Hormonal Regulation of Epithelial Organization in a Three-Dimensional Breast Tissue Culture Model

Abstract

The establishment of hormone target breast cells in the 1970's resulted in suitable models for the study of hormone control of cell proliferation and gene expression using two-dimensional (2D) cultures. However, to study mammogenesis and breast tumor development in vitro, cells must be able to organize in three-dimensional (3D) structures like in the tissue. We now report the development of a hormone-sensitive 3D culture model for the study of mammogenesis and neoplastic development. Hormone-sensitive T47D breast cancer cells respond to estradiol in a dose-dependent manner by forming complex epithelial structures. Treatment with the synthetic progestagen promegestone, in the presence of estradiol, results in flat epithelial structures that display cytoplasmic projections, a phenomenon reported to precede side-branching. Additionally, as in the mammary gland, treatment with prolactin in the presence of estradiol induces budding structures. These changes in epithelial organization are accompanied by collagen remodeling. Collagen is the major acellular component of the breast stroma and an important player in tumor development and progression. Quantitative analysis of second harmonic generation of collagen fibers revealed that collagen density was more variable surrounding budding and irregularly shaped structures when compared to more regular structures; suggesting that fiber organization in the former is more anisotropic than in the latter. In sum, this new 3D model recapitulates morphogenetic events modulated by mammogenic hormones in the breast, and is suitable for the evaluation of therapeutic agents.

Rapid three-dimensional quantification of voxel-wise collagen fiber orientation

“…It was immersed in phosphate buffered saline to remove the OCT compound before use. Images were also acquired from an engineered 3D breast tissue model described previously [40]. Briefly, human T47D cells were seeded in 3D culture using a 1 mg/ml collagen type 1 matrix and treated with mammogenic hormones (17β estradiol in combination with promegestone, an analogue of progesterone) for two weeks.…”

“…Briefly, human T47D cells were seeded in 3D culture using a 1 mg/ml collagen type 1 matrix and treated with mammogenic hormones (17β estradiol in combination with promegestone, an analogue of progesterone) for two weeks. The gels were wholemounted and stained with Carmine Alum to visualize the epithelial structures [40]. Images of both samples were acquired using two-photon excitation fluorescence (TPEF) (ex: 800 nm , em: 525 ± 25 nm ), together with SHG which resolved the collagen fiber structure (ex: 800 nm , em: 400 ± 10 nm ).…”

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

“…Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action 12 . This model uses the commercially available hormone-responsive human breast epithelial cell line, T47D 3,11,13 , which were originally derived from a pleural effusion obtained from a 54 year old female patient with an infiltrating ductal carcinoma of the breast.…”

“…This 3D culture model is appropriate for the study of the action of the three main mammotropic hormones (estradiol, promegestone (an analogue of progesterone), and prolactin) on human breast epithelial cells. Hormone-induced epithelial morphology can be assessed quantitatively over time by morphometric analysis 12 .…”

“…Additionally, this model is suitable to test the effects of a sequential hormonal treatment; for example, after treatment with estradiol during the first week and replacement with other hormone/combination of hormones during the following week. The effect of estrogenic compounds and antiestrogens, such as ICI 182,780, can also be studied using this 3D culture model 12 .…”