Perimenopausal and Menopausal Mammary Glands In A 4-Vinylcyclohexene Diepoxide Mouse Model

Abstract

As both perimenopausal and menopausal periods are recognized critical windows of susceptibility for breast carcinogenesis, development of a physiologically relevant model has been warranted. The traditional ovariectomy model causes instant removal of the entire hormonal repertoire produced by the ovary, which does not accurately approximate human natural menopause with gradual transition. Here, we characterized the mammary glands of 4-vinylcyclohexene diepoxide (VCD)-treated animals at different time points, revealing that the model can provide the mammary glands with both perimenopausal and menopausal states. The perimenopausal gland showed moderate regression in ductal structure with no responsiveness to external hormones, while the menopausal gland showed severe regression with hypersensitivity to hormones. Leveraging the findings on the VCD model, effects of a major endocrine disruptor (polybrominated diphenyl ethers, PBDEs) on the mammary gland were examined during and after menopausal transition, with the two exposure modes; low-dose, chronic (environmental) and high-dose, subacute (experimental). All conditions of PBDE exposure did not augment or compromise the macroscopic ductal reorganization resulting from menopausal transition and/or hormonal treatments. Single-cell RNA sequencing revealed that the experimental PBDE exposure during the post-menopausal period caused specific transcriptomic changes in the non-epithelial compartment such as Errfi1 upregulation in fibroblasts. The environmental PBDE exposure resulted in similar transcriptomic changes to a lesser extent. In summary, the VCD mouse model provides both perimenopausal and menopausal windows of susceptibility for the breast cancer research community. PBDEs, including all tested models, may affect the post-menopausal gland including impacts on the non-epithelial compartments.

Keywords: 4-Vinylcyclohexene Diepoxide (VCD); Breast Cancer; Mammary Gland; PBDEs; Single-Cell RNA Sequencing; Windows of Susceptibility.

Fig. 1

Effects of the VCD treatment on the mouse mammary gland. A The overview of the perimenopause and menopause VCD models. VCD treatment was performed at 9 weeks old. The VCD-treated mice were considered to be completely menopaused at 23 weeks old (100 days after the VCD treatment) according to the previous study. B Representative images of the mammary gland from the Perimenopause and Menopause VCD models

Fig. 2

Quantitative analysis of the mouse mammary gland from the Perimenopause and Menopause VCD models. Comparisons of the total ductal length per arbitrary unit area (A), the number of the branching points per arbitrary unit area (B), and the number of end buds per arbitrary unit area (C) between the experimental groups. The box-plot elements were defined as follows: center line, median; box limits, upper and lower quartiles; whiskers, 1.5 × interquartile range; points, outliers. The numbers above brackets indicate p-values

Fig. 3

Effects of the PBDEs exposure on the VCD-induced perimenopausal and menopausal mouse mammary gland. A The overview of the three different VCD-PBDE experiments. B-D Comparisons of the total ductal length per arbitrary unit area (B), the number of the branching points per arbitrary unit area (C), and the number of end buds per arbitrary unit area (D) between the experimental groups. The box-plot elements were defined as follows: center line, median; box limits, upper and lower quartiles; whiskers, 1.5 × interquartile range; points, outliers. The numbers above brackets indicate p-values

Fig. 4

Single-cell RNA sequencing analysis of the whole mammary gland after the VCD treatment, the High-Subacute or the Low-Chronic PBDEs exposure, the hormone replacement treatments, and/or the co-treatment with fluvestrant. A The overview of the sample labeling (column) and the experimental interventions (row). B The results of dimension reduction and clustering on a UMAP plot with the putative cell type label. C The hierarchal clustering of the pseudo RNA seq data. The sample names are accompanied by shapes, which indicate different PBDEs treatments (High-Subacute, Low-Chronic, or None), and color-coded by additional hormonal treatments (+ E2, + E2 + ICI, + E2 + P4, or None). D-E The expression of a gene of interest visualized on the violin plot (left) and the UMAP plots (right) stratified and color-coded by the PBDEs exposure. The None group; VCD, VCD + E2, and VCD + E2 + P4. the Low-Chronic group; VCD + LP, VCD + E2 + LP, and VCD + E2 + P4 + LP. The High-Subacute group; VCD + HP, VCD + E2 + HP, and VCD + E2 + P4 + HP. DErrfi1. The violin plot shows expression on Errfi1 in the fibroblast clusters. EAY036118