Lucia Speroni

Estrogens in the wrong place at the wrong time: Fetal BPA exposure and mammary cancer.

Iatrogenic gestational exposure to diethylstilbestrol (DES) induced alterations of the genital tract and predisposed individuals to develop clear cell carcinoma of the vagina as well as breast cancer later in life. Gestational exposure of rodents to a related compound, the xenoestrogen bisphenol-A (BPA) increases the propensity to develop mammary cancer during adulthood, long after cessation of exposure. Exposure to BPA during gestation induces morphological alterations in both the stroma and the epithelium of the fetal mammary gland at 18 days of age. We postulate that the primary target of BPA is the fetal stroma, the only mammary tissue expressing estrogen receptors during fetal life. BPA would then alter the reciprocal stroma-epithelial interactions that mediate mammogenesis. In addition to this direct effect on the mammary gland, BPA is postulated to affect the hypothalamus and thus in turn affect the regulation of mammotropic hormones at puberty and beyond.

From Single Cells to Tissues: Interactions between the Matrix and Human Breast Cells in Real Time

Background Mammary gland morphogenesis involves ductal elongation, branching, and budding. All of these processes are mediated by stroma - epithelium interactions. Biomechanical factors, such as matrix stiffness, have been established as important factors in these interactions. For example, epithelial cells fail to form normal acinar structures in vitro in 3D gels that exceed the stiffness of a normal mammary gland. Additionally, heterogeneity in the spatial distribution of acini and ducts within individual collagen gels suggests that local organization of the matrix may guide morphogenesis. Here, we quantified the effects of both bulk material stiffness and local collagen fiber arrangement on epithelial morphogenesis. Results The formation of ducts and acini from single cells and the reorganization of the collagen fiber network were quantified using time-lapse confocal microscopy. MCF10A cells organized the surrounding collagen fibers during the first twelve hours after seeding. Collagen fiber density and alignment relative to the epithelial surface significantly increased within the first twelve hours and were a major influence in the shaping of the mammary epithelium. The addition of Matrigel to the collagen fiber network impaired cell-mediated reorganization of the matrix and increased the probability of spheroidal acini rather than branching ducts. The mechanical anisotropy created by regions of highly aligned collagen fibers facilitated elongation and branching, which was significantly correlated with fiber organization. In contrast, changes in bulk stiffness were not a strong predictor of this epithelial morphology. Conclusions Localized regions of collagen fiber alignment are required for ductal elongation and branching suggesting the importance of local mechanical anisotropy in mammary epithelial morphogenesis. Similar principles may govern the morphology of branching and budding in other tissues and organs.

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

Defining fiber orientation at each voxel within a 3D biomedical image stack is potentially useful for a variety of applications, including cancer, wound healing and tissue regeneration. Current methods are typically computationally intensive or inaccurate. Herein, we present a 3D weighted orientation vector summation algorithm, which is a generalization of a previously reported 2D vector summation technique aimed at quantifying collagen fiber orientations simultaneously at each voxel of an image stack. As a result, voxel-wise fiber orientation information with 4° to 5° accuracy can be determined, and the computational time required to analyze a typical stack with the size of 512x512x100 voxels is less than 5 min. Thus, this technique enables the practical extraction of voxel-specific orientation data for characterizing structural anisotropy in 3D specimens. As examples, we use this approach to characterize the fiber organization in an excised mouse mammary gland and a 3D breast tissue model.

New insights into fetal mammary gland morphogenesis: differential effects of natural and environmental estrogens

An increased breast cancer risk during adulthood has been linked to estrogen exposure during fetal life. However, the impossibility of removing estrogens from the feto-maternal unit has hindered the testing of estrogen’s direct effect on mammary gland organogenesis. To overcome this limitation, we developed an ex vivo culture method of the mammary gland where the direct action of estrogens can be tested during embryonic days (E)14 to 19. Mouse mammary buds dissected at E14 and cultured for 5 days showed that estrogens directly altered fetal mammary gland development. Exposure to 0.1 pM, 10 pM, and 1 nM 17 β-estradiol (E2) resulted in monotonic inhibition of mammary buds ductal growth. In contrast, Bisphenol-A (BPA) elicited a non-monotonic response. At environmentally relevant doses (1 nM), BPA significantly increased ductal growth, as previously observed in vivo, while 1 μM BPA significantly inhibited ductal growth. Ductal branching followed the same pattern. This effect of BPA was blocked by Fulvestrant, a full estrogen antagonist, while the effect of estradiol was not. This method may be used to study the hormonal regulation of mammary gland development, and to test newly synthesized chemicals that are released into the environment without proper assessment of their hormonal action on critical targets like the mammary gland.

