61
Comer Letters, 63 (1992) 61- 66 Elsevier Scientific Publishers Ireland Ltd.
Monoclonal antibody MON-114: detection of a marker neuroendocrine differentiation in human lung cancer Hans L.P. van Duijnhoven”, Ellen H.M. Moorsa, Arie Groeneveld”, Julia M. Polakd Lou F.M.H. de Leijb, Sjoerd SC. Wagenaar’, and Wim J.M. van de VenaTe
for
Erika D.J. Timmera,
“Molecular Oncology Section, Department of Biochemistry, Uniuersity of Nijmegen. P.O. Box 9101, 6500 HB Nijmegen (The Netherlands) and bDepartment of Clinical Immunology, University Hospital Groningen, Oostersingel 59, 9713 EZ Groningen (The Netherlands) and ‘Department of Pathology, St. Anton& Hospital, P.O. Box 2500, 3430 EM Nieutuegein (The Netherlands) and dDepartment of Histochemistry, Royal Postgraduate Medical School, Du Cane Road, London WI2 ONN (UK) and ‘Molecular Oncology Section, Center of Human Genetics, University of Leuuen, Herestraat 49, B-3000 Leuven (Belgium) (Received 10 October 1991) (Revision received 15 January (Accepted 20 January 1992)
1992)
Summary
Introduction
Mouse monoclonal antibody MON-114 was generated upon immunization with a human small cell lung carcinoma cell line GLC-19. Immunohistochemical analysis of normal tissues with MON-114 showed staining of the adrenal gland, brain and peripheral nerves. With respect to human lung carcinomas, 7 out of 8 small cell lung carcinomas were positively stained as well as 5 out of 5 carcinoid tumors, whereas only 4 out of 31 squamous celi carcinomas and 3 out of 19 adenocarcinomas were weakly stained. Furthermore, 1 large cell carcinoma was negative for MON-114 staining. Apparently, MON-114 stains cells of neuroendocrine differentiation.
Lung cancer is a leading cause of death, especially among men in the Western world, and the incidence is significantly increasing in women 191. According to the World Health Organization [14], a pathologic subclassification of human lung cancer can be made on histological and morphological criteria. Four major subtypes of lung cancer can be distinguished: squamous cell carcinoma, adenocarcinoma, large cell carcinoma (these three subtypes are collectively referred to as non-small cell lung carcinoma (NSCLC)), and small cell lung carcinoma (SCLC). The subdivision of carcinoma of the lung into SCLC and NSCLC is also made because of the different response of these tumors to radiation and cytotoxic therapy. SCLCs, which comprise about 25% of all new cases, are the most sensitive to radio- and chemotherapy [6,7]. In general, SCLCs are identified by their neuroendocrine phenotype, since NSCLCs generally do not express neuroendocrine features.
Keywords: monoclonal antibody; neuroendocrine; lung cancer; immunohistochemistry Correspondence to: Hans L.P. van Duijnhoven, cology Section, megen.
Department
P.O. Box 9101,
0304-3835/92/$05,00 Printed
and Published
of Biochemistry,
6500 HB Nijmegen,
0
Molecular OnUniversity of Nij-
The Netherlands.
1992 Elsevier Scientific Publishers
in Ireland
Ireland
Ltd
62
The neuroendocrine characteristics are most often preserved in SCLC-derived cell lines. SCLC cell lines can be subdivided into two major categories: classic-type (c-SCLC, 70% of all SCLC cell lines) and variant-type (v-SCLC, 30%) cell lines [5]. Of these, classic cell lines exhibit the most pronounced neuroendocrine phenotype, which is lost to some extent in variant cell lines. A wide variety of biomarkers for different subtypes of lung cancer have been identified. They include enzymes, hormones, cell surface markers, as well as DNA and cytogenetic markers [9]. Some of these markers can be used in a serum assay to identify SCLC [4]. In addition, a large number of monoclonal antibodies (MoAbs) have been raised against lung cancer cells. Some of these antibodies appeared to be useful in the diagnosis of various subtypes of lung cancer and possibly also in therapy. The First and Second International Workshops on Lung Cancer Antigens have brought together numerous MoAbs in order to classify reactivity and to identify antigens recognized by these antibodies [3,11]. In this way, clusters of MoAbs were identified which appeared to detect similar antigens. The MoAbs of the largest cluster, cluster 1, recognized the human neural cell adhesion molecule (NCAM) [lo]). The antigen is expressed on small cell lung cancer, carcinoid tumor, neuroblastoma, renal carcinoma, thyroid epithelium, and peripheral nerve [ll]. As an approach to raise antibodies against markers for small ceil lung tumors, we developed MoAbs against the SCLC cell line GLC-19, a drug-resistant member of a panel of three cell lines established from primary and recurrent tumors of a patient during longitudinal clinical follow-up [2]. Apart from obtaining MoaAbs that are capable of distinguishing different subtypes of lung cancer, this approach may also lead to the development of MoAbs that might be instrumental in the identification and characterization of phenotypes of (multi)drug resistant lung cancer cells.
