A Novel Monoclonal Antibody Against Human S100A4

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Jan 29, 2006 - High levels of S100A4 protein are strongly associated with tumor progression and recent studies have suggested that it could act as a ...
[Calcium Binding Proteins 1:4, 219-223, October/November/December 2006]; ©2006 Landes Bioscience

Research Paper

A Novel Monoclonal Antibody Against Human S100A4 Its Production, Characterization and Application Nan Zhang1 Min Dumin2 Angela Platt-Higgins2 Xiangyang Hu3 Yinying Ye3 Daowei He3 Guozheng Wang2 Roger Barraclough2 Philip S. Rudland2 Xiaoming Huang3,*

Abstract

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RIB

3Laboratory

of Medical Biotechnology; The Medical School; Southeast University; Nanjing P.R. China

Introduction

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*Correspondence to: X.M. Huang; Laboratory of Medical Biotechnology; The Medical School; Southeast University; Nanjing 210009 P.R. China; Tel.: 0086.258.3272555; Email: [email protected]

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IST

and Polio Research Fund Laboratories; School of Biological Science; The University of Liverpool; Liverpool UK

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2Cancer

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of Clinical Medicine; The Medical School; Southeast University; Nanjing P.R. China

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1Department

S100A4 is a calcium binding protein of the S100 family1 and is overexpressed in various types of human cancers including breast,2 lung,3 gastric,4 oesophageal,5 bladder,6 colorectal7‑9 and pancreatic cancer.10,11 The strong correlation of the levels of S100A4 with poor patient prognosis has been shown in several types of cancers, including breast,2,12 colorectal,13 bladder,9 and prostate cancers.14 In rodent mammary tumor cells and transgenic mouse model systems, the upregulation of S100A4 can induce directly metastatic phenotypes.15‑18 These studies suggest strongly that S100A4 is a prognostic marker with the potential for future clinical applications. However, the only antibody presently commercially available for S100A4 protein is a polyclonal one. Therefore, variations between different batches of antiserum are difficult to control and its reproducibility cannot be fully guaranteed. In this study we have produced a monoclonal antibody using recombinant human S100A4 as immunogen. The monoclonal antibody has been characterized using ELISA, Western blotting, immunohistochemistry and immunofluorescent staining. A small group of clinical tumour specimens were stained immunohistochemically using both this monoclonal antibody and the commercial polyclonal antibody to S100A4. The results show that the monoclonal antibody is more specific than that of the commercial polyclonal antibody, at least in Western blotting, suggesting that the monoclonal antibody will be more reliable for future clinical use.

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Original manuscript submitted: 01/29/06 Manuscript accepted: 10/05/06

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Previously published online as a Calcium Binding Proteins E-publication: http://www.landesbioscience.com/journals/cbproteins/abstract.php?id=3495

Key words

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S100A4, monoclonal antibody, immunohistochemistry Acknowledgements

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See page 223. Note

High levels of S100A4 protein are strongly associated with tumor progression and recent studies have suggested that it could act as a prognostic marker for a number of human cancers. Objective: To establish a reliable method to detect S100A4 protein in tumor specimens. The widely accepted method for most routine pathology applications is immunohistochemical staining and the key reagent is a specific antibody. Since only a rabbit anti-S100A4 polyclonal antibody is commercially available, a more specific monoclonal antibody is required. Methods: A standard hybridoma method was used to generate monoclonoal antibodies against recombinant human S100A4. ELISA, Western blotting, immunofluorescent staining and immunohistochemistry were used to validate this antibody and the antibody is used to examine specimens of human breast and colon cancers. Results: The monoclonal antibody is shown to be more specific for S100A4 protein than the polyclonal antibody, at least in Western blotting. The monoclonal antibody is suitable for detecting the expression of S100A4 in specimens from human tissues and gave results consistent with those of a commercially available polyclonal antibody in a small group of breast and colorectal carcinomas. Conclusion: The monoclonal antibody is specific for S100A4 and can be produced on a large scale; therefore it will be more reproducible for future clinical applications.

