Aggressiveness and Metastatic Potential of Breast Cancer Cells Co-Cultured with Preadipocytes and Exposed to an Environmental Pollutant Dioxin: An in Vitro and in Vivo Zebrafish Study

Background: Breast cancer (BC) is a major public health concern, and its prognosis is very poor once metastasis occurs. The tumor microenvironment and chemical pollution have been suggested recently to contribute, independently, to the development of metastatic cells. The BC microenvironment consists, in part, of adipocytes and preadipocytes in which persistent organic pollutants (POPs) can be stored. Objectives: We aimed to test the hypothesis that these two factors (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an extensively studied, toxic POP and the microenvironment) may interact to increase tumor aggressiveness. Methods: We used a co-culture model using BC MCF-7 cells or MDA-MB-231 cells together with hMADS preadipocytes to investigate the contribution of the microenvironment and 2,3,7,8-tetrachlorodibenzo-p-dioxin TCDD on BC cells. Global differences were characterized using a high-throughput proteomic assay. Subsequently we measured the BC stem cell–like activity, analyzed the cell morphology, and used a zebrafish larvae model to study the metastatic potential of the BC cells. Results: We found that coexposure to TCDD and preadipocytes modified BC cell properties; moreover, it induced the expression of ALDH1A3, a cancer stem cell marker, and the appearance of giant cancer cells with cell-in-cell structures (CICs), which are associated with malignant metastatic progression, that we demonstrated in vivo. Discussion: The results of our study using BC cell lines co-cultured with preadipocytes and a POP and an in vivo zebrafish model of metastasis suggest that the interactions between BC cells and their microenvironment could affect their invasive or metastatic potential. https://doi.org/10.1289/EHP7102

. Cell death, senescence, proliferation, or autophagy measurements. MCF7 cells were grown with hMADS and/or treated with 25nM TCDD for 48h (Control (vehicle MCF-7 cells, alone), TCDD (MCF-7 cells treated with 25nM TCDD), coculture (MCF-7 co-cultured with hMADS) and coexposure (co-culture with TCDD)). (A) Quantification of MCF7 apoptosis using annexin V-FITC (y-axis)/PI staining(x-axis). (a) Flow cytometer results: cells were classified as early apoptotic cells (Annexin V+, PI−), late apoptotic cells (Annexin V+, PI+), and damaged cells (Annexin V−, PI+). (b) Percentage of cells from (a) under each different experimental condition. Graph represent means ± SEM of 5 measurements. (B) q-RT-PCR experiments quantifying apoptosis and autophagy biomarkers. Relative levels compared to the control of mRNA expression of ATG5, ATG7 and BAX by real-time qPCR. Graph represent means ± SEM of 5 measurements. (C) Quantification of MCF-7 proliferation using the AlamarBlue assay. Mean absorbance is indicated for each condition as a horizontal bar and statistically significant differences were determined by ANOVA and indicated as P values above the comparison, bars, SD. N=3. The numerical information mean +/-SEM and p-values are provided in Table S5, S6 and S7. (Kruskal-Wallis's H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series, ** p<0.01; * p<0.05). (D) Senescence-associated β-galactosidase activity. The blue-stained cells and the total number of cells were observed.10X magnification (1 field par well). The pictures are representative of at least 3 different experiments. Table S1. Numerical data mean +/-SEM and p-values for Figure 1C: Cell index and pvalues for MCF-7 cells under control, TCDD, coculture, or coexposure conditions using the xCELLigence system.
[a] Data presented are mean of 6 experiments.
[b] p-values calculated via Kruskal-Wallis's H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series), All conditions compared to control. Table S2. Numerical data mean +/-SEM and p-values for Figure 2B: ALDH enzymatic analysis for MCF-7 cells under control, TCDD, coculture, or coexposure conditions.
[a] Data presented are mean of 6 experiments.
[b] p-values calculated via Kruskal-Wallis's H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series), All conditions compared to control.  [a] Data presented are mean of 14-39 measurements on 3 experiments.
[b] p-values calculated via Kruskal-Wallis's H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series), All conditions compared to control.  [a] Data presented are mean of 3 field in 3 experiments.
[b] p-values calculated via Kruskal-Wallis's H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series), All conditions were compared to control.  [a] Data presented are mean of 5 experiments.
[b] p-values calculated via Kruskal-Wallis's H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series), All conditions were compared to control. Figure S2B: mRNA expression of ATG7, ATG5, and Bax in MCF-7 cells under control, TCDD, coculture, or coexposure conditions as determined by q-RT-PCR.

