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. 2012 Dec;14(12):1260–1268. doi: 10.1593/neo.121554

Anti-S100A4 Antibody Suppresses Metastasis Formation by Blocking Stroma Cell Invasion1,2

Jörg Klingelhöfer *,†, Birgitte Grum-Schwensen *, Mette K Beck *, Rikke Stagaard Petersen Knudsen *, Mariam Grigorian *,†, Eugene Lukanidin *,†, Noona Ambartsumian *,†
PMCID: PMC3540950  PMID: 23308057

Abstract

The small Ca-binding protein, S100A4, has a well-established metastasis-promoting activity. Moreover, its expression is tightly correlated with poor prognosis in patients with numerous types of cancer. Mechanistically, the extracellular S100A4 drives metastasis by affecting the tumor microenvironment, making it an attractive target for anti-cancer therapy. In this study, we produced a function-blocking anti-S100A4 monoclonal antibody with metastasis-suppressing activity. Antibody treatment significantly reduced metastatic burden in the lungs of experimental animals by blocking the recruitment of T cells to the site of the primary tumor. In vitro studies demonstrated that this antibody efficiently reduced the invasion of T cells in a fibroblast monolayer. Moreover, it was capable of suppressing the invasive growth of human and mouse fibroblasts. We presume therefore that the antibody exerts its activity by suppressing stroma cell recruitment to the site of the growing tumor. Our epitope mapping studies suggested that the antibody recognition site overlaps with the target binding interface of human S100A4. We conclude here that this antibody could serve as a solid basis for development of an efficient anti-metastatic therapy.

Introduction

More than 90% of cancer-related deaths are caused by dissemination of cancer cells to distant organs with subsequent formation of secondary tumors, known as metastases. Metastatic dissemination of cancer cells in the body occurs through interaction with cancer-associated stroma cells that play a crucial role in stimulation of cancer cell dissemination, survival, and colonization of secondary organs [1]. In contrast to the primary tumor, metastasis is especially challenging to treat because of its systemic nature and frequent association with resistance to existing therapeutic agents [2]. Despite substantial progress in targeted cancer treatments, development of a therapy, which specifically targets molecules of the metastatic process, is still at a very early stage. However, progress in identification of molecules involved in metastasis has helped to identify new targets, thereby creating novel opportunities to prevent or treat metastasis.

Recently, the metastasis-promoting S100A4 protein was suggested as a therapeutic target to prevent metastasis [3]. S100A4 belongs to the S100 family of small Ca-binding proteins. It plays a regulatory role in a variety of cellular processes, such as cell motility and differentiation [4]. In clinic, S100A4 has gained attention because of its up-regulation in different types of human cancers, which has been correlated to a bad prognosis for patients (reviewed in [5]).

Numerous experimental approaches, including studies of xenograft and genetically modified mouse models, have verified a causal role of S100A4 in promoting metastatic disease (reviewed in [5,6]). Mechanistically, metastasis-stimulating activity could be attributed to different extracellular and intracellular functions of the S100A4 protein. For instance, S100A4 stimulates cancer cell motility and invasion through interaction with intracellular targets such as nonmuscle myosin [7–9]. As an extracellular protein, S100A4 affects different signaling pathways. It has been shown that S100A4 modulates epidermal growth factor receptor signaling by interacting with epidermal growth factor receptor ligands [10] and activates mitogen-activated protein (MAP) kinase and nuclear factor kappa-light chain-enhancer of activated B-cells (NF-γB) pathways in a variety of cell types [11,12]. Downstream, the S100A4-dependent activation leads to the remodeling of the extracellular matrix, induces angiogenesis, and attracts different immune cells to the growing tumor [13–16].

Accumulated data suggested that S100A4 is an attractive candidate for anti-metastatic therapy. Analysis of the tumor secretome revealed that S100A4 is accumulated in human breast tumor microenvironment [17]. Similarly, increased levels of S100A4 were detected in early stage tumors in a spontaneous metastatic mouse mammary cancer model [16].