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Since the hybridoma cells from which the antibody was been generated are difficult to resurrect from frozen stocks, the coding sequences for both heavy and light chains have been subcloned and a recombinant antibody is currently being developed.

Materials and Methods Cells and tissues. SP2/o myeloma cell line, rat tumour cell lines Rama 37, KP‑1, human tumour cell lines MDA‑MB 231, T47D, Huma 7, Huma 62, HeLa, were maintained in RPMI 1640 medium containing 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. For MDA‑MB 231 and T47D, 1 mg/ml insulin, and 1 ng/ml EGF were also included. Paraffin‑embedded sections (4 mm) of primary human breast and

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colorectal cancers were obtained from the Department of Pathology, Southeast University, China. Expression and purification of recombinant proteins. Recombinant human S100A4 was expressed in E. coli and purified as previously described.19 Since nontagged S100A4 cannot properly block both polyclonal and monoclonal anti‑S100A4 antibodies in immunohistochemical staining, a GST‑S100A4 fusion protein was expressed in E. coli and purified using glutathione agarose (Sigma, St Louis, USA). The fusion protein was then dialyzed against 20 mM Tris‑HCl pH 7.5 with 100 mM NaCl before use. Recombinant GST‑S100A120 was used as a control. Immunizations and monoclonal antibody production. Male Balb/c mice were immunized with recombinant human S100A4 protein suspended in Freund’s complete adjuvant at a concentration of 1 mg/mL. A total of 200 mg was injected subcutaneously every two weeks, until the serum titer of the mouse was half‑maximal at a dilution of 2 x 10‑3, as judged by ELISA. Spleens were removed from the mice, dissociated, and fused with SP2/o myeloma cells using 50% (v/v) polyethylene glycol (molecular weight, 1,300 to 1,600; Sigma). The fused cells were plated in 96‑well plates and selected in HAT (hypoxanthine, aminopterin, and thymidine) medium for 10–14 days to allow growth of cell clones. The supernatants of the cultures were screened for secreted antibodies to S100A4 using ELISA. Sera from the immunized mice were used as positive controls, and sera from healthy mice were used as negative controls. Positive hybridomas were subcloned twice by limiting dilution. The anti‑S100A4 monoclonal antibody was purified to homogeneity using HiTrapTM protein G HP column (Amersham Pharmacia Biotech. Bucks, UK). The purified antibody was stored at a concentration of 5 mg/mL at ‑80˚C in buffered saline containing 50% (v/v) glycerol. Enzyme‑linked immunosorbent assay (ELISA). Microtiter plates were coated with 10 mg/mL of the recombinant S100A4 protein and incubated overnight at 4˚C. Nonspecific binding sites were blocked with 1% BSA (w/v) in phosphate‑buffered saline plus 0.2% (v/v) Tween‑20 (PBS‑T). Mouse sera diluted 1:1000 in PBS‑T or undiluted hybridoma supernatant media were added to each well and incubated for 2 h at room temperature. After extensive washes, HRP conjugated goat anti‑mouse IgG as a secondary antibody was added and the mixture was incubated at room temperature for 1 h. After washing, tetramethylbenzidine (TMB) as the substrate was added and incubated for 30 min, and the absorbance was measured at a wavelength of 650 nm. Western immunoblot analysis. Purified human recombinant S100A4 and other S100 proteins including S100A1, S100A2, S100A6, S100B, and S100P as well as lysates of cell lines were separated by SDS 15% (w/v) polyacrylamide gel electrophoresis and electroblotted onto PVDF membranes. The membranes were incubated with the purified monoclonal antibody (1:1000 dilution) or commercial polyclonal antibody against S100A4 (DAKO; Denmark) (1:2000 dilution). After washing, the blots were incubated with HRP‑conjugated anti‑mouse IgG (Amersham Pharmacia Biotech.) at 1:2000 dilution or HRP conjugated anti‑rabbit IgG (Sigma) at 1:16,000 for 1 h, respectively. The bound antibodies were detected using the ECL system (Pierce, Tattenhall, Cheshire, UK) and exposure against Kodak XAR‑5 film. Fluorescence immunocytochemistry. Immunofluorescent detection of S100A4 protein was performed as described previously.20 About 500 cells were plated per well of chambered slides and cultured for 24 h. The cells were fixed with 4% (w/v) paraformadehyde 220