SAMA: A Method for 3D Morphological Analysis

Three-dimensional (3D) culture models are critical tools for understanding tissue morphogenesis. A key requirement for their analysis is the ability to reconstruct the tissue into computational models that allow quantitative evaluation of the formed structures. Here, we present Software for Automated Morphological Analysis (SAMA), a method by which epithelial structures grown in 3D cultures can be imaged, reconstructed and analyzed with minimum human intervention. SAMA allows quantitative analysis of key features of epithelial morphogenesis such as ductal elongation, branching and lumen formation that distinguish different hormonal treatments. SAMA is a user-friendly set of customized macros operated via FIJI (http://fiji.sc/Fiji), an open-source image analysis platform in combination with a set of functions in R (http://www.r-project.org/), an open-source program for statistical analysis. SAMA enables a rapid, exhaustive and quantitative 3D analysis of the shape of a population of structures in a 3D image. SAMA is cross-platform, licensed under the GPLv3 and available at http://montevil.theobio.org/content/sama.

3D organizational mapping of collagen fibers elucidates matrix remodeling in a hormone-sensitive 3D breast tissue model

Hormones play an important role in normal and diseased breast tissue development. However, they can also disrupt cell-matrix interactions and their role in extracellular matrix reorganization during epithelial morphogenesis remains poorly understood, partly due to a lack of sensitive approaches for matrix characterization. Here, we assess the hormonal regulation of matrix reorganization in a three-dimensional (3D) breast tissue culture model using a novel metric, i.e., 3D directional variance, to characterize the 3D organization of collagen fibers visualized via high-resolution, second harmonic generation imaging. This metric enables resolving and quantifying patterns of spatial organization throughout the matrix surrounding epithelial structures treated with 17β-estradiol (E2) alone, and E2 in combination with either promegestone, a progestogen, or prolactin. Addition of promegestone results in the most disorganized fibers, while the E2 alone treatment leads to the most organized ones. Location-dependent organization mapping indicates that only the prolactin treatment leads to significant heterogeneities in the regional organization of collagen fibers, with higher levels of alignment observed at the end of the elongated epithelial structures. The observed collagen organization patterns for all groups persist for tens of micrometers. In addition, a comparison between 3D directional variance and typical 2D analysis approaches reveals an improved sensitivity of the 3D metric to identify organizational heterogeneities and differences among treatment groups. These results demonstrate that 3D directional variance is sensitive to subtle changes in the extracellular micro-environment and has the potential to elucidate reciprocal cell-matrix interactions in the context of numerous applications involving the study of normal and diseased tissue morphogenesis.

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

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

Evaluating cell matrix mechanics using an integrated nonlinear optical tweezer-confocal imaging system

Biomechanics plays a central role in breast epithelial morphogenesis. In this study we have used 3D cultures in which normal breast epithelial cells are able to organize into rounded acini and tubular ducts, the main structures found in the breast tissue. We have identified fiber organization as a main determinant of ductal organization. While bulk rheological properties of the matrix seem to play a negligible role in determining the proportion of acini versus ducts, local changes may be pivotal in shape determination. As such, the ability to make microscale rheology measurements coupled with simultaneous optical imaging in 3D cultures can be critical to assess the biomechanical factors underlying epithelial morphogenesis. This paper describes the inclusion of optical tweezers based microrheology in a microscope that had been designed for nonlinear optical imaging of collagen networks in ECM. We propose two microrheology methods and show preliminary results using a gelatin hydrogel and collagen/Matrigel 3D cultures containing mammary gland epithelial cells.