S100A4-neutralizing antibody suppresses spontaneous tumor progression, pre-metastatic niche formation and alters T-cell polarization balance

RPMI 1640, PBS and FCS were from (Gibco Life Technologies). Protease inhibitors were from (Roche). Recombinant mouse IL2 was from (Miltenyi Biotech). Mouse IgG and rabbit IgG were from (Sigma-Aldrich, USA). Isolation of the oligomeric form of the S100A4 protein and the S100A4 mutant (G47W) was described earlier [25,26]. Isolation and characterization of 6B12 anti-S100A4 mouse monoclonal antibody was described in [24].

Virgin female PyMT mice (Polyoma-middle T spontaneous metastatic mammary cancer model) of A/Sn genetic background were used for the experiments. Breeding and genotyping was performed as described earlier [13]. 6-week-old PyMT female mice were injected either with a loading dose (7.5 mg/kg) of 6B12 (n = 20), or IgG control (n = 20), intra-peritoneal. Injections of antibodies were repeated three times a week. 5 animals of each experimental group were sacrificed at age of 12 weeks. For the rest animals were sacrificed and processed as described earlier when the biggest tumor reached 1 cm³ [13].

CSML100 metastatic mouse mammary carcinoma cells (1×10⁶) [27] were injected subcutaneously into S100A4(-/-) mice of A/Sn genetic background [11] followed by intravenous injection of 2.5×10⁵ S100A4(+/+) or S100A4(-/-) mouse embryonic fibroblasts (MEFs). Experiment was repeated twice.

For the analysis of the antibody effect, CSML100 cells (1×10⁶) were injected subcutaneously followed by intravenous injection of 2.5×10⁵ S100A4(+/+) or S100A4(-/-) mouse embryonic fibroblasts (MEFs) mixed with either 100 μg of 6B12, or IgG control. Then the mice were injected with a loading dose (7.5 mg/kg) of 6B12, or IgG control intra-peritoneally three times a week. Injections of MEFs mixed with antibodies were repeated three times with a one-week interval. Animals were sacrificed when the tumor reaches 5-6 mm in diameter (pre-metastatic phase) [13]. Experiment was repeated twice.

Pre-metastatic lungs were incubated for 2 hours in PBS at 37°C. Conditioned media (CM) from five individual lung cultures were combined in equal ratio and applied to RayBio Mouse Cytokine Antibody Arrays 3 and 4, allowing simultaneous analysis of 96 cytokines (RayBiotech Inc.). The assay was performed according to the manufacturer’s instructions.

The mouse Th1-Th2-Th3 RT Profiler™ PCR Array (SABiosciences) was used to determine the relative expression of 84 genes related to CD4⁺ T-helper cells, according to the manufacturer’s instructions. RayBio Mouse Cytokine Antibody Array 1 for detecting 22 cytokines was used to test the cytokine profile in the T-cell CM.

Formalin-fixed paraffin embedded tissue sections were stained with antibodies against CD3 (rabbit polyclonal, catalogue #ab5690; Abcam), mouse α-smooth muscle actin (clone 1A4; Sigma-Aldrich), anti-Fibronectin (FN) (clone AB-10; Neomarkers), anti-CD31 (clone MEC 13.3) and anti-CD45 (clone 30 F11), both from BD Biosciences according to the manufacturer’s protocols.

Corresponding secondary horse-radish peroxidase-conjugated antibodies (DAKO, Glostrup, Denmark) were used, followed by incubation with chromogenic substrate 3,3′-diaminobenzidine (DAKO).

For double staining, secondary antibodies coupled to Alexa Fluor 488 or 568 (1:1500) were purchased from Molecular Probes. Sections were examined by means of confocal microscopy on a LSM 510 (Carl Zeiss Inc).

The blood vessel density was determined by quantifying CD31+ capillaries in 3-4 fields from two different sections of the tumor (magnification, ×200).

Quantification of CD3+ T-cells in tumors from 12-week-old PyMT mice and in pre-metastatic lungs was performed as described [13].

Proteins isolated from the pre-metastatic lungs were resolved by SDS-PAGE. FN expression was analyzed using a standard Western-blot procedure with anti-FN (DAKO) antibodies. Mouse anti-tubulin-α (clone AA13; Sigma-Aldrich) was used as a loading control. MultiGauge software was used for data analysis (Fujifilm).

To test the Jak-Stat signalling pathway activation, purified T-cells were starved in RPMI 1640 for 3 h and stimulated for 5 and 10 minutes with S100A4 protein (1 μg/ml), or mixed with 6B12 antibody (6 μg/ml).