The potential efficiency of S100A4 as a therapeutic target was demonstrated by suppression of metastasis in S100A4-deficient mice, which was associated with aberrant stroma development, in particular deficiency in T cell accumulation [16,18,19]. Recently, an inhibitor of S100A4 transcription, niclosamide, was identified as a suppressor of metastasis formation in a colon cancer xenograft model [20].

Among prospective biologically targeted therapies, antibody-based therapies are regarded as a mainstream of the future cancer treatment strategy [21,22]. Compared with traditional treatment options, an antibody-targeted therapy is more specific, less toxic, and may be more effective [23,24].

In the present work, we generated and selected an anti-S100A4-neutralizing antibody with the purpose of blocking metastasis formation. The selected anti-S100A4 antibody efficiently recognized mouse and human S100A4 protein and blocked metastasis formation in a mouse xenograft model. The antibody was proficient in blocking not only the invasion of mouse and human fibroblasts but also the attraction of mouse T cells to the fibroblast monolayer, indicating that the metastasis-neutralizing activity of this antibody is associated with prevention of stroma cell invasion of the primary tumor.

Materials and Methods

Cell Lines

The CSML100 mouse mammary adenocarcinoma cell line was derived from spontaneous tumors in A/Sn mice [25]. Isolation of mouse embryo fibroblast (MEF) cell lines has been described earlier [19]. All other cells were obtained from the American Type Culture Collection (ATTC) collection. Cell lines were propagated in a suitable basal medium (Gibco BRL) supplemented with 10% FBS (Life Technology LTD, Paisley, United Kingdom), penicillin (100 units/ml), and streptomycin (100 units/ml) in a humidified 5% CO2 atmosphere.

Mice

A/Sn strain of mice was used in all mouse experiments. All animals were maintained according to the Federation of European Laboratory Animal Science Associations guidelines for the care and use of laboratory animals.

Peptides

The following nine 10- to 12-mer peptides corresponding to the entire 101 amino acids of human S100A4 protein were synthesized by solid-phase synthesis (Alpha Diagnostic International, San Antonio, TX) and used for epitope mapping by ELISA and in inhibition experiments: #1: MACPLEKALD (10-mer: 1.10), #2: VMVSTFHKYS (10-mer: 11–20), #3: GKEGDKFKLNK (11-mer: 21–31), #4: SELKELLTREL (11-mer: 32–42), #5: PSFLGKRTDEA (11-mer: 43–53), #6: AFQKLMSNLDSN (12-mer: 54–65), #7: RDNEVDFQEYCV (12-mer: 66–77), #8: FLSCIAMMCNEF (12-mer: 78–89), #9: FEGFPDKQPRKK (12-mer: 90–101).

Enzyme-linked Immunosorbent Assay

ELISA was performed as described by Kosmac et al. [26]. The concentrations of peptides for coating were between 0.1 and 1000 nM. Serial dilutions of the monoclonal antibody (mAb) ranged from 0.032 to 32 nM. The absorbance was quantified spectrophotometrically at 405 nm using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA).

Western Blot

Western blot was performed as described by Klingelhöfer et al. [10]. Mouse monoclonal anti-S100A4 antibodies (3B1C4, 11F8.3, and 6B12, all IgG1γ isotypes) or polyclonal rabbit anti-S100A4 produced in our laboratory were used as primary antibodies. As secondary antibodies, rabbit anti-mouse HRP-conjugated or goat anti-rabbit HRP-conjugated antibodies were used. Both antibodies were obtained from Dako (Glostrup, Denmark).

To detect endogenous S100A4 protein from different cell lines, 0.4 x 105 cells per well were seeded on a six-well tissue culture plate and grown until the cells reached confluence under standard tissue culture conditions. Protein extracts were made by lysing cells in 300 µl of 1x sodium dodecyl sulfate-gel loading buffer and subsequent boiling at 95°C for 5 minutes. Twenty microliters of cell extract were loaded per lane.