Materials and methods Cell lines
Cell lines were obtained from Dr. L. de Leij, Dr. G. Bepler, Dr. D. Carney and the American Type Culture Collection. Cells were grown in either RPM1 1640 or Dulbecco’s modified Eagle medium, supplemented with 10% fetal bovine serum (HyClone), 2 mM Lglutamine, I mM sodium pyruvate, streptomycin (100 pg/ml) and penicillin (100 units/ml). The cell line SCLC-16HVA was established from the parent cell line SCLC-16HV after repetitive selection for adherent cells. Cell lines GLC-14, GLC-16 and GLC-19 were developed respectively from the primary and recurrent tumors of the same patient [2], and still maintain the initial drug sensitivity. Generation
of hybridomas
A BALB/c mouse was immunized intraperitoneally (i.p.) with 5 x lo6 GLC-19 cells using Freund’s complete adjuvant. Subsequently, the mouse was rechallenged i.p. five times at three week intervals with 5 x lo6 GLC-19 cells using Freund’s incomplete adjuvant. Three days before the fusion 3 x lo6 GLC-19 cells in PBS were injected intravenously into a tail vein. Spleen cells were fused with SP2/0-Ag14 cells as described earlier [ 131. Screening
procedure
and
isotype
deter-
mination
Tissue culture supernatants of growing clones were tested in an immunofluorescence assay (IFA) and by Western blot analysis. For IFA, GLC-14, GLC-19 and H23 cells were applied on glass slides, dried and subsequently fixed in cold methanol (-20°C) and acetone. After two washes for 5 min with PBS/0.05% Tween-20 (PBS-T), the cells were incubated with hybridoma tissue culture supernatant for 2 h at room temperature. The slides were washed twice for 10 min with PBS-T and the cells were incubated with rabbit anti-mouse FITC
(diluted 1:50 in PBS) for 2 h at room temperature. This was followed by two 10 min washes with PBS-T and the slides were coverslipped. For Western blot analysis, blots of GLC-14, GLC-19 and HZ3 were prepared and incubated as described earlier [U] . The isotype of the MoAbs was determined using a dipstick isotyping kit (Holland biotechnology bv) . lmmunocytochemistry For testing of reactivity of the MoAbs in immunocytochemistry, an immunofluorescence assay was used (see screening procedure). Immunohistochemistry Sections were cut from frozen material (4 pm) and allowed to dry at room temperature for at least 30 min. The sections were fixed in cold methanol (-20°C) and acetone and subsequently washed three times in PBS for 5 min. After an incubation with normal horse serum, diluted 1:30 in PBS, the sections were incubated with MON-114 tissue culture supernatant for 1 h at room temperature. After washes with PBS (3 x 5 min), biotinylated horse anti-mouse (Vector Labs., diluted 1: 100 in PBS) was applied for 30 min. This was followed by washing in PBS as before and an incubation with avidin-biotin-peroxidase complex (Vector Labs., diluted 1:lOO in PBS) for 60 min. The peroxidase was revealed using either 3-amino-g-ethyl carbazole solution (0.2 mg/ml in 5 mM sodium acetate buffer, pH 4.85) or 0.05% diaminobenzidine solution and nickel enhancement [8]. The sections were rinsed twice in distilled water and counterstained lightly with hematoxylin.
Results Immunization of mice with GLC-19 lung tumor cells and subsequent cell fusion experiments resulted in the isolation of several hybridomas. One of them, MON-114 (IgGl), showed a strong cytoplasmic reactivity in the assay described in immunofluorescence
Fig.1. lmmunofluorescence v-SCLC cell line SCLC-16HVA MOC-1 (B).
