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

Abstract

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.

Figure 1.

Co-culture model and real time MCF7 cells analysis. (A) Presentation of the 2D co-culture system and the protocol. (B) xCELLigence dynamic monitoring of MCF-7 cells, A representative graph from xCELLigence system: cell index (CI) profiles are the $\text{mean}\pm \text{SD}$ (duplicate) of each condition: Control (*, vehicle MCF-7 cells, alone), TCDD ($\$$, MCF-7 cells treated with $25\text{\hspace{0.17em}nM}$ TCDD), co-culture ($€$, MCF-7 co-cultured with hMADS) and coexposure (&, co-culture with TCDD). (C) The evolution of the CI for each condition was determined by analyzing the slope of the line in the interval (26–48 h). Each graph represents the mean slope (in bold) compared with the $\text{control}\pm \text{SEM}$ for six measurements. The numerical information $\text{mean}\pm \text{SEM}$ and $p$-values are provided in Table S1. (Kruskal–Wallis’s H test (nonparametric comparison of $k$ independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series), **$p<0.01$; *$p<0.05$). Note: ANOVA, analysis of variance; SEM, standard error of the mean; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Figure 2.

Proteomics analysis and ALDH enzymatic analysis of MCF7 cells growth in co-culture with hMADS and exposed to $25\text{\hspace{0.17em}nM}$ TCDD for 48 h. (A) High-throughput proteomic analysis of MCF7 cells [Control (vehicle MCF-7 cells, alone), TCDD (MCF-7 cells treated with $25\text{\hspace{0.17em}nM}$ TCDD), co-culture (MCF-7 co-cultured with hMADS), and coexposure (co-culture with TCDD)]. The plots show the mean of biological quadruplicates and technical triplicates for each sample. CYP1A1, CYP1B1, and ALDH1A3 were induced when they appeared in the upper right part of the representation. (B) ALDH (aldehyde dehydrogenase) enzymatic activity was detected in MCF7 cells using the ALDEFLUOR assay (FACS analysis). DEAB was used to inhibit the reaction of ALDH with the ALDEFLUOR reagent, providing a negative control. Percentage of ALDH positive cell was set according to the gate of DEAB control cells. Graph represents means of the percentage of ALDH positive cells (in bold) compared with the $\text{control}\pm \text{SEM}$ of six measurements. The numerical information $\text{mean}\pm \text{SEM}$ and p-values are provided in Table S2. (Kruskal–Wallis’s H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series, **$p<0.01$; *$p<0.05$). Note: ANOVA, analysis of variance; SEM, standard error of the mean; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Figure 3.

Sphere formation assay. (A) Representative images of sphere formation taken on day 7 on two consecutive generations. Only breast stem/progenitor cells can self-renew and grow into a spheroid structure. Control (vehicle MCF-7 cells, alone), TCDD (MCF-7 cells treated with $25\text{nM}$ TCDD), co-culture (MCF-7 co-cultured with hMADS) and coexposure (co-culture with TCDD). Scale bar $10\mathrm{\mu }\mathrm{m}$. (B) Spheroid area (in ${\mathrm{\mu }\mathrm{m}}^2$) in passage 1 using media from the various conditions. Graph represents $\text{means}\pm \text{SEM}$ of n (see figure) measurements. The numerical information $\text{mean}\pm \text{SEM}$ and p-values are provided in Table S3. Untreated MCF-7 cells alone represent the control condition (Kruskal–Wallis’s H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series, ****$p<0.0001$). Note: ANOVA, analysis of variance; SEM, standard error of the mean; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Figure 4.

Morphological differences of MCF7 cells and MDA-MB-231 cells following exposure to TCDD, co-culture, or coexposure. Cells were grown with hMADS and/or treated with $25\text{\hspace{0.17em}nM}$ TCDD [Control (vehicle MCF-7 cells, alone), TCDD (MCF-7 cells treated with $25\text{nM}$ TCDD), co-culture (MCF-7 co-cultured with hMADS), and coexposure (co-culture with TCDD)]. After 48 h treatment, cells were fixed and stained for paxillin, actin, and nucleus (blue). (A) Staining of MCF7 cells. Scale bar $20\mathrm{\mu }\mathrm{m}$. (B) Quantitation of MCF7 cell number and giant cells (very large cells with multiple nuclei) per field ($n=3$) field per conditions. Graph represents $\text{means}\pm \text{SEM}$ of three experiments. The numerical information $\text{mean}\pm \text{SEM}$ and p-values are provided in Table S4. Kruskal–Wallis’s H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series, *$p<0.05$). (C) Staining of MDA-MB-231. Scale bar $20\mathrm{\mu }\mathrm{m}$. Symbols were used to point out the focal adhesions (arrow), lamellipods (*) and giant cells (#). Note: ANOVA, analysis of variance; SEM, standard error of the mean; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Figure 5.

Protein (A-actin, B-beta-catenin) localization & nucleus staining in MCF7 cells and MDA-MB-231 cells. MCF7 cells were grown with hMADS and/or treated with $25\text{\hspace{0.17em}nM}$ TCDD for 48h [Control (vehicle MCF-7 cells, alone), TCDD (MCF-7 cells treated with $25\text{nM}$ TCDD), co-culture (MCF-7 co-cultured with hMADS), and coexposure (co-culture with TCDD)]. (A) MCF7 cells were stained for actin (green) and nucleus (blue). Scale bar $20\mathrm{\mu }\mathrm{m}$. (B) MCF7 cells were stained for beta-catenin (green) and nucleus (blue). One representative cell with cell-in-cell structures was marked with an asterisk (*). Scale bar $20\mathrm{\mu }\mathrm{m}$. Note: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Figure 6.

Measurement of metastatic spread of MCF7 and MDA-MB231 cells in zebrafish larvae in vivo models Human RFP-MCF7 (left) or RFP-MDA-MB-231 (right) cells cultured with conditioned media from different conditions, were injected into the perivitelline space of 2-day-old zebrafish larvae [Control (vehicle MCF-7 cells, alone), TCDD (MCF-7 cells treated with $25\text{\hspace{0.17em}nM}$ TCDD), co-culture (MCF-7 co-cultured with hMADS), and coexposure (co-culture with TCDD)]. Fish were imaged 24 h later at $8.5\times$ magnification by fluorescence microscopy. (A) Representative images from 21–46 fish/group. (B) Quantitation of (a) the number of fish with one or more metastases and (b) the average number of $\text{metastases}/\text{fish}+\text{SE}$. Graph represents $\text{means}\pm \text{SEM}$ of three measurements. The numerical information $\text{mean}\pm \text{SEM}$ and p-values are provided in Table S8. (Kruskal–Wallis’s H test (nonparametric comparison of k independent series) followed by a 1-factor ANOVA test (parametric comparison of k independent series, * $p<0.05$). (C) The percentage of fish with metastasis in the head region. Each experiment was repeated in triplicate (MCF-7) or quadruplicate (MDA-MB-231) with 7–12 fish per condition in each experiment. Note: ANOVA, analysis of variance; SEM, standard error of the mean; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.