Pull-down and Peptide Competition Experiments

For peptide competition experiments, 1 µg of antibody was preincubated with 250 ng of peptides in the interaction buffer [TBS, 0.1 mM CaCl2, 0.5% blocking solution (Roche Diagnostics A/S, Hvidovre, Denmark)] for 1 hour at room temperature, followed by 1-hour incubation with 250 ng of recombinant S100A4. The antibodies were pulled down with 0.05 mg of Pierce Protein A/G magnetic beads (Thermo Scientific, Slangerup, Denmark) for 30 minutes followed by washing in TBS/0.05% Tween-20. The amount of co-precipitated S100A4 was analyzed by Western blot.

For the target binding site competition experiment, 500 ng of S100A4 was pre-incubated with a C-terminal fragment of p53 or the p53Δ1 in 2.5- and 10-fold molar excess in the interaction buffer for 1 hour at room temperature followed by addition of 2 µg of 6B12 mAb for 1 hour. The pull-down was performed as described for the peptide competition experiment.

T-lymphocyte Invasion Assay

Invasion of primary mouse T lymphocytes into fibroblast monolayers was tested using a modification of methods described elsewhere [16,27]. MEFs were grown to confluency in 12-well plates; T lymphocytes were labeled with Vybrant DID cell-labeling solution (Life Technology LTD) according to the manufacturer's instructions. Labeled T lymphocytes (4.5 x 105) were added to the wells and incubated for 2 hours. Noninvaded cells were removed by washing and mechanical agitation three times in phosphate-buffered saline. The infiltrated cells were counted using a fluorescence microscope in 10 random fields (original magnification, x100). Invasion assays were performed in RPMI 1640 containing 10% fetal calf serum with or without 6 µg/ml rabbit IgG (Sigma-Aldrich, St Louis, MO) or 6 µg/ml 6B12 antibodies. The experiments were performed in quadruplicate and repeated three times.

Three-dimensional Matrigel Invasion Assay

Detailed protocol for invasion assay in three-dimensional (3D) Matrigel is described elsewhere [28]. Briefly, 6 x 104 cells were incubated in a hanging drop of medium to form an aggregate, which was then placed on a layer of Matrigel containing Dulbecco's modified Eagle's medium and 10%fetal calf serum. The clump was covered with a drop of Matrigel and incubated at 37°C for polymerization. Invasive growth was stimulated by addition of conditioned medium from cancer cells with or without S100A4. Extent of the outgrowth was monitored in an inverted microscope using x10 objective for 120 hours.

Immunofluorescence and Immunohistochemical Analyses

Tumor tissue sections were stained with affinity-purified rabbit polyclonal antibodies against CD3 (Abcam, Cambridge, United Kingdom) and rat mAbs against CD31 (clone MEC 13.3; BD Biosciences, Albertslund, Denmark), as described elsewhere and according to the manufacturer's protocols [16]. For immunofluorescence staining, MEF-/- or MEF+/+ were grown overnight on an eight-chambered glass coverslip (Greiner Bio-One, Frickenhausen, Germany), fixed, and stained as described earlier by Olsen et al. [29].

Animal Experiments

A/Sn mice were subcutaneously injected with 1 x 106 CSML100 cells in a volume of 200 µl/mouse, and on the same day, the loading dose (7.5 mg/kg in a volume of 100 µl) of antibodies was injected intraperitoneally. Injections of antibodies were repeated three times a week. The animals were sacrificed when the first tumor reached the maximal allowed size (1 cm3) by an intraperitoneal injection of pentobarbital (Euthanyl) followed by perfusion with phosphate-buffered saline. The tumor tissue and lungs were paraffin-embedded, sectioned (4 µm), and stained with hematoxylin and eosin. The total metastatic burden was quantified by calculating the percentage area of each lung section occupied by metastases as described elsewhere [16].

Statistical and Computerized Analyses

Data are presented as average ± SEM. The confidence level was calculated using Student's t distribution. For the prediction of nonsolvent. exposed amino acids of S100A4, we used the Epitopia server (http://epitopia.tau.ac.il/index.html) [30]. As input sequence, we used the calcium-bound dimeric human S100A4 (PDB ID: 2Q91) [31]. Analyses and imaging were performed with the Molegro Molecular Viewer software v. 2.2.0/mac (Molegro ApS, Aahus, Denmark).