staining of the adherent using MON- 114 (A) and
Table 1. Reactivity of MON.114 with cell lines Cell line
Type
Reactivity of MON-114:
SCLC- 16HC SCLC-21H
cmSCLC c-SCLC c-SCLC c-SCLC C~SCLC C~SCLC c-SCLC v-SCLC v-SCLC v-SCLC v-SCLC v-SCLC Adenocarcinoma lung Adenocarcinoma lung Epitheloid cervix carcinoma Myelocytic leukemia Promyelocytic leukemia Histiocytic lymphoma Pheochromocytoma, rat Pituitary tumor, mouse
+ + ++ ++ + ++ + + + + -
GLC- 14 GLC- 16 GLC- 19 GLC-l-Ml3 GLC-28 SCLC- 16HV SCLC16HVA GLC-1 GLC-4 H82 H23 A549 HeLa ML-1 HL-60 u937 PC-12 AtT20
+ _ + + + +
64
Materials and Methods, but no reactivity was observed in Western blot analysis. Initially, the expression pattern of the antigen detected by MON-114 was studied in a panel of cell lines by immunocytochemistry. In these experiments, 11 out of 12 SCLC cell lines were positively stained. Of the SCLC cell lines, GLC-4 was negative for MON-114 staining. Furthermore, 2 out of 2 adenocarcinomas cell lines of the lung and 4 out of 4 non-lung tumor cell lines (HeLa, ML-l, HL-60 and U937) were also negative, as shown in Table 1. Figure 1A shows the reactivity of MON- 114 with cell line SCLC-16HVA. As a control, the same cell line was stained using the MOC-1 antibody, which recognizes NCAM (Fig. 1B). The strong reactivity with PC-12 and AtT-20 cells suggested that MON-114 might detect a neuroendocrine antigen which was conserved in rat and mouse. Subsequently, the expression pattern of MON-114 was studied in a small panel of normal human tissues (see Table 2). The observation that all neural cells of the brain and cells of the adrenal medulla were positively stained supported the idea that MON-114 might specifically detect an antigen expressed on neuroendocrine cells. In Fig. 2B reactivity of MON-114 with a peripheral nerve is shown as an illustration. In liver, spleen, kidneys, ovaries and pancreas no reactivity could be observed. It should be noted that MON-114 reactivity can only be observed with cryo-sections (data not shown).
Table 2. tissues.
Reactivity of MON-114
with normal
To evaluate the usefulness of MON-114 in human lung cancer pathology, reactivity of the antibody was tested in an immunohistochemical study of a series of human lung tumors. The results are summarized in Table 3. Positive staining was observed in 7 out of 8 SCLCs and very strong staining in 5 out of 5 carcinoid tumors (Fig. 2A). With respect to NSCLCs, in 4 out of 31 squamous cell carcinomas and in 3 out of 19 adenocarcinomas weak staining was observed, however, in all cases only focal-
human Table 3. Reactivity of MON-114 lung tumors.
with primary human
Tissue
Reactivity
Liver Colon Spleen Kidney Adrenal medulla Brain Ovary Pancreas
-
Tumor type
Reactivity of MON-114
-
SCLC Squamous cell carcinoma Adeno carcinoma Large cell carcinoma Carcinoid
7/8 4/31 3/19 O/l 5/5
+ ++
of MON-114
Fig. 2. Immunohistochemical analysis of a carcinoid tumor (A) and staining of peripheral nerve in a primary squamous cell carcinoma (B) using MON-114.
l
‘Number of positive cases of total tested tumors.
65
ly. No reactivity was found in the only case of large cell carcinoma that was studied. In 3 out of 3 cases, MON-114 also positively stained SCLC metastases in lymph nodes (data not shown). Occasionally, a weak staining of stroma cells was observed. Discussion The reactivity pattern of monoclonal antibody MON-114 with cell lines, normal tissues and primary human lung tumors indicated that the antigen detected by this antibody was preferentially expressed in cells and tissues with neuroendocrine characteristics. The intensity of staining observed in a panel of three SCLC cell lines established from one patient during longitudinal clinical follow-up [Z] , GLC-14, GLC-16 and GLC-19, was similar. Only a small decrease in expression was observed in the drug-resistant cell line GLC-19 as compared to GLC-14, which was established before therapy. Apparently, expression of the antigen is not affected during the development of the drug-resistant phenotype of these cells. While v-SCLC cell lines seem to lose neuroendocrine characteristics to some extent