Figure 1. Comparison of S100A4 polyclonal and monoclonal antibody by western blotting. One mg of S100A1, S100A2, S100A4, S100A6, S100B and S100P proteins were subjected to electrophoresis on 15% (w/v) polyacrylamide gels and the proteins were transferred to PVDF membranes. The membranes were incubated with rabbit polyclonal anti‑S100A4 antibody (Panel A), and mouse anti‑S100A4 monoclonal antibody (Panel B), as described in Materials and Methods. Protein size markers in kDa are shown alongside.

in phosphate‑buffered saline for 10 min and permeabilized with 0.1% (w/v) Triton X‑100 in phosphate‑buffered saline for 5 min. After a 30‑min incubation in blocking buffer (2% (w/v) BSA in PBS), the cells were incubated with mouse monoclonal anti‑human S100A4 (1:500) and rabbit anti‑S100A4 (1:500) in blocking buffer. After extensive washes, the cells were incubated with fluorescein isothiocyanate (FITC)‑conjugated anti‑mouse IgG and tetramethylrhodamine‑isothiocyanate (TRITC)‑conjugated anti‑rabbit IgG for 1 h. After washing, the slides were dried and mounted. Photographs were recorded using a Zeiss LSM 510 confocal microscope. To test their specificity, the polyclonal antiserum/monoclonal antibodies were preincubated for 1 h at room temperature with recombinant GST‑S100A4 in a molar ratio of 1:5 and then the mixture was used in fluorescent staining. Immunohistochemistry. Four‑mm sections from formalin‑fixed, paraffin‑embedded tissues were deparaffinized and rehydrated, endogenous peroxidase activity was blocked with 0.35% (v/v) hydrogen peroxide in 50% (v/v) methanol for 15 min at room temperature and antigen retrieval was performed by microwave heating in citrate buffer (pH 6.0). Following incubation with 2% (v/v) normal horse serum in PBS‑1% (w/v) BSA for 30 min, sections were incubated overnight with the affinity‑purified monoclonal antibody (1:1000 dilution in PBS containing 1% (w/v) BSA) or polyclonal antibody (1:500 dilution) at 4˚C. The sections were then incubated with biotin‑ labelled secondary antibody and streptavidin‑peroxidase conjugate for 20 min each. Slides were incubated for 5 min with 0.05% (w/v) 3,3‑diaminobenzidine tetrahydrochloride freshly prepared in 0.05 M Tris‑HCl buffer (pH 7.6) containing 0.025% (v/v) hydrogen peroxide and then counterstained with haematoxylin, dehydrated, and mounted in DPX (Merck Ltd,). For a negative control, the monoclonal antibodies/polyclonal anti‑serum was incubated with

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Figure 2. Western blotting with rabbit polyclonal anti‑S100A4. One mg of recombinant human S100A4 (hrS100A4) and 50mg of cell lysates from T47D, MDA MB‑231, KP1‑Rama 37, Huma 7, Rama 37, Huma 62 and HeLa cells were separated by electrophoresis on 15% (w/v) polyacrylamide gels, and the S100A4 proteins were detected by rabbit polyclonal S100A4 antiserum as described in Materials and Methods�������������������������� . ������������������������ Protein size markers in kDa are shown alongside. ����������������������������������������������� The position of the S100A4 band and the higher molecular weight band referred to in the text (extra band) are shown by arrows.

recombinant human GST‑S100A4 or GST‑S100A1 protein in a molar ratio 1:10 at room temperature for 1 h prior to the immunohistochemical staining. For a positive control, sections of rat kidney were stained. The stained sections were microscopically examined and scored by two observers independently. For carcinoma specimens, those which had over 5%, (+), 1‑5% (±) or less than 1 % (‑) of the carcinoma cells positively stained, were categorized as positive, borderline and negative for S100A4 expression, respectively.