Results

Production and Characterization of a Panel of S100A4-specific mAbs

Recent data have indicated that S100A4 promotes metastasis via modulating host immune cells [15,16]. This limited us to testing the anti-metastatic efficiency of S100A4-neutralizing antibody in immunocompetent mice. Furthermore, to obtain a function-neutralizing antibody with the prospective to develop a humanized antibody for therapeutic use, we selected antibodies that recognized both human and mouse proteins. We used recombinant mouse S100A4 protein for screening the hybridoma clones to identify and select only those hybridomas that produce antibodies of appropriate specificity. From this panel of S100A4-specific antibodies, three mAbs (3B1C4, 11F8.3, and 6B12) with the highest affinity in the ELISA screen were selected (data not shown).

First, we used a Western blot assay to test whether the antibodies recognize human S100A4 protein or cross-react with other members of the S100 family. As shown in Figure 1A, 3B1C4 and 6B12 mAbs recognized both mouse and human S100A4 proteins, whereas 11F8.1 only reacted with the mouse protein. Importantly, all three antibodies showed no cross-reactivity to other S100 family members.

Figure 1.

Figure 1

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The specificity of 6B12 mAb. (A) 6B12 mAb recognizes mouse and human S100A4 proteins and shows no cross-reactivity to other S100 family members as shown by Western blot analysis. Lower panel amido black staining of the polyvinylidene difluoride (PVDF) membrane is serving as a loading control. (B) Immunofluorescence staining of MEF(+/+) and MEF(-/-) using 6B12 antibody (green). F-actin is stained by rhodamine-phalloidin (red) and the nucleus by 4′,6-diamidino-2-phenylindole (DAPI, blue). (C) Western blot analysis membrane stained with 6B12 mAb comparing cell extract from MEF(+/+) and MEF(-/-) cells. Coomassie blue staining and α-tubulin serve as loading controls. (D and E) Detection of endogenous S100A4 protein by 6B12 mAb from cell extracts of mouse and human cancer cell lines: mouse mammary adenocarcinoma, 4T1 and CSML100; human breast cancer, MCF7, MDA-MB-231, MDA-MB-468, T-47D; human colon cancer, SW-480, SW-620, colo205, LoVo, HT-29.

Second, immunocytochemical analysis showed that all three antibodies detected S100A4 protein expressed in MEFs. S100A4 immunoreactivity was observed in the cytoplasm in the perinuclear area with similar distribution as reported previously [32]. S100A4(-/-) MEFs, which served as a negative control, showed no immunofluorescence staining and revealed no cross-reactivity (Figure 1B). We then tested the recognition of S100A4 protein in total cell extracts from S100A4(+/+) and S100A4(-/-) MEFs as well as from human and mouse cancer cell lines (Figure 1, C and D). Data in Figure 1A show that 11F8.3 antibody recognized only the mouse protein, whereas the two other antibodies were able to recognize both mouse and human S100A4. In contrast to the other clones, 3B1C1 also recognized some unidentified proteins with an approximate size of 55 and 90 kDa. This cross-reactivity makes the 3B1C1 antibody less prospective. Finally, 6B12 mAb also recognized S100A4 in a panel of human cancer cell lines from breast cancer, MDA-MB-468, as well as from various human colon cancers (Figure 1E).

Evaluation of the Function-Blocking Capability of Different Anti-S100A4 Antibodies

Because the purpose of this study was to isolate an antibody with metastasis-blocking activity, we decided to perform preliminary analyses, including in vitro and in vivo assays. The in vitro assay was based on the ability of the S100A4 protein to stimulate cell invasion in a 3D Matrigel [33]. As shown in Figure 2A, the addition of S100A4 stimulated the invasion of MEF(+/+) into the matrix. 6B12 Antibody added to the culture medium successfully blocked the invasion (Figure 2A, left panel). The blocking ability of the antibodies was measured by semiquantitative assessment of the extent of the invasion. 11F83 Antibody was not able to block the S100A4-stimulated invasion of fibroblasts into the Matrigel (Figure 2B), 6B12 and 3B1C4 mAbs blocked the invasion to a similar extent. Because we were interested in selection of a metastasis-blocking antibody applicable for human cancer, we further analyzed the ability of 6B12 antibody to block invasion of human mammary fibroblasts (HMF3s). Figure 2C shows that 6B12 antibody blocked the invasion of HMF3 cells (P = .04), indicating that it is equally efficient in blocking the physiological activity of both human and mouse proteins.