as compared to c-SCLC cell lines, only one of the v-SCLC cell lines, GLC-4, was negative for MON-114 staining. GLC-4 cells grew as a loosely adherent monolayer which made it different from other SCLC cell lines which all grew in suspension. Austin Doyle et al. [l] reported that NCAM expression was high in SCLC cells that grew in suspension but is down regulated in adherent v-SCLC cell lines. It might be possible that MON-114 recognized a molecule that is down-regulated similarly in adherent v-SCLC cell lines. However, no difference in expression of the antigen recognized by MON-114 was observed in the adherent cell line SCLC-16HVA and the parent cell line SCLC-16HV. Furthermore, anti-NCAM staining by MOC-1 was also not affected (data not shown). The neuroendocrine reactivity pattern of MON-114 in a series of lung tumors, SCLCs as well as NSCLCs, is somewhat similar to that of cluster 1 lung cancer antibodies [ll], In 7 out
of 50 adenocarcinomas and squamous cell a weak positive staining with carcinomas, MON-114 was observed; in 4 out of these, also MOC-1 reactivity was observed and electron microscopy revealed the presence of dense core granules (data not shown), which is indicative for neuroendocrine characteristics. However, there are also differences. As shown in Fig. lB, anti-NCAM staining of membranes by MOC-1 is clearly different from cytoplasmic MON-114 staining (Fig. 1A). Furthermore, MON-114 did not react with D243 cells transfected with the gene encoding the 140-kDa transmembrane isoform of NCAM from human skeletal muscle (Dr. J. Ledermann, personal communication). All cluster 1 antibodies submitted to the Second International Workshop on Lung Cancer Antigens showed binding to these D243 cells [ll]. Altogether, the results suggests that MON-114 recognizes an epitope of a neuroendocrine differentiation marker that has a similar expression pattern as NCAM. However, the antigen of MON-114 is still unknown. Western blotting analysis and immunoprecipitation experiments did not uncover the nature of the antigen, and screening of a cDNA library using MON-114 did also not reveal the coding sequence of the corresponding gene. Nevertheless, MON-114 might prove to be a useful discriminating tool in lung cancer pathology. Acknowledgements We would like to thank Dr. J. Ledermann for testing MON-114 on NCAM-transfectants. In addition, we thank F. Schmitz du Moulin for excellent technical assistance and Dr. K. Pate1 for helpful discussion. Part of this work is supported by the Dutch Cancer Society. References Austin Doyle, L., Borges, M., Hussain, A., Elias, A. and Tomiyasu. T. (1990) An adherent subline of a unique small-cell lung cancer cell line downregulates antigens of the neural ceil adhesion molecule. J. Clin. Invest., 86, 1848 - 1854. Berendsen, H.H., De Leij, L., De Vries, E.G., Mesander, G., Mulder,
N.H.,
De Jong,
B., Buys,
C.H.,
Postmus.
66 P.E., Poppema, S., Sluiter, H.J. and The, H.T. (1988) Characterization of three small cell lung carcinoma cell lines established from one patient during longitudinal follow-up. 3
4
5
6
7
8
Cancer Res., 48, 6891- 6899. Beverley, P.C., Souhami, R.L. and Bobrow, L.G. (1988) Results of the central data analysis. Lung Cancer, 4, 15-36.
9
10
Bork, E., Hansen, M., Urdal, P., Paus, E., Hoist, J.J., Schifter, S., Fenger. M. and Engbaek, F. (1988) Early detection of response in small cell bronchogenic carcinoma by changes in serum concentration of creatine kinase, neuron specific enolase, calcitonin, ACTH, serotonin and gastrin releasing peptide. Eur. J. Cancer Clin. Oncol., 24, 1033 - 1038. Carney, D., Gazdar, A.F., Bepler, G., Guccion, J.G.,
11
Marangos, P.J., Moody, T.W., Zweig, M.H. and Minna, J.D. (1985) Establishment and identification of small cell lung cancer cell lines having classic and variant features. Cancer Res., 4.5, 2913 - 2923.
12
Duchesne, G., Cassoni, A. and Pera, M. (1988) Radiosensitivity related to neuroendocrine and endodermal differentiation in lung carcinoma lines. Radiother. Oncol., 13, 153- 161.
13
its application to double immunohistochemistry. Histochem. Cytochem., 30, 1079 - 1082.
J.