Results Using human recombinant S100A4 as the antigen, a positive hybridoma clone (D101) that secretes monoclonal antibody against S100A4 was obtained. The specificity of this monoclonal antibody for S100A4 amongst a number of S100 proteins was tested using Western blotting (Fig. 1B), The monoclonal antibody recognized human S100A4 but did not cross‑react with the closely‑related

Figure 3. Western blotting with mouse monoclonal anti‑S100A4. One mg of recombinant human S100A4 protein (rhS100A4) and 50 mg of cell lysates from Huma 7, Huma 62, KP1‑Rama 37, HeLa and MDA MB‑231 cells were subjected to electrophoresis on 15% (w/v) polyacrylamide gels, and the S100A4 proteins were detected by mouse monoclonal anti‑S100A4 antibody, as described in Materials and Methods. Protein size markers in kDa are shown alongside.

human calcium‑binding proteins, S100A1, S100A2, S100A6, S100B and S100P at the same loading on the gel (Fig. 1B); the same results were obtained with the commercial polyclonal antibody to S100A4 (Fig. 1A). Both the polyclonal antiserum (Fig. 2) and monoclonal antibody (Fig. 3) recognized a 9kDa band from the cell lysates, identical in size to that of S100A4. However, the polyclonal antiserum also recognized a 60kDa band in the MDA MB‑231, HeLa, Huma 7 and Huma 62 cell lines, which did not correspond to the size of S100A4. This band is not a polymer of S100A4 because T47D and KP‑1 cells express high levels of S100A4 but no extra band was detected (Fig. 2) while Huma 7 and Huma 62 cells do not express S100A4 but present a strong extra band (Fig. 2). In contrast, the monoclonal antibody does not recognize any extra band in both S100A4 positive and negative cell lines (Fig. 3). This result shows that the monoclonal antibody, in contrast to the commonly used polyclonal antiserum, is specific for the 9 kDa subunit of S100A4. Using the monoclonal antibodies/polyclonal antiserum, the intracellular localization of endogenous S100A4 in the MDA‑MB‑231 cells was examined by immunofluorescent staining (Fig. 4). S100A4 protein was mainly localized to the perinuclear region and to the cytoskeleton as previously reported.21 The images from the monoclonal antibody (Fig. 4A) and polyclonal antiserum (Fig. 4B) can be superimposed (Fig. 4C) particularly in the perinuclear region. The immunofluorescent staining was specific, since Figure 4. Immunofluorescent staining of MDA‑MB‑231 cells. The cells were grown in chambered slides and stained as described in Materials and Methods. Cells in (A and B) were stained with either monoclonal (green) or polyclonal (red) anti‑S100A4, respectively. (C) is the superimposed image of (A and B). Cells in (D and E) were stained with either monoclonal or polyclonal anti‑S100A4 that was preincubated with rGST‑S100A4. (F) is the phase contrast image of the same field as that of (D and E). Bars: (A–C), 20 mm; (D–F), 50 mm.

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Table 1

Summary of the immunochemical staining of human breast and colorectal carcinomas

Specimen Number Type of Cancer

Polyclonal Antiserum

Monoclonal Antibody

1

Breast

+

+

2

Breast

+

+

3

Breast

±

±

4

Breast ‑ ‑

5

Breast ‑ ‑

6

Colorectal

+

+

7

Colorectal

+

+

8

Colorectal ‑ ‑

9

Colorectal ‑ ‑

10

Colorectal ‑ ‑

+, positive staining, >5% of carcinoma cells were stained positively; ±, borderline staining, 1–5% cells stained positively; ‑, negative staining,