The Endocrine Disruptor Compound Bisphenol-A (BPA) Regulates the Intra-Tumoral Immune Microenvironment and Increases Lung Metastasis in an Experimental Model of Breast Cancer

Abstract

Breast cancer (BC) metastasis represents the main physiopathology leading to poor prognosis and death. Bisphenol A (BPA) is a pollutant, classified as an endocrine-disrupting chemical compound with estrogenic properties, their exposure in the early stages of neonatal life leads to an increase in the size and weight of breast tumors and induces cellular changes in the tumoral immune microenvironment where cytokines play a key role. Thus, we used female BALB/c mice exposed neonatally to a single dose of BPA. Once mice reached sexual maturity, a mammary tumor was induced, injecting 4T1 cells in situ. After 25 days of injection, we evaluated endocrine alterations, cytokine expression, tissue alterations denoted by macro or micro-metastasis in the lung, and cell infiltration induced by metastasis. We found that BPA neonatal treatment did not show significant endocrine alterations. Noteworthy, BPA led to an augmented rate of metastasis to the lung associated with higher intratumoral expression of IL-1β, IL-6, IFN-γ, TNF-α, and VEGF. Our data suggest that cytokines are key players in the induction of BC metastasis and that BPA (an environmental pollutant) should be considered as a risk factor in the clinical history of patients as a possible inductor of BC metastasis.

Keywords: Bisphenol A; breast cancer; cytokines; endocrine disruptors; metastasis; tumor microenvironment.

Figure 1

Intratumoral expression of cytokines. Representative images belonging to the intratumoral expression of IL-1β, IL-4, IL-6, and IL-10 corresponding to the three experimental groups, namely control, vehicle, and BPA, are shown. The staining was performed with rhodamine and pictures were taken in a confocal microscope, using Newarsky contrast, being red the cytokine expressed and gray the not stained tumor.

Figure 2

Intratumoral expression of cytokines. Representative images belonging to the intratumoral expression of TNF-α, IFN-γ, and VEGF corresponding to the three experimental groups, namely control, vehicle, and BPA, are shown. The staining was performed with rhodamine and pictures were taken in a confocal microscope, using Newarsky contrast, being red the cytokine expressed and gray the non-stained tumor.

Figure 3

Macro metastasis at the pulmonary level. Representative images of the macro metastasis (tissue lesions) identified in the lungs belonging to the control, vehicle, and BPA-treated groups; millimetric grid as the background.

Figure 4

Histological examination of the lungs of female mice without tumors. Different magnification as indicated of lung tissues of different experimental groups. There is a normal histological appearance in intact or vehicle lungs, while BPA induces a slight infiltration of lymphocytes.

Figure 5

Histological examination of the lungs of female mice exposed to BPA without tumors. Representative images of the lungs in female mice exposed to BPA without tumor induction at different magnifications (4×, 20×, 40×, and 100×).

Figure 6

Histological examination of the BPA-treated animals with tumors. Parenchymal (A–F) and subpleural (G,H) micrometastasis in the lungs of mice neonatally exposed to BPA and orthotopically implanted at eight weeks old with tumor cell line 4T1. Representative images of the lungs of female mice with tumor induction neonatally exposed to BPA at 100× magnification.