Oncology, 3rd Edition, pp. 591-705. Editors: V.T. De Vita, S. Hellman, and S.A. Rosenberg. J.B. Lippencott Company, Philadelphia. Patel, K., Moore, S.E., Dickson, G., Rossell, R.J., Beverley, P.C., Kemshead, J.T. and Walsh, F.S. (1989) Neural cell adhesion molecule (NCAM) is the antigen recognized by monoclonal antibodies of similar specificity in small cell lung carcinoma and neuroblastoma. Int. J. Cancer, 44, 573- 578. Souhami, R.L., Beverley, P.C.L., Bobrow, L.G. and Ledermann, J.A. (1991) Antigens of lung cancer: results of the second international workshop on lung cancer antigens. J. Natl. Cancer Inst., 83, 609-612. Van Duijnhoven J.L.P., Ayoubi T.A.Y., Timmer E.D.J., Braks A.A.M., Roebroek A.J.M., Martens, G.J.M. and Van de Ven W.J.M. (1989) Development of a monoclonal antibody against recombinant neuroendocrine 7B2 protein. FEBS Len., 255, 372-376. Van Duijnhoven, J.L.P., Verschuren, M.C.M., Timmer, E.D.J., Vissers, P.M.A.M., Groeneveld, A., Ayoubi. T.A.Y., Van den Ouweland, A.M.W. and Van de Ven, W.J.M. (1991) Application of recombinant DNA technology in epitope mapping and targeting. Development and characterization of a panel of monoclonal an-
Graziana, S.L., Mazid, R., Newman, N., Tatum, A., Oler, A,, Mortimer. J.A., Gullo, J.J., DiFino, S.M. and Scalzo, A.J. (1989) The use of neuroendocrine immunoperoxydase markers to predict chemotherapy response in patients with non-small cell lung cancer. J. Clin. Oncol., 7, 1398 - 1406. Hsu, S.M. and Soban, E. (1982) Colour modification of diaminobenzidine (DAB) precipitation by metalic ions and
Minna, J.D., Pass, H., Glatstein, E. and Ihde, D.C. (1989) Cancer of the lung. In: Cancer: Principles and Practice in
14
tibodies against the 7B2 neuroendocrine protein. J. Immunol. Methods, in press. World Health Organization (1982) The World Health Organization histological typing of lung tumors, 2nd Edition. Am. J. Clin. Pathol.,
77, 123-
136.
Comer Letters, 63 (1992) 61- 66 Elsevier Scientific Publishers Ireland Ltd.
Monoclonal antibody MON-114: detection of a marker neuroendocrine differentiation in human lung cancer Hans L.P. van Duijnhoven”, Ellen H.M. Moorsa, Arie Groeneveld”, Julia M. Polakd Lou F.M.H. de Leijb, Sjoerd SC. Wagenaar’, and Wim J.M. van de VenaTe
for
Erika D.J. Timmera,
“Molecular Oncology Section, Department of Biochemistry, Uniuersity of Nijmegen. P.O. Box 9101, 6500 HB Nijmegen (The Netherlands) and bDepartment of Clinical Immunology, University Hospital Groningen, Oostersingel 59, 9713 EZ Groningen (The Netherlands) and ‘Department of Pathology, St. Anton& Hospital, P.O. Box 2500, 3430 EM Nieutuegein (The Netherlands) and dDepartment of Histochemistry, Royal Postgraduate Medical School, Du Cane Road, London WI2 ONN (UK) and ‘Molecular Oncology Section, Center of Human Genetics, University of Leuuen, Herestraat 49, B-3000 Leuven (Belgium) (Received 10 October 1991) (Revision received 15 January (Accepted 20 January 1992)
1992)
Summary
Introduction
Mouse monoclonal antibody MON-114 was generated upon immunization with a human small cell lung carcinoma cell line GLC-19. Immunohistochemical analysis of normal tissues with MON-114 showed staining of the adrenal gland, brain and peripheral nerves. With respect to human lung carcinomas, 7 out of 8 small cell lung carcinomas were positively stained as well as 5 out of 5 carcinoid tumors, whereas only 4 out of 31 squamous celi carcinomas and 3 out of 19 adenocarcinomas were weakly stained. Furthermore, 1 large cell carcinoma was negative for MON-114 staining. Apparently, MON-114 stains cells of neuroendocrine differentiation.
Lung cancer is a leading cause of death, especially among men in the Western world, and the incidence is significantly increasing in women 191. According to the World Health Organization [14], a pathologic subclassification of human lung cancer can be made on histological and morphological criteria. Four major subtypes of lung cancer can be distinguished: squamous cell carcinoma, adenocarcinoma, large cell carcinoma (these three subtypes are collectively referred to as non-small cell lung carcinoma (NSCLC)), and small cell lung carcinoma (SCLC). The subdivision of carcinoma of the lung into SCLC and NSCLC is also made because of the different response of these tumors to radiation and cytotoxic therapy. SCLCs, which comprise about 25% of all new cases, are the most sensitive to radio- and chemotherapy [6,7]. In general, SCLCs are identified by their neuroendocrine phenotype, since NSCLCs generally do not express neuroendocrine features.
Keywords: monoclonal antibody; neuroendocrine; lung cancer; immunohistochemistry Correspondence to: Hans L.P. van Duijnhoven, cology Section, megen.
Department
P.O. Box 9101,
0304-3835/92/$05,00 Printed
and Published
of Biochemistry,
6500 HB Nijmegen,
0
Molecular OnUniversity of Nij-
The Netherlands.