Figure 2.

Figure 2

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In vitro blocking activity of 6B12 mAb. (A) Representative phase-contrast images of fibroblasts invading the Matrigel in response to conditioned media from VMR mouse adenocarcinoma cells alone, after addition of S100A4 (1 µg/ml) or in the presence of S100A4-neutralizing mAb 6B12 (8 µg/ml) after 48 hours of incubation. (B) 6B12 mAb inhibits significantly the invasion of mouse fibroblasts and (C) in the presence of MCF7 breast carcinoma cell conditioned media HMF3s under 3D culture conditions (P = .0274 and P = .0400, respectively).

We further characterized antibodies by their ability to neutralize metastasis-stimulating activity of the S100A4 protein in vivo. The ability of the three antibodies to block tumor growth and metastasis formation by the CSML100 cell line was tested in a spontaneous metastasis assay [25]. This assay allowed us to test changes both in tumor growth and metastasis formation. At the end of the experiment, the average tumor size showed no statistically significant differences between different groups (Table 1). The treatment did not show signs of toxicity, as the mean body weight showed only a marginal, less than 3%, difference (Table 1). The assessment of the metastatic burden in the lungs of mice treated with the three antibodies, however, exhibited substantial relative difference. Mice treated with 11F8.3 antibody showed the highest metastatic burden, indicating that the antibody was not efficient. This result corroborates the findings of the invasion assay analysis, which showed inability of 11F3 to block invasion (Figure 2B). 3B1C4 Antibody showed only a weak tendency in metastasis-neutralizing activity (Table 1), whereas 6B12 antibody substantially suppressed metastasis. Comparison of the 6B12- and the 11F8.3-treated groups displayed a 10-fold lower metastatic burden, although this difference was not statistically significant (P = .052). In summary and on the basis of these results, 6B12 mAb was chosen for a more detailed study.

Table 1.

Effect of Different Anti-S100A4 mAbs on Tumor Growth and Metastasis Formation.

α-S100A4 mAb 3B1C4 11F8.3 6B12
Number of mice (N) 7 6 7
Average tumor size (mm3) 200.7 ± 39.05 230.3 ± 21.90 176.6 ± 23.91
Metastatic burden (%) 3.246 ± 1.707 4.878 ± 2.170 0.4997 ± 0.2345
Average weight loss before/after (g/g) 26.6/25.6 ≙ 3.01% 26.4/25.6 ≙ 4.55% 26.1/24.6 ≙ 5.75%
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The effect of 3B1C4, 11F.8.3, and 6B12 mAbs on the tumor size, the amount of metastasis-free animals, and the metastatic burden in CSML100 xenograft mice.

Anti-S100A4 mAb Significantly Reduces the Metastatic Burden in Lungs and Suppresses T Cell Accumulation in Primary Tumor

On the basis of the strong tendency of 6b12 mAb to suppress metastasis and the small number of animals used in the pilot study, we decided to perform a large-scale mouse experiment to assess in detail the metastasis-suppressing activity of the antibody. Following the same experimental setup as the previous experiment, the antibodies were injected intraperitoneally into mice grafted with CSML100 cells. Similar to the previous experiment, the extent of tumor growth of the 6B12 mAb-treated group did not substantially differ from the control group (Figure 3A). Autopsy analysis revealed that the overall amount of metastasis-free animals was more than two times higher in the 6B12-treated group (45%) than in the control (19%). Moreover, assessment of the metastatic burden in lung tissue sections showed that this parameter was significantly (P = .02) reduced in the 6B12-treated group (Figure 3, A and B).

