other tissues. These

b Fig. 4. Immunohistochemical staining of esophageal cancer tissue by the enzyme labeled antibody method (X160) a: Original tumor tissue from which KYSE-50 was established: b: Tumor tissue of another esophageal cancer,

Fig. 5. High magnification of Fig. 4b (X320) Staining of the cytoplasm in a granular pattern.

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Table 2. Reactivity of KYMN-28,5 to Human Culture Cell Lines as Determined by ELISA Cell Lines of Squamous Cell Carcinoma Derived from Esophageal Cancer Well differentiated type (TE-1, TE-3, KYSE-30, NYSE-180) Moderately differentiated type (TE-8, KYSE-170, KYSE-220) Poorly differentiated type (TE-2, KYSE-50, KYSE-110, KYSE-150, LMEK-2) Total

1~

~

+

--

Total

3

0

1

0

4

1

2

0

0

3

3

1

1

0

5

7

3

2

0

12

1

0

0

0

1

0

1

0

0

1

0

1

0

0

1

0

1

0

0

1

0

1

0

0

1

0

1

0

0

1

0

1

0

0

1

0

1

0

0

1

0

1

0

0

1

0

1

0

0

1

l

9

0

0

10

Others MIA-PaCA-2 (Pancreatic cancer) SK-MES-1 (Lung squamous cell carcinoma) QG-90 (Lung small cell carcinoma) SK-LU-1 (Lung small cell carcinoma) A-549 (Lung adenocarcinoma A-431 (Vulvar cancer) SW-1222 (Colon cancer) AZ-521 (Gastric cancer) T-24 (Bladder cancer) MEWO (Malignant melanoma) Total

--: absorbance (500 nm) strongly stained areas_~20%

nized by this m o n o c l o n a l antibody is a carbohydrate chain. From western blotting, KYMN-28-5 was shown to recognize antibodies which had reacted to bands with molecular weights of 53K and 56K. These findings show that the epitope is also present in glycoproteins, although the carbohydrate chain c o m p o n e n t of glycoproteins and glycolipids is r e l a t i v e l y c o m m o n . CA19-9,1 CA50, ~1,12 and sialyl SSEA-1, a3 typical cancerassociated carbohydrate antigens, are present in both glycoproteins and glycolipids of cancer cells. Similarly, KYMN-28-5 seems to recognize a carbohydrate chain that is comm o n to both glycoproteins and glycolipids. T L C - i m m u n o s t a i n i n g revealed that the

2) 90%>strongly stained areas_~50% 4) 20%>strongly stained areas

neutral glycolipid fraction extracted from teratoma contains glycolipids with a carbohydrate chain which reacts strongly with KYMN-28-5. A comparison with HPTLCs from the same a m o u n t o f these glycolipids treated with orcinol sulfuric acid demonstrated KYMN-28-5 recognition of neutral glycolipids extracted from O-type red blood cells with the same distances as CTH and nLc4. However, TLC-immunostaining and ELISA of glycolipids showed that KYMN-28-5 did not react with CTH or nLc4 in the lane of neutral glycolipids extracted from O-type red b l o o d cells. T h e r e f o r e , the carbohydrate antigen recognized by this monoclonal antibody has a structure unlike that of CTH and

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Monoclonal ab to human esophageal ca

nLc4. Since CTH consists of 3 carbohydrate residues, namely; GA1 a 1-4GA1fll-4Gacfil1Cer, and nLc4 consists of 4 carbohydrate residues, namely; Gal/31-4GlcNAcf11-4Gal/314Glc/31-1Ceri the carbohydrate antigen recognized by KYMN-28-5 consists of 3-4 carbohydrate residues. Tissue staining showed that KYMN-28-5 does not always react specifically to tissue derived from esophageal carcinoma. In fact this monoclonal antibody showed a marked reaction to all tumor derived cells, except for those derived from colorectal cancer. However, the antibody reacted only weakly or not at all with normal tissues, apart from the normal squamous epithelial layer of the esophageal mucosa and the epidermal squamous epithelial layer obtained from patients with cancer. MoreoVer, light microscopy under high magnification demonstrated that KYMN-28-5 reacts with granules contained in the cytoplasm. These findings suggest not only that the antigen recognized by this monoclonal antibody is produced by cells, being present in large amounts in squamous cell carcinoma and normal squamous cells, but that it is also produced in other glandular epithelial cells when malignant changes occur. ELISA of the cell lines showed no difference in the reactivity among cell lines established from esophageal squamous cell carcinomas of various differentiation types, indicating that the antigen recognized by KYMN-28-5 is present regardless of the degree of differentiation. Moreover, ELISA of the cell lines established from malignant tumors other than squamous cell carcinoma revealed a moderate to strong reactiOn of this monoclonal antibody in each case. A commercial kit for measuring SCC antigenTM is clinically used as a tumor marker for squamous cell carcinoma, and has proven Useful in the diagnosis of squamous cell carcinoma of the head and neck, 1~,16 oral squamous cell carcinoma, ~7 lung squamous cell carcinoma,la,19esophageal carcinoma, 26,2a squamous cell carcinoma of the uterine cervix,~2-24 and squamous cell carcinoma of