1992 Elsevier Scientific Publishers
in Ireland
Ireland
Ltd
62
The neuroendocrine characteristics are most often preserved in SCLC-derived cell lines. SCLC cell lines can be subdivided into two major categories: classic-type (c-SCLC, 70% of all SCLC cell lines) and variant-type (v-SCLC, 30%) cell lines [5]. Of these, classic cell lines exhibit the most pronounced neuroendocrine phenotype, which is lost to some extent in variant cell lines. A wide variety of biomarkers for different subtypes of lung cancer have been identified. They include enzymes, hormones, cell surface markers, as well as DNA and cytogenetic markers [9]. Some of these markers can be used in a serum assay to identify SCLC [4]. In addition, a large number of monoclonal antibodies (MoAbs) have been raised against lung cancer cells. Some of these antibodies appeared to be useful in the diagnosis of various subtypes of lung cancer and possibly also in therapy. The First and Second International Workshops on Lung Cancer Antigens have brought together numerous MoAbs in order to classify reactivity and to identify antigens recognized by these antibodies [3,11]. In this way, clusters of MoAbs were identified which appeared to detect similar antigens. The MoAbs of the largest cluster, cluster 1, recognized the human neural cell adhesion molecule (NCAM) [lo]). The antigen is expressed on small cell lung cancer, carcinoid tumor, neuroblastoma, renal carcinoma, thyroid epithelium, and peripheral nerve [ll]. As an approach to raise antibodies against markers for small ceil lung tumors, we developed MoAbs against the SCLC cell line GLC-19, a drug-resistant member of a panel of three cell lines established from primary and recurrent tumors of a patient during longitudinal clinical follow-up [2]. Apart from obtaining MoaAbs that are capable of distinguishing different subtypes of lung cancer, this approach may also lead to the development of MoAbs that might be instrumental in the identification and characterization of phenotypes of (multi)drug resistant lung cancer cells.
Materials and methods Cell lines
Cell lines were obtained from Dr. L. de Leij, Dr. G. Bepler, Dr. D. Carney and the American Type Culture Collection. Cells were grown in either RPM1 1640 or Dulbecco’s modified Eagle medium, supplemented with 10% fetal bovine serum (HyClone), 2 mM Lglutamine, I mM sodium pyruvate, streptomycin (100 pg/ml) and penicillin (100 units/ml). The cell line SCLC-16HVA was established from the parent cell line SCLC-16HV after repetitive selection for adherent cells. Cell lines GLC-14, GLC-16 and GLC-19 were developed respectively from the primary and recurrent tumors of the same patient [2], and still maintain the initial drug sensitivity. Generation
of hybridomas
A BALB/c mouse was immunized intraperitoneally (i.p.) with 5 x lo6 GLC-19 cells using Freund’s complete adjuvant. Subsequently, the mouse was rechallenged i.p. five times at three week intervals with 5 x lo6 GLC-19 cells using Freund’s incomplete adjuvant. Three days before the fusion 3 x lo6 GLC-19 cells in PBS were injected intravenously into a tail vein. Spleen cells were fused with SP2/0-Ag14 cells as described earlier [ 131. Screening
procedure
and
isotype
deter-
mination
Tissue culture supernatants of growing clones were tested in an immunofluorescence assay (IFA) and by Western blot analysis. For IFA, GLC-14, GLC-19 and H23 cells were applied on glass slides, dried and subsequently fixed in cold methanol (-20°C) and acetone. After two washes for 5 min with PBS/0.05% Tween-20 (PBS-T), the cells were incubated with hybridoma tissue culture supernatant for 2 h at room temperature. The slides were washed twice for 10 min with PBS-T and the cells were incubated with rabbit anti-mouse FITC
(diluted 1:50 in PBS) for 2 h at room temperature. This was followed by two 10 min washes with PBS-T and the slides were coverslipped. For Western blot analysis, blots of GLC-14, GLC-19 and HZ3 were prepared and incubated as described earlier [U] . The isotype of the MoAbs was determined using a dipstick isotyping kit (Holland biotechnology bv) . lmmunocytochemistry For testing of reactivity of the MoAbs in immunocytochemistry, an immunofluorescence assay was used (see screening procedure). Immunohistochemistry Sections were cut from frozen material (4 pm) and allowed to dry at room temperature for at least 30 min. The sections were fixed in cold methanol (-20°C) and acetone and subsequently washed three times in PBS for 5 min. After an incubation with normal horse serum, diluted 1:30 in PBS, the sections were incubated with MON-114 tissue culture supernatant for 1 h at room temperature. After washes with PBS (3 x 5 min), biotinylated horse anti-mouse (Vector Labs., diluted 1: 100 in PBS) was applied for 30 min. This was followed by washing in PBS as before and an incubation with avidin-biotin-peroxidase complex (Vector Labs., diluted 1:lOO in PBS) for 60 min. The peroxidase was revealed using either 3-amino-g-ethyl carbazole solution (0.2 mg/ml in 5 mM sodium acetate buffer, pH 4.85) or 0.05% diaminobenzidine solution and nickel enhancement [8]. The sections were rinsed twice in distilled water and counterstained lightly with hematoxylin.