Figure 3.

Figure 3

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Anti-S100A4 mAb 6B12 suppresses metastasis and inhibits T lymphocytes attraction to the tumor site. (A) Testing the effect of 6B12 mAb in a spontaneous metastasis model. The amount of metastasis-free animals in the 6B12-treated group was increased compared to the control group, 45% to 19.1%. (B) Significant (P = .0197) reduction of metastatic burden in the lung of mice treated with 6B12 mAb compared to the control. (C) 6B12 mAb inhibits the attraction of T lymphocytes to the tumor site compared to the IgG-treated control group (T cells total: 326 vs 550, P = .0003; N = 5 per group; 10 fields per tumor were analyzed). (D) S100A4 stimulated the attraction of T lymphocytes to the fibroblast monolayer (control), which is inhibited in the presence of the neutralizing S100A4-specific mAb 6B12; in contrast, the mouse IgG control did not display inhibiting function (P = .0231).

We extended our studies to determine whether neutralization of the S100A4 activity by 6B12 antibody also affected the composition of tumor-associated stroma. Immunohistochemical staining of sections of the primary tumors with antibodies specific for endothelial cells (CD31) and T cells (CD3) revealed that treatment with 6B12 antibody did not affect vessel density. In contrast, the amount of T cells accumulated in the vicinity of the growing tumor was reduced by 40% (Figure 3C). This confirms our previous observations that S100A4 deficiency leads to changes in the tumor stroma compartment, particularly in the recruitment of immune cells to the site of the growing tumor [16].

To further support the argument that 6B12 mAb mechanistically acts to reduce T cell accumulation in the primary tumor, we performed an in vitro T cell invasion assay. Previously, we have shown that S100A4(+/+) but not S100A4(-/-) fibroblasts substantially stimulate the ability of T cells to invade the fibroblast monolayer [16]. Using the same approach, we demonstrate here that 6B12 antibody reduced T cell invasion into the fibroblast monolayer by approximately 30% (Figure 3D). This result supports our initial notion that 6B12 antibody manifests its metastatic activity by inhibiting the recruitment of T cell to the primary tumor site.

Determining of the Binding Site of 6B12 Antibody

To further investigate the mechanism of 6B12's neutralizing activity, we performed an epitope mapping analysis by testing the affinity of 6B12 antibody to nonoverlapping 10- to 12-mer peptides of the entire human S100A4 protein with an ELISA. 6B12 Antibody binds to peptide 7 (RDNEVDFQEYCV) and peptide 8 (FLSCIAMMCNEF) but not to the others, as shown for peptide 9 (Figure 4A). Competitive immunoprecipitation assay revealed that peptides 7 and 8 block the interaction of 6B12 antibody with the S100A4 protein, whereas peptide 9, chosen as a negative control, had no effect on 6B12 antibody interaction (Figure 4B). Peptide 7 forms the second EF-hand [amino acid (aa) 63–74] for Ca2+ coordination and peptide 8 partly forms the α-helix IV (aa 72–88) of the S100A4 protein [31].

Figure 4.

Figure 4

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The epitope of 6B12 mAb is located at the C terminus of S100A4 protein overlapping with the target binding interface. (A) ELISA showing 6B12 affinity to peptides 7 and 8 but not peptide 9 to the human S100A4 sequence. (B) The competitive immunoprecipitation assay showing the pull-down of S100A4 by 6B12 antibody in the absence (line 1) and the presence of peptides 7, 8, and 9 (P7, P8, and P9). As control, lane 5 shows a pull-down without added 6B12 mAb. (C) Diagram of the p53 fragments, which were used for competitive pull-down experiments. The recombinant C-terminal 100 aa of human p53 fusion construct, including a His-tag, a V5 epitope, and a TEV protease cleavage site, was used to show competition between 6B12 mAbs. Fragment p53Δ1 is partially lacking the binding site of S100A4 (bs-S100A4). (D) Binding of the C-terminal p53 fragment to S100A4 compromises the pull-down by 6B12 mAb. The p53 fragments were added in 2.5- and 10-fold molar excess. As control, lane 1 shows a sample without added 6B12. As positive control, lane 2 shows a pull-down of 500 ng of human S100A4 by 6B12 mAb (2 µg).

