Zoledronic acid blocks the interaction between breast cancer cells and regulatory T-cells

Abstract

Background: Zoledronic acid (ZA), a nitrogen-containing bisphosphonate, inhibits osteoclastogenesis. Emerging evidence suggests that ZA has anti-tumor and anti-metastatic properties for breast cancer cells. In a mouse model of ZA-related osteonecrosis of the jaw, ZA administration was found to suppress regulatory T-cells (Tregs) function. Our previous reports also demonstrated ZA acted as an immune modulator to block Tregs. Manipulation of Tregs represents a new strategy for cancer treatment. However, the relationship among ZA, Tregs, and cancer cells remains unclear. In this study, we investigated the effects of ZA on the interaction of breast cancer cells and Tregs.

Methods: The anti-tumor effect of ZA on triple negative breast cancer cell lines were validated by XTT, wound healing and apoptosis analysis. A flow cytometry-based assay was used to analyze the immunosuppressive effect of Tregs treated with media conditioned by breast cancer cells, and a transwell assay was used to evaluate the chemotactic migration of Tregs. Differential gene expression profile on MDA-MB-231 treated with ZA (25 μM) was analyzed by. microarrays to describe the molecular basis of actions of ZA for possible direct anti-tumor effects. Enzyme-linked immunosorbent assays and quantitative real-time PCR were used to investigate the effect of ZA on the expression of cytokines/factors by breast cancer cells.

Results: ZA was found to inhibit the proliferation and migration of breast cancer cells. Media conditioned by the MDA-MB-231 cells promoted the expansion, chemotactic migration, and immunosuppressive activity of Tregs, and these effects were attenuated in a dose-dependent manner by ZA treatment, and the attenuation was due to reduced expression of selected breast cancer cell factors (CCL2, CCL5, and IDO).

Conclusions: ZA can significantly affect the interaction between breast cancer cells and Tregs. Our findings indicate that ZA is a potential therapeutic agent that can be used to reduce cancer aggressiveness by abolishing the supportive role of Tregs.

Keywords: Breast cancer; Immunomodulation; Regulatory T-cells; Zoledronic acid.

Fig. 1

Effects of ZA treatment on proliferation of MDA-MB-231 cells. MDA-MB-231 cells were treated with various concentrations ZA for 72 h. Cell proliferation was determined by the XTT assay. Each data point represents the mean ± standard deviation based on quadruplicate determinations in three to five independent experiments. Significant difference was determined using one-way ANOVA; **P < 0.01, *** P < 0.001

Fig. 2

Effects of ZA treatment on migration of MDA-MB-231 cells. a Representative pictures of migration (wound closing) of MDA-MB-231 cells grown in the presence of various concentrations of ZA at 0, 12 and 24 h after wounding (100x magnifications). Quantification of migration distance as a percentage of the control value (ZA absent) in graphs of % wound closure after (b) 12 h and (c) 24 h of migration. The statistical analysis was performed using Prism version 5.00 software (GraphPad Software, USA). Data were analyzed using one-way ANOVA; *** P < 0.001

Fig. 3

Effects of ZA treatment on apoptosis detected by annexin V/PI ataining. a The MDA-MB-231 cells were treated without or with various concentration of ZA for 48 h. Annexin V-FITC/PI staining was carried out and determined using flow cytometry. b The distribution of MDA-MB-231 cells undergoing early (annexin V+ PI-) and late apoptosis (annexin V+ PI+) were qualified, after treatment with ZA

Fig. 4

Effects of C.M. from ZA-pretreated MDA-MB-231 cells on Tregs proliferation. Tregs were cultured in the presence of 100 U/ml rIL-2 and Dynabeads® Human Treg Expander with or without C.M from ZA-pretreated MDA-MB-231 stimulation. Proliferation was expressed as the percentage of cell numbers relative to that at day 1 (100%). Proliferation percentages were calculated at day 5 and 14. Data are representative of three independent experiments. One-way ANOVA was applied to analyze the results. * P < 0.05, ** P < 0.01, *** P < 0.001