Results Immunization of mice with GLC-19 lung tumor cells and subsequent cell fusion experiments resulted in the isolation of several hybridomas. One of them, MON-114 (IgGl), showed a strong cytoplasmic reactivity in the assay described in immunofluorescence
Fig.1. lmmunofluorescence v-SCLC cell line SCLC-16HVA MOC-1 (B).
staining of the adherent using MON- 114 (A) and
Table 1. Reactivity of MON.114 with cell lines Cell line
Type
Reactivity of MON-114:
SCLC- 16HC SCLC-21H
cmSCLC c-SCLC c-SCLC c-SCLC C~SCLC C~SCLC c-SCLC v-SCLC v-SCLC v-SCLC v-SCLC v-SCLC Adenocarcinoma lung Adenocarcinoma lung Epitheloid cervix carcinoma Myelocytic leukemia Promyelocytic leukemia Histiocytic lymphoma Pheochromocytoma, rat Pituitary tumor, mouse
+ + ++ ++ + ++ + + + + -
GLC- 14 GLC- 16 GLC- 19 GLC-l-Ml3 GLC-28 SCLC- 16HV SCLC16HVA GLC-1 GLC-4 H82 H23 A549 HeLa ML-1 HL-60 u937 PC-12 AtT20
+ _ + + + +
64
Materials and Methods, but no reactivity was observed in Western blot analysis. Initially, the expression pattern of the antigen detected by MON-114 was studied in a panel of cell lines by immunocytochemistry. In these experiments, 11 out of 12 SCLC cell lines were positively stained. Of the SCLC cell lines, GLC-4 was negative for MON-114 staining. Furthermore, 2 out of 2 adenocarcinomas cell lines of the lung and 4 out of 4 non-lung tumor cell lines (HeLa, ML-l, HL-60 and U937) were also negative, as shown in Table 1. Figure 1A shows the reactivity of MON- 114 with cell line SCLC-16HVA. As a control, the same cell line was stained using the MOC-1 antibody, which recognizes NCAM (Fig. 1B). The strong reactivity with PC-12 and AtT-20 cells suggested that MON-114 might detect a neuroendocrine antigen which was conserved in rat and mouse. Subsequently, the expression pattern of MON-114 was studied in a small panel of normal human tissues (see Table 2). The observation that all neural cells of the brain and cells of the adrenal medulla were positively stained supported the idea that MON-114 might specifically detect an antigen expressed on neuroendocrine cells. In Fig. 2B reactivity of MON-114 with a peripheral nerve is shown as an illustration. In liver, spleen, kidneys, ovaries and pancreas no reactivity could be observed. It should be noted that MON-114 reactivity can only be observed with cryo-sections (data not shown).
Table 2. tissues.
Reactivity of MON-114
with normal
To evaluate the usefulness of MON-114 in human lung cancer pathology, reactivity of the antibody was tested in an immunohistochemical study of a series of human lung tumors. The results are summarized in Table 3. Positive staining was observed in 7 out of 8 SCLCs and very strong staining in 5 out of 5 carcinoid tumors (Fig. 2A). With respect to NSCLCs, in 4 out of 31 squamous cell carcinomas and in 3 out of 19 adenocarcinomas weak staining was observed, however, in all cases only focal-
human Table 3. Reactivity of MON-114 lung tumors.
with primary human
Tissue
Reactivity
Liver Colon Spleen Kidney Adrenal medulla Brain Ovary Pancreas
-
Tumor type
Reactivity of MON-114
-
SCLC Squamous cell carcinoma Adeno carcinoma Large cell carcinoma Carcinoid
7/8 4/31 3/19 O/l 5/5
+ ++
of MON-114
Fig. 2. Immunohistochemical analysis of a carcinoid tumor (A) and staining of peripheral nerve in a primary squamous cell carcinoma (B) using MON-114.
l
‘Number of positive cases of total tested tumors.