In addition, peptides 7 and 8 contribute to the hydrophobic cleft, which forms the S100A4 target binding interface [34]. We therefore studied whether 6B12 antibody can interfere with S100A4-p53 interaction. To study this, we used an S100A4 pull-down assay with a p53 C-terminal fragment that contains the S100A4 binding interface [35]. As a negative control, we used the C-terminal fragment of p53 (p53Δ1) with a 30-amino acid deletion lacking the binding site (Figure 4C). As shown in Figure 4D, the amount of S100A4 precipitated by 6B12 mAb was substantially reduced when the S100A4 protein was pre-incubated with p53 C-terminal fragment, compared to the control. In contrast, the C-terminal p53 deletion mutant (p53Δ1) did not interfere with 6B12 binding (Figure 4D). In accordance with the previous result, this strongly suggests that the 6B12 epitope at least partially overlaps with the target binding interface.

We next performed a computer-assisted analysis to predict the recognition epitope on the S100A4 protein structure. We used the published structure of calcium-bound dimeric human S100A4 in a resolution of 1.63 Å (PDB ID: 2Q91) and the Epitopia Web server [30]. On the basis of the data presented above, the 24 amino acid-long sequence of peptides 7 and 8 were used as core region of the antibody-S100A4 interface. It is worth noting that the sequences of peptides 7 and 8 are identical in the mouse and human proteins. Amino acids, which could not be part of the epitope surface because they are either hidden in the protein structure or at a different planar level, were excluded from the potential epitope (see details in Table W1). This analysis suggests a planar structure on the surface of S100A4 for the possible 6B12 epitope with a diameter of 30.9 Å (Figure 4A and 5A). The surface of the planar patch is formed by four amino acids from peptide 7 (Arg66, Asn68, Gln73, and Val77) and seven amino acids from peptide 8 (Cys81, Ile82, Met84, Met85, Asn87, Glu88, and Phe89; Figure 5B). The suggested epitope consists of the following sequence: 66R-N-Q-V-CI-MM-NEF89.

Figure 5.

Figure 5

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Epitope mapping revealed a planar patch on the dimer of S100A4 governing the epitope for 6B12 antibody. (A) The ribbon diagram represents the molecular structure of the calcium-bound S100A4 dimer (2Q91). The amino acids of S100A4, which contribute to the potential 6B12 epitope, are indicated in green (only A chain is colored). The arrow indicates the patch size in diameter (30.9 Å). The α-helices of S100A4 are shown in red and calcium atoms are shown in orange. (B) Molecular surface visualization of S100A4 shows the predicted epitope region located in/or close to the hydrophobic cleft for target protein interaction (arrows). The surface of the suggested epitope is colored in green and its amino acids are labeled.

Discussion

The efficacy of mAbs as cancer therapeutics has been proven in recent years. Antibodies are used both to target surface antigens on tumor cells and to enhance anti-tumor immune responses by targeting immune cells [36]. Here, we report the isolation and characterization of an S100A4-blocking antibody that exhibits a remarkable anti-metastatic effect. Even though metastases are the most serious complication of cancer, the success in development of anti-metastatic therapies is very limited and mostly directed toward suppression of the primary tumor growth [21].

A number of studies carried in the last decades showed that S100A4 plays a key role in stimulation of metastasis [37]. The exact mechanism of its metastasis-stimulating function is not clear. Recently, several studies suggested the extracellular form as major trigger of metastasis formation [10,12,16,33,38], making it an easily accessible target for therapy. Moreover, the expression pattern of S100A4 in a normal organism is quite restricted [39,40], and mice with inactivated S100A4 gene are viable and fertile [41].

Recently, by using high-throughput screening technology, the S100A4 transcription inhibitor, niclosamide, was identified. Consistent with its inhibitory activity toward S100A4, niclosamide led to inhibition of metastasis formation in a colon cancer xenograft model [20].