Cell lysates were prepared in the presence of protease- and phosphatase-inhibitors. Activation of the Jak3/Stat3 signalling pathway was analysed using a standard Western blot procedure with phospho-Janus Kinase 3 (Jak3; Tyr⁹⁸⁰/Tyr⁹⁸¹, clone D44E3), phospho-Signal Transducer and Activator of Transcription 3 (Stat3; Tyr⁷⁰⁵, clone D3A7) and Jak3 (clone D7B12) and Stat3 (clone D3Z2G), antibodies (Cell Signaling Technology). Each experiment was reproduced 3 times using independent primary T-cell isolations.

T-cells purified as described in [13] were stimulated with S100A4 (1 μg/ml) for 19 h in presence of 10 μg/ml Polymyxin B (Invitrogen). Total RNA was isolated using a NucleoSpin® TriPrep kit (Macherey-Nagel). First-strand cDNA synthesis was performed using Super-Script III RT according to the manufacturer instructions. qRT-PCR was performed using a LightCycler 2.0 instrument (Roche Applied Science, USA). The expression level relative to the housekeeping GAPDH gene, as a control, was calculated. The PCR analysis was repeated 3 times using independent primary T-cell isolation. The primers used in this work are presented in Table 1.

Spleen, thymus and inguinal and brachial lymph nodes were dissected from 8-week-old A/Sn S100A4(+/+) and S100A4(-/-) mice [11]. Single-cell suspensions were prepared from the organs. Cells were counted, adjusted to 1 × 10⁷cell/ml and plated in 96-well plates (100 μl/well). Distribution and activation status of T/B cells was assessed using a cocktail of primary conjugated antibodies: anti-CD4 (clone RM4-5), anti-CD8a (clone 53-6.7), anti-CD19 (clone ID3), anti-TCRαβ (clone H57-597), anti-CD25 (clone PC61), anti-CD44 (clone IM7), or anti-CD62L (clone MEL-14) (BD Biosciences). Cells were incubated with the antibodies (1:100) in ice-cold PBS containing 2% FCS and 0.1% NaN₃ for 30 min on ice. Data acquisition and analysis were performed on aFACSCalibur (BD Biosciences) using FlowJo software (Tree Star).

Primary T-cells were isolated from 3-4 mouse spleens by negative selection on magnetic bids using the Pan T cell Isolation Kit II (Miltenyi Biotech), see Grum-Schwensen et al. for details [13]. Purified T-cells were maintained in RPMI for 3 or 6 days with anti-CD3 or a combination of anti-CD3 and anti-CD28 antibodies, coupled to MACSibeads (Miltenyi Biotec) plus 10 ng/ml recombinant IL2 as described in [28].

Activated T-cells were stimulated with S100A4 protein (1 μg/ml) or S100A4 protein mixed with 6B12 antibody (6 μg/ml). Before fixation, PMA/Ionomycin and Golgistop™ (BD Biosciences) were added for 5 hours. Cells were washed with PBS and Fixable Viability Stain 450 (BD Biosciences) was added to discriminate between viable and dead cells.

Cells were fixed using the Cytofix/Cytoperm™ kit (BD Biosciences) and stained with the mouse Th1/Th2/Th17 phenotyping kit (BD Biosciences) containing antibodies against CD4, IL17A, IFNγ and IL4 according to the manufacturer’s instructions. Data acquisition and analysis were performed on a FACSVerse (BD Biosciences) using FlowJo software (Tree Star). All experiments were repeated 3-5 times.

Cell viability and proliferation were measured by the LDH (Roche) and CyQuant® cell proliferation assay kit according to the manufacturer’s instructions.

The concentration of the cytokines IL2, IL4, IFNγ and IL4 in the CM from the T-cell cultures was measured using the Mouse Th1/Th2 ELISA assay (eBioscience) according to the manufacturer’s instructions. The experiment was performed twice.

As we have shown earlier, the treatment of T-cells with S100A4 protein in vitro led to the production of distinct cytokines [13]. This opened up the possibility that similar changes occurred in the pre-metastatic lungs in vivo. To test this idea, we implemented the recently developed mouse model of pre-metastatic niche formation [13].

Briefly, the metastatic CSML100 mammary carcinoma cells displayed a suppressed ability to form metastases when grafted to S100A4(-/-) mice. However, they could be primed to metastasize by intra-venous administration of S100A4 expressing fibroblasts (S100A4(+/+)MEFs), but not by S100A4(-/-) MEFs. In this model we defined the ‘metastatic stage’ as the point when tumor-bearing mice contained pulmonary metastases, and the ‘pre-metastatic stage’ when tumors were small (5-6 mm) and lungs contained only solitary cancer cells. Notably, S100A4(+/+), but not S100A4(−/−) MEFs, stimulated the accumulation of T-cells in the lung parenchyma, suggesting a potential role of T-cells in the formation of a pro-metastatic milieu [13].