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the skin. 25 SCC antigen is a subfraction (molecular weight, 42-48K) of the glycoprotein TA-4 antigen extracted by Kato et al.26 from squamous cell carcinoma of the uterine cervix, TA-4 being the general term for subfractions with a molecular weight of 42-48K obtained by electrophoresis. However, the fine structures still remain to be clarified, 27 although since the molecular weights of the glycoproteins identified by KYMN-28-5 were 53 and 56K, this antigen must be different from any subfraction of TA-4. Histologically, this antigen is present in the cell, as is TA-4. Our results show that immunohistologicaUy, the organ specificity of this antigen to malignant tumors is lower than that of SCC, but its specificity to the malignant tumors themselves is higher. However the cross reaction between the antigen recognized byu KYMN-28-5 and SCC antigen remains to be determined. KYMN28-5 reacted with normal squamous epithelial tissue to a similar extent as the antibody that recognizes SCC antigen?s The carbohydrate antigen recognized by KYMN-28-5 is present in both malignant and normal squamous epithelial cells. It is also present in small amounts in other normal glandular epithelial cells and increases with malignant changes. The application of this monoclonal antibody to serological diagnosis has yet to be investigated. Since the combined use of various tumor markers has improved the accuracy of diagnosis for adenocarcinoma, KYMN-28-5 may prove to be clinically useful in the future for enhancing th e accuracy of serologicaUy diagnosing squamous cell carcinoma when combined with the measurement of SCC antigen. ACKNOWLEDGEMENTS

We express deep gratitude to Yutaka Shimada and Yasuaki Hattori of our Department and Masayuki Miyake of the Surgical Department, Chest Disease Research Institute, Kyoto University for much cooperation.

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W e w o u l d a l s o l i k e to t h a n k T o s h i h i r o Adachi, of the Pathological Department, Kyoto U n i v e r s i t y a n d K u n i h i r o U e d a o f t h e Department of Clinical Laboratory Medicine, K y o t o U n i v e r s i t y f o r t h e i r d i r e c t i o n , as well as Testuro Nishihira, of the Second Surgical Department, Tohoku University for provis i o n o f cells.

( R e c e i v e d f o r p u b l i c a t i o n o n Apr. 19, 1989) REFERENCES 1. Koprowski H, Heflyn M, Steplewski Z. Specific antigen in serum of patient with colon carcinoma. Science 1981; 212: 53-55. 2. Komatsu H, Kawamura M, Yoneda R. A study on serum tumor markers (CEA, NSE, SCC and CA199) in cases with bronchopulmonary carcinoma. Nippon Kyobu Rinsho (]pnJ Chest Diseases) 1987; 46: 213-218. (in Japanese with English Abst.) 3. Torigoshi T, Takeuchi M, Sugawa K, Takamizawa Y, Hashimoto M, Shinagawa N, Nishitani I, Ishii K, Kato K, Kasamatsu T, Yamada T, Masubuchi K, Okura H, Kufihara S, Nozawa S, Fujiwara Y, Sato Y, Yaganti N, Chihara T, Katsuya tL Sugiyama Y, Noda K, Kurachi K, Inoue Y, Ohga Y, Noda K, Mochizuki M, Shibata K, Sekiba K, Tanimura T, Kato H0 Mori T, Yamabe T. Clinical use of squamous cell carcinoma related antigen TA-4 RIA kit. Sanka to Fujinka (Obstet Gynecol (Tokyo)) 1984; 51: 1199-1207. (in Japanese ) 4. Yagi H, Danno K, Maruguchi Y, Yamamoto M, Imamura S. Significance of squamous cell carcinoma (SCC)-related antigens in cutaneous SCC. Arch Dermatol 1987; 123: 902-906. 5. Koehler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975; 256: 495-497. 6. Shimada Y, Imamura M, Naito M, Tsuboi K, Tobe T, Yamamoto I, Ueno S, Ri H, Takahashi K, Hatanaka M, Yamaguchi N. Establishment and characterization of two cell lines from cancer of the esophagus; KYSE-30 with hyperexpression of epidermal growth factor (EGF) receptors and KYSE-50 producing parathyroid hormone (PTH) like substance. Dai 46 kai Nihon Gan Gakjkai Soukai Kiji (GANN proceedings of 46th annual meeting) 1987; 222. (in Japanese) 7. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and applications. Proc Natl Acad Sci USA 1979; 76: 4350-4354.

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Production of monoclonal antibody to human esophageal cancer cell line.

The immunization of Balb/C mice with esophageal cell line KYSE-50 established from poorly-differentiated esophageal squamous cell carcinoma, resulted ...
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