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

Abstract

BackgroundMammary 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.ResultsThe 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.ConclusionsLocalized 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.

Anisotropy of Solid Breast Lesions in 2D Shear Wave Elastography is an Indicator of Malignancy

“…For ductal elongation local collagen fibre alignment is necessitated which leads to local mechanical anisotropy in the mammary gland [17]. Provenzano et al have shown that the orientation of collagen fibres changes during tumour growth: first regions of dense collagen develop in the tissue, then the collagen fibres are aligned parallel to the tumour boundary while during further tumour growth, collagen fibres become reorganized orthogonal to the tumour boundary to enable invasion [18].…”

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

“…To acquire the 3D analysis results, we calculated the θ distribution of each voxel within these 150 150 150 × × voxel stacks using the 3D vector summation technique. Regarding the 2D analysis, we calculated the θ orientation using the 2D vector summation technique in a frame-by-frame manner, which was previously reported [33]. Specifically, we applied the 2D algorithm to 150 frames of each stack sequentially, and acquired the θ orientation for every voxel of these stacks too.…”

“…As a result, pixel-specific fiber orientation information can be provided faster than pixel-wise Fourier analysis with comparable accuracy by an order of magnitude. This technique has been successfully applied for studying interactions between the matrix and human breast cells [33], and mapping scar formation in histological tissue sections [34,35]. Compared to edge detection algorithms that have been developed for rapidly detecting fiber orientation in micrographs, such as algorithms based on Sobel or Canny operators [36][37][38], this technique has improved accuracy.…”

Theoretical approach of ductal morphogenesis

“…The bulk mechanical properties of gels are not sufficient to explain these different morphogenetic processes because gels with collagen and 0% or 5% matrigel have very similar macroscopic mechanical properties but lead nevertheless to different epithelial shapes 10 . However, these different gels have very different microscopic properties, as shown by electron micrographs.…”

“…By contrast, the addition of matrigel leads to a more globular matrix, where fibers are less accessible, depending on the amount of matrigel added. 10 .…”