Table S6. Numerical data means +/-SEM and p-values for
[a] Data presented are mean of 5 experiments.
[b] p-values calculated via Kruskal-Wallis's H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series), All conditions were compared to control. Table S7. Numerical data means +/-SEM and p-values for Figure S2C: Quantification of proliferation of MCF-7 cells under control, TCDD, coculture, or coexposure conditions as determined using the AlamarBlue assay. Absorbance measured at 540 nm.
[a] Data presented are mean of 3 experiments.
[b] p-values calculated via Kruskal-Wallis's H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series), All conditions were compared to control. Table S8. Numerical data means +/-SEM and p-values for figure 6B-b.
[b] p-values calculated via Kruskal-Wallis's H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series), All conditions were compared to control. Figure S1.

Table S9. Numerical data means +/-SEM and p-values for
[a] Data presented are mean of 5 experiments.
[b] p-values calculated via Kruskal-Wallis's H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series), All conditions were compared to control.

Additional File-Excel Document
Figure S1: semi-quantitation of E-Cadherin mRNA in MCF-7 cells. MCF7 cells were grown with hMADS and/or treated with 25nM TCDD for 48h (Control (vehicle MCF-7 cells, alone), TCDD (MCF-7 cells treated with 25nM TCDD), coculture (MCF-7 cocultured with hMADS) and coexposure (co-culture with TCDD)). Relative levels (compared to the control) of E-Cadherin mRNA were measured by real-time qPCR. Graph represent means ± SEM of 5 measurements. The numerical information mean +/-SEM and p-values are provided in Table S9. (Kruskal-Wallis's H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series), ** p<0.01, *p<0.05). Relative levels compared to the control of mRNA expression of ATG5, ATG7 and BAX by real-time qPCR. Graph represent means ± SEM of 5 measurements. (C) Quantification of MCF-7 proliferation using the AlamarBlue assay. Mean absorbance is indicated for each condition as a horizontal bar and statistically significant differences were determined by ANOVA and indicated as P values above the comparison, bars, SD. N=3. The numerical information mean +/-SEM and p-values are provided in Table S5, S6 and S7. (Kruskal-Wallis's H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series, ** p<0.01; * p<0.05). (D) Senescence-associated β-galactosidase activity. The blue-stained cells and the total number of cells were observed.10X magnification (1 field par well). The pictures are representative of at least 3 different experiments. Table S1. Numerical data mean +/-SEM and p-values for Figure 1C: Cell index and p-values for MCF-7 cells under control, TCDD, coculture, or coexposure conditions using the xCELLigence system [a] Data presented are mean of 6 experiments [b] p-values calculated via Kruskal-Wallis's H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series), All conditions compared to control.    Table S4.

Numerical data mean +/-SEM and p-values for Figure 4: Quantitation of cell number and giant cells (very large cells with multiple nuclei) per field (N=3 field per conditions) in MCF-7 cells under control, TCDD, coculture, or coexposure conditions
[a] Data presented are mean of 3 field in 3 experiments [b] p-values calculated via Kruskal-Wallis's H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series), All conditions were compared to control.  [a] Data presented are mean of 5 experiments [b] p-values calculated via Kruskal-Wallis's H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series), All conditions were compared to control     Table S7 : Numerical data means +/-SEM and p-values for Figure S2C: Quantification of proliferation of MCF-7 cells under control, TCDD, coculture, or coexposure conditions as determined using the AlamarBlue assay. Absorbance measured at 540 nm [a] Data presented are mean of 3 experiments [b] p-values calculated via Kruskal-Wallis's H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series), All conditions were compared to control. Table S8: Numerical data means +/-SEM and p-values for figure 6B-b [a] Data presented are mean of 3 experiments. The total number of fish injected with CM-Dillabelled MCF-7 was: control (N=32), TCDD (N=21), Coculture (N=36) and Coexposure (N=34). The total number of fish injected with RFP-labelled MDA-MB-231 cells was: control (N=46), TCDD (N=41), Coculture (N=24) and Coexposure (N=42).
[b] p-values calculated via Kruskal-Wallis's H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series), All conditions were compared to control