Fig. 5

Inhibiting effects of C.M. from ZA-pretreated MDA-MB-231 cells on Tregs migration. Tregs (5 × 10 ⁴) were placed in the upper chambers for measurement of migration of Tregs into the lower chambers containing DMEM with 2% FBS with and without ZA and C.M. of MDA-MB-231 cells with and without ZA pretreatment. The relative percentages of migrated cells are shown compared with that of C.M. of MDA-MB-231 cells without ZA. Values indicate means ± SEM of results from three independent experiments performed in duplicate. Significant differences in percentage were determined using one-way ANOZA; ** P < 0.01; *** P < 0.001

Fig. 6

Inhibition effects of C.M. from ZA-pretreated MDA-MB-231 cells on Tregs immunosuppression function. a The percentage of responding effector T cells expressing activation marker CD69 in PBMC stimulated with anti-CD3/CD28 beads. b Tregs suppressed the percentage of responding effector T cells expressing activation marker CD69 in PBMC stimulated with anti-CD3/CD28 beads. c Tregs were cultured with C.M. of MDA-MB-231 cells and enhance the immunosuppression of Tregs to decrease responding effector T cells expressing activation marker CD69 in PBMC stimulated with anti-CD3/CD28 beads. d The percentage of immunosuppression was calculated using the following formulas: 100 – [(% CD69-positive in the presence of Tregs/% CD69-positive in the absence of Tregs) × 100]. Values shown are means ± SEM of results from three independent experiments. Significant differences were compared using one-way ANOVA; ** P < 0.01

Fig. 7

Treatment with ZA affects gene expression profile in MDA-MB-231cells and IFN-γ activated MDA-MB-231cells. a-b The human global gene expression profiles of two independent RNA samples were analyzed by human oligonucleotide DNA microarray at 48 h post-transfection. c Corrected microarray signal values of genes involved in different biological process, clustered by Biological process of MDA-MB-231 cells treated for 24 h with 25 μM ZA in comparison to control cells. d Corrected microarray signal values of genes involved in different biological process, clustered by Biological process of IFN-γ activated MDA-MB-231 cells treated for 24 h with 25 μM ZA in comparison to control cells. e Of the genes commonly down-regulated by ZA in MDA-MB-231cells and IFN-γ activated MDA-MB-231cells. f Of the genes commonly down-regulated by ZA in MDA-MB-231cells and IFN-γ activated MDA-MB-231cells

Fig. 8

Influence of ZA on IDO mRNA expression by MDA-MB-231 and MDA-MB-468 cells. The mRNA levels of IDO were evaluated by qRT-PCR in 100 ng/ml IFN-γ  activated MDA-MB-231 and MDA-MB-468 cells treated with 0, 5, 10 and 25 μM ZA for 24 h. Gene expression values were normalized to GAPDH expression. Relative mRNA expression of IDO was calculated from cell treatment with ZA compared with cells only with IFN-γ stimulation. Values indicate means ± SEM of results from three independent experiments performed in duplicate (n = 3). * P < 0.05, *** P < 0.001

Fig. 9

Chemokines expression in triple negative breast cancer cells treated with ZA. Relative mRNA levels of (a) CCL2 and (b) CCL5 were evaluated by qRT-PCR in MDA-MB-231 and MDA-MB-468 cells with or without 100 ng/ml IFN-γ activated in the presence of ZA (0, 5, 10 and 25 μM) for 24 h. Gene expression values were normalized to GAPDH expression. Concentrations of (c) CCL2 and (d) CCL5 were measured in the C.M. of 100 ng/ml IFN-γ treated MDA-MB-231 cells in the presence of ZA (0, 5, 10 and 25 μM) for 48 h by ELISA. Data were analyzed using one-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001

Fig. 10

Inhibition of C.M. of MDA-MB-231 induced migration of Tregs following treatment with monoclonal antibody of CCL2 or CCL5. C.M. with or without addition of anti-CCL2 antibody (10 μg/ml), anti-CCL5 antibody (20 μg/ml), alone or in combination on the migration of Tregs was applied. The relative percentages of migrated cells were compared with that of C.M. of MDA-MB-231 cells without monoclonal antibody (control). Values indicate means ± SEM of results from three independent experiments. Significant differences were compared using one-way ANOVA; ** P < 0.01; *** P < 0.001