Characterization of estrogen and the endocrine-disrupting chemical-induced mouse mammary gland reorganization via single-cell RNA-sequencing

Exposure to estrogen and its mimics during menopausal transition is thought to modify the structure of mammary gland. Using a surgical menopausal (ovariectomized) mouse model, we assessed how mammary gland tissue was affected by both 17ß-estradiol (E2) and polybrominated diphenyl ethers (PBDEs). Three analyzed flame retardants, BDE-47, BDE-100 and BDE-153, are most frequently detected in human serum from all over the world. During physiologically-relevant exposure to E2, PBDEs enhanced E2-mediated regrowth of mammary glands with terminal end bud-like structures. According to sequencing analysis of mammary gland RNA, PBDEs both augmented E2-facilitated cell proliferationgene expression changes and modulated immune regulation. To better elucidate the effects of E2 and PBDEs, single-cell RNA sequencing (scRNAseq) analysis was performed. In a definitive manner, E2 was found to induce Pgr expression in both Esr1+ and Esr1- cells. Significantly, molecular evidence was available to show functional differences among cells with differential expression of Esr1 and Pgr. The PBDEs + E2 treatment increased the number of double positive (Esr1+Pgr+) and Pgr only (Esr1-Pgr+) cells in both mature luminal epithelial and progenitor cells of luminal epithelialcells. Moreover, the combination increased the mammary gland population of M2 macrophages. The results help clarify how exposure to both E2 and endocrine disrupting chemicals like PBDEs during menopausal transition impact mammary gland structure. Ultimately, the findings advance understanding of how exposure can increase the risk of developing breast cancer. Overall design: Female BALB/cj mice were exposed to E2 and a mixture of PBDEs for 1 week. Single-cell RNA sequencing was done to evaluate gene expression changes among treatment groups. Mice were subjected to 3 different treatments (Vehicle, E2, E2+PBDE).

学界普遍认为，围绝经期暴露于雌激素及其模拟物，会改变乳腺组织的结构。本研究采用手术绝经（卵巢切除）小鼠模型，评估了17β-雌二醇（17ß-estradiol, E2）与多溴二苯醚（polybrominated diphenyl ethers, PBDEs）对乳腺组织的影响。本次分析的三种阻燃剂——BDE-47、BDE-100与BDE-153，是全球人类血清中检出率最高的多溴二苯醚同系物。在符合生理相关浓度的E2暴露条件下，PBDEs可增强E2介导的、带有末端芽样结构的乳腺组织再生。通过乳腺组织RNA测序分析发现，PBDEs既可强化E2诱导的细胞增殖与基因表达谱改变，也可调控免疫相关通路。为进一步阐明E2与PBDEs的作用机制，本研究开展了单细胞RNA测序（single-cell RNA sequencing, scRNAseq）分析。研究明确证实，E2可在Esr1阳性（Esr1+）与Esr1阴性（Esr1-）两类细胞中诱导Pgr基因的表达。值得注意的是，本研究通过分子证据证实，Esr1与Pgr表达模式不同的细胞间存在功能差异。PBDEs联合E2处理，可增加成熟腔上皮细胞与腔上皮祖细胞中双阳性（Esr1+Pgr+）以及仅表达Pgr（Esr1-Pgr+）的细胞数量。此外，联合处理还可提升乳腺组织中M2型巨噬细胞的占比。本研究结果阐明了围绝经期同时暴露于E2与PBDEs这类内分泌干扰物（endocrine disrupting chemicals）时，乳腺结构的改变机制。最终，本研究成果加深了人们对暴露相关乳腺癌风险升高机制的理解。实验设计：将雌性BALB/cj小鼠暴露于E2与PBDEs混合液中，处理周期为1周；通过单细胞RNA测序分析不同处理组间的基因表达差异。本次实验设置3种处理方案：溶剂对照（Vehicle）、E2处理组、E2与PBDEs联合处理组。