To further characterize the changes that occur in the pre-metastatic lungs we compared the cytokines released in pre-metastatic lung organ cultures from S100A4(+/+) MEF versus S100A4(-/-) MEF boosted tumor-bearing mice using a cytokine antibody array. This experiment revealed altered expression of a number of cytokines including elevated levels of G-CSF and eotaxin-2 (Table 2). We also detected an increase in the level of IL4 and IL9 and a decrease in the level of IFNγ and IL1 compared to control. These might reflect a modulation in the pattern of T-cells in pre-metastatic lungs. Also noted was the change in expression of cytokines not involved in T-cell differentiation, suggesting the complexity of changes that occur in pre-metastatic lungs.

We further performed gene expression profiling of S100A4-stimulated primary T-cells that revealed changes in the expression of genes responsible for the differentiation of CD4⁺ T-cells (Additional file 2: Table S1). The differential expression of selected genes was validated by independent experiments using qRT-PCR (Figure 1A). While S100A4 had little effect on the transcription of GATA3 and TGF-β, the transcription of IL6, IL10, Jak3, Tyk2, CD40 and CTLA4 was substantially increased in S100A4-treated primary T-cells. To further validate the results of gene expression profiling, we examined the level of selected cytokines produced by S100A4-stimulated T-cells. This analysis confirmed the previously observed increase in IL6, IL2 and IL10 levels. We also detected an increase in the production of IL9, IL17, IL13 and decrease in IFNγ. The complete list of differentially expressed cytokines is presented in the Additional file 2: Table S2.

The resulting data also demonstrated that S100A4 modulates the expression of transcription factor and signal transduction pathway genes involved in determining the fate of T-helper lineage. Alteration of T-cell polarization could be predicted based on the up-regulation of Jak3, Stat1, Socs3, Tyk2 as well as down-regulation of Tbx21 (t-bet), GATA3, Socs1 and Socs5 [29].

We next tested whether S100A4 could activate the Jak/Stat pathway which is known for its implication in the lineage differentiation of T-cells [30]. Western blot analyses of Jak3 and Stat3 phosphorylation of T-cell protein extracts showed strong activation of Jak3 and modest activation of Stat3 upon S100A4 treatment, whereas the S100A4 mutant protein did not activate the phosphorylation (Figure 1B and C). Moreover, the S100A4-neutralizing 6B12 antibody efficiently blocked phosphorylation of both, Jak3 and Stat3 (Figure 1B and C). These results opened the possibility that S100A4 affects T-cell differentiation via activation of the Jak/Stat pathway.

Analysis of cytokine antibody and gene expression arrays directed us to study the effect of S100A4 on the T-cell differentiation.

We next assessed the long-term effect of S100A4 on T-cells. Isolated primary T-cells were primed to proliferate by CD3 and propagated for 3 and 6 days with or without S100A4. The percentage of live cells (data not shown) as well as the percentage of CD4⁺ in both cultures at 3 and 6 days was similar (Figure 2A), suggesting that S100A4 did not alter the population of CD4⁺ T-cells. The CD4⁺ T-cell fraction was further analysed to establish changes in the T-helper cell populations. The Th1/Th2 ratio at 3 days and Th2/Th17 ratio at 3 and 6 days remained unchanged by S100A4-treatment (data not shown). In contrast, at 6-days the percentage of Th1-polarized cells in S100A4-treated cultures was significantly reduced while the fraction of Th2-polarized cells remained unaffected (Figure 2B and C). Priming S100A4-treated T-cell with CD3/CD28/IL2, to stimulate production of Th1-polarized cells, did not reverse the situation (Additional file 2: Figure S1). Relative Th1/Th2 ratios showed a significant shift towards prevalence of Th2 polarized cells in the S100A4-treated cultures (Figure 2D). Treatment of the cultures with the 6B12, the S100A4-neutralizing antibody, partially restored the Th1/Th2 balance in the S100A4-treated population indicating that the effect was S100A4 dependent (Figure 2E).

To confirm changes of the Th1/Th2 balance in the S100A4-treated cultures, we determined the level of IFNγ, IL4 and IL10 signature cytokines in the CM. Sandwich ELISA analyses showed a reduction of IFNγ and increase in the IL10 in the CM of S100A4-challenged T-cells (Figure 2F). The concentration of IL4 in samples was below limit necessary for detection (data not shown).

The anti-S100A4 monoclonal antibody, 6B12, exhibited anti-metastatic activity by blocking T-cell infiltration in a tumor graft mouse model [24]. Injecting fully transformed cancer cells does not allow for studying the early steps of cancerogenesis. In order to develop the antibody as a potential therapeutic treatment, the assessment of its efficacy during pre-malignant steps of metastatic cancer development is especially important. For this purpose we used a PyMT-spontaneous metastatic mammary cancer mouse model [31].