The purpose of the present study was to identify an anti-S100A4 mAb with metastasis-blocking activity. We also intended to reveal the corresponding epitope. End point assessment of the anti-metastatic activity of 6B12 antibody in a spontaneous metastasis assay showed significant suppression of metastasis. Curiously, analysis of the dynamics of tumor growth showed no difference in this parameter. This observation confirms data obtained earlier from analysis of spontaneous mammary tumor formation in the PyMT-S100A4 knockout mouse model [16] and once more supports the notion that S100A4 protein is a metastasis- rather than a tumor-promoting protein.

T cell accumulation in the primary tumors of the 6B12 antibody-treated group was substantially suppressed. A decreased accumulation of T cells of yet an unidentified subclass was recently observed in tumors of S100A4(-/-) PyMT double transgenic mice [16], which is in a good agreement with the above-mentioned data. In recent years, it has been shown that tumor-infiltrating T cells can promote tumor cell invasion, metastasis, and aggressive behavior [42]. However, the functional significance of leukocytes in regulating pro-tumor immunity is poorly understood. Studies in mice demonstrated that a subpopulation of CD4+ T lymphocytes promotes the invasion and metastasis of breast cancer cells by inducing the ability of tumor-associated macrophages to activate pro-tumorigenic pathway in mammary epithelial cells [43]. Increased presence of immune cells, in particular T regulatory cells in primary tumors, correlates with low tumor grade and poor overall survival of patients with breast cancer [44].

Human and mouse S100A4 sequences differ in six amino acids. The recognition sequence of 6B12 mAb was mapped to peptides 7 and 8 of S100A4, which are identical between human and mouse. Computer-assisted analysis of the 3D structure of human S100A4 dimer localized the interactive epitope to an area that is recognized as a target binding site of S100 proteins [45]. A characteristic of this interaction site is a hydrophobic cleft, which has recently been shown by crystal structure analysis to interact with a 45-residue fragment of nonmuscle myosin heavy chain IIA [46]. Previously, we have demonstrated p53 as another target for S100A4 [35]. Here, we show that a fragment of p53, containing the target binding site, competed with 6B12 antibody in binding with S100A4. Therefore, one could speculate that interference of the antibody with target binding is the putative mechanism of 6B12 mAb's blocking activity.

However, a combination of different mechanisms, documented for other mAb therapies, cannot be excluded [21]. For instance, binding by antibody to S100A4 in the blood and tumor microenvironment could lead to a faster body clearance of the S100A4 molecule. Because the function-blocking antibody was tested in immunocompetent mice, neither one can exclude innate immune effector mechanisms that engage the Fc portion of antibodies through Fc receptors [47]. Such mechanisms include antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity. For instance, antibody-dependent cellular cytotoxicity effects have been well described as an accompanying cytotoxic mechanism for therapeutic mAbs, like trastuzumab, rituximab, alemtuzumab, ofatumumab, and cetuximamb [47–50]. 6B12 Antibody could potentially provoke immunologic anti-tumor effects. However, the fact that tumor development was not affected by the antibody treatment argues against this proposition.

In summary, this work shows that a specific anti-S100A4 antibody, 6B12, efficiently inhibited the metastasis formation directed by grafted tumor cells in a spontaneous metastasis assay. Identification of the blocking epitope paves the way for development of an efficient anti-metastatic therapy.

Supplementary Material

Supplementary Figures and Tables
neo1412_1260SD1.pdf (31.7KB, pdf)

Acknowledgments

We thank Inge Skibshøj, Therese Brøndsted Sten, and Lene Bregnholt Larsen for careful technical assistance. We also thank our former Masters student Henrik Devitt Møoller for cloning and purification of the p53-protein fragments.

Footnotes

1

This work was supported by European Union grants, FP7-Tumic, Health-F2-2008-201662, and FP7-INCO-2010-6-265128 (INARMERA).

2

This article refers to supplementary material, which is designated by Table W1 and is available online at www.neoplasia.com.

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