65
ly. No reactivity was found in the only case of large cell carcinoma that was studied. In 3 out of 3 cases, MON-114 also positively stained SCLC metastases in lymph nodes (data not shown). Occasionally, a weak staining of stroma cells was observed. Discussion The reactivity pattern of monoclonal antibody MON-114 with cell lines, normal tissues and primary human lung tumors indicated that the antigen detected by this antibody was preferentially expressed in cells and tissues with neuroendocrine characteristics. The intensity of staining observed in a panel of three SCLC cell lines established from one patient during longitudinal clinical follow-up [Z] , GLC-14, GLC-16 and GLC-19, was similar. Only a small decrease in expression was observed in the drug-resistant cell line GLC-19 as compared to GLC-14, which was established before therapy. Apparently, expression of the antigen is not affected during the development of the drug-resistant phenotype of these cells. While v-SCLC cell lines seem to lose neuroendocrine characteristics to some extent as compared to c-SCLC cell lines, only one of the v-SCLC cell lines, GLC-4, was negative for MON-114 staining. GLC-4 cells grew as a loosely adherent monolayer which made it different from other SCLC cell lines which all grew in suspension. Austin Doyle et al. [l] reported that NCAM expression was high in SCLC cells that grew in suspension but is down regulated in adherent v-SCLC cell lines. It might be possible that MON-114 recognized a molecule that is down-regulated similarly in adherent v-SCLC cell lines. However, no difference in expression of the antigen recognized by MON-114 was observed in the adherent cell line SCLC-16HVA and the parent cell line SCLC-16HV. Furthermore, anti-NCAM staining by MOC-1 was also not affected (data not shown). The neuroendocrine reactivity pattern of MON-114 in a series of lung tumors, SCLCs as well as NSCLCs, is somewhat similar to that of cluster 1 lung cancer antibodies [ll], In 7 out
of 50 adenocarcinomas and squamous cell a weak positive staining with carcinomas, MON-114 was observed; in 4 out of these, also MOC-1 reactivity was observed and electron microscopy revealed the presence of dense core granules (data not shown), which is indicative for neuroendocrine characteristics. However, there are also differences. As shown in Fig. lB, anti-NCAM staining of membranes by MOC-1 is clearly different from cytoplasmic MON-114 staining (Fig. 1A). Furthermore, MON-114 did not react with D243 cells transfected with the gene encoding the 140-kDa transmembrane isoform of NCAM from human skeletal muscle (Dr. J. Ledermann, personal communication). All cluster 1 antibodies submitted to the Second International Workshop on Lung Cancer Antigens showed binding to these D243 cells [ll]. Altogether, the results suggests that MON-114 recognizes an epitope of a neuroendocrine differentiation marker that has a similar expression pattern as NCAM. However, the antigen of MON-114 is still unknown. Western blotting analysis and immunoprecipitation experiments did not uncover the nature of the antigen, and screening of a cDNA library using MON-114 did also not reveal the coding sequence of the corresponding gene. Nevertheless, MON-114 might prove to be a useful discriminating tool in lung cancer pathology. Acknowledgements We would like to thank Dr. J. Ledermann for testing MON-114 on NCAM-transfectants. In addition, we thank F. Schmitz du Moulin for excellent technical assistance and Dr. K. Pate1 for helpful discussion. Part of this work is supported by the Dutch Cancer Society. References Austin Doyle, L., Borges, M., Hussain, A., Elias, A. and Tomiyasu. T. (1990) An adherent subline of a unique small-cell lung cancer cell line downregulates antigens of the neural ceil adhesion molecule. J. Clin. Invest., 86, 1848 - 1854. Berendsen, H.H., De Leij, L., De Vries, E.G., Mesander, G., Mulder,
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Oncology, 3rd Edition, pp. 591-705. Editors: V.T. De Vita, S. Hellman, and S.A. Rosenberg. J.B. Lippencott Company, Philadelphia. Patel, K., Moore, S.E., Dickson, G., Rossell, R.J., Beverley, P.C., Kemshead, J.T. and Walsh, F.S. (1989) Neural cell adhesion molecule (NCAM) is the antigen recognized by monoclonal antibodies of similar specificity in small cell lung carcinoma and neuroblastoma. Int. J. Cancer, 44, 573- 578. Souhami, R.L., Beverley, P.C.L., Bobrow, L.G. and Ledermann, J.A. (1991) Antigens of lung cancer: results of the second international workshop on lung cancer antigens. J. Natl. Cancer Inst., 83, 609-612. Van Duijnhoven J.L.P., Ayoubi T.A.Y., Timmer E.D.J., Braks A.A.M., Roebroek A.J.M., Martens, G.J.M. and Van de Ven W.J.M. (1989) Development of a monoclonal antibody against recombinant neuroendocrine 7B2 protein. FEBS Len., 255, 372-376. Van Duijnhoven, J.L.P., Verschuren, M.C.M., Timmer, E.D.J., Vissers, P.M.A.M., Groeneveld, A., Ayoubi. T.A.Y., Van den Ouweland, A.M.W. and Van de Ven, W.J.M. (1991) Application of recombinant DNA technology in epitope mapping and targeting. Development and characterization of a panel of monoclonal an-
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