Administration of 6B12 to PyMT tumor-bearing mice led to a delay in the development of tumors and reduction in the rate of tumor growth (Figure 3A). This delay in tumor growth could be caused by the suppression of the pro-angiogenic function of S100A4 [10,21]. We therefore determined tumor vessel density by staining tissue sections with the endothelial-specific anti-CD31 antibody. Indeed, quantifying capillaries in the tissue sections, demonstrated a substantial decrease in vessel density in tumors of 6B12-treated animals compared to the control (Figure 3B).

We next analysed whether the 6B12 antibody was able to block T-cell infiltration in pre-malignant tumors. The resulting data showed that 6B12 significantly reduced the accumulation of T-cells in primary tumors at the adenoma (MIN)/early carcinoma stage (Figure 3C).

Finally, end-point analyses of the metastatic burden as well as the overall number of metastases in the lungs of 6B12-treated mice, showed a significant reduction of both parameters when compared with the control group (Figure 3D and E). This data indicated that 6B12 was instrumental in manipulating tumor growth as well as metastasis formation when applied during early tumor development.

The reduced number of metastatic nodules in lungs of 6B12 treated PyMT-mice suggests that it could affect the formation of the pre-metastatic niche. We therefore analysed the efficacy of the 6B12 antibody in blocking the pre-metastatic niche by monitoring hallmarks of pre-metastatic niche formation, such as the deposition of FN, the production of G-CSF and the accumulation of T-cells [13,32,33]. Tumor-bearing mice at the pre-metastatic stage were treated with the 6B12 antibody or with the IgG control. The status of the pre-metastatic niche was analysed by immunohistochemical staining of lung tissue sections, which showed increased FN deposits as well as accumulation of T-cells around the blood vessels in pre-metastatic lungs, of mice boosted by S100A4(+/+)MEFs compared to the control S100A4(-/-)MEFs (Figure 4A). Quantitative analysis of FN transcripts in the lungs confirmed increased FN gene expression in tumor-bearing mice compared to control mice injected only with S100A4(+/+) MEFs. Inducing pre-metastatic niche formation by boosting mice with S100A4(+/+) MEFs further increased FN transcription, although the results were not statistically significant (Figure 4B). The 6B12 antibody showed a tendency to suppress FN transcription (P = 0.093) (Figure 4C), whereas analysis of FN protein expression showed a significant reduction of FN in the 6B12- treated group (n = 6, Figure 4D). Similar to FN, the transcription of G-CSF in pre-metastatic lungs decreased in response to 6B12 treatment (P = 0.033, Figure 4C). Quantifying the amount of T-cells accumulated in the vicinity of blood vessels within pre-metastatic lungs showed that the treatment with the 6B12 antibody led to a substantial reduction in T-cell numbers compared to the non-treated control (Figure 4E).

The suppression of tumor development and metastasis in S100A4(-/-) mice was associated with a decrease in T-cell infiltration [12,13], suggesting that S100A4 could affect T-cell production in general. To explore this possibility we performed a detailed analysis of the T-cell compartment of lymphoid organs of S100A4(-/-) mice.

Analysis of thymus, spleen and lymph nodes of S100A4(+/+) and S100A4(-/-) mice (n = 12) showed that the overall cellularity of organs was similar, with a slight tendency towards less cellularity in S100A4(-/-) spleens, which was not statistically significant (Additional file 2: Table S3).

The T-cell population in the thymus of S100A4(-/-) and S100A4(+/+) mice within the double negative (DN) compartment, based on the expression of CD44 and CD25, was not affected by S100A4. Furthermore, the proportion of CD4⁺ and CD8⁺ T-cells was similar (data not shown).

In contrast, the absolute number of CD4⁺ and CD8⁺ T-lymphocytes in S100A4(-/-) spleens was significantly reduced compared to the control, whereas the proportion of CD8⁺ and CD4⁺ cells remained unchanged (Figure 5A,B). We did not observe any differences in the proportion of naïve (CD62L⁺CD44^-) and memory (CD62L^-CD44⁺) CD4⁺ T-cells, as well as naïve (CD62L⁺CD44^-) and memory (CD62L^-CD44⁺) CD8⁺ T-cells (data not shown).

The reduction in the absolute number of T-cells in the spleen of S100A4(-/-) mice could reflect the effect of S100A4 on the survival of T-cells. This proposition was of interest since it has been shown earlier that extracellular S100A4 stimulated survival of neurons and the viability of cardiac myocytes [18,34]. We therefore analyzed growth and survival of T-cell populations in vitro in the presence of S100A4 for 6 days. As shown in Figure 5C, S100A4 did not affect the viability of cells as judged by a LDH cell viability assay. The proliferation rate of T-cells in culture was also similar.