Mechanical regulation of mammary epithelial branching morphogenesis

Abstract

Spatial patterning of cell behaviors establishes the regional differences within tissues that collectively develop branched organs into their characteristic tree-like shapes. Although a variety of endocrine, paracrine, autocrine and extracellular matrix (ECM) signals have been implicated as global regulators of mammary epithelial branching morphogenesis, comprehensive understanding of the mechanisms of pattern formation during the process is lacking. Here we show that the pattern of branching morphogenesis of three-dimensional (3D) engineered mammary epithelial tissues is controlled in part by gradients of endogenous mechanical stress.

We used microfabrication to build model mammary epithelial tissues of defined geometry that branched in a stereotyped pattern when induced with growth factors. We combined continuum mechanics with computational modeling, atomic force microscopy and confocal reflectance microscopy to define the experimental parameters required to directly measure the mechanical stress profile of the tissues. We found that calculating stresses accurately in these settings required accounting for cell-induced mechanical heterogeneities within the ECM. Using this technique, we measured endogenous traction forces at the epithelial surface and resolved qualitative and quantitative patterns of mechanical stress throughout the tissue. We discovered that the mechanical profile of the tissues was dictated by the epithelial geometry, with cells within certain geometric features consistently experiencing higher forces. In addition to quantifying tissue-induced forces, this method allowed us to define the parameters which govern epithelial force generation and subsequently fabricate tissues with precisely tuned mechanical profiles.

Using this platform, we demonstrated that mammary epithelial branches initiated from sites of high mechanical stress within the preexisting tissue; the extent of branching correlated with the local magnitude of stress. Branch sites were defined by activation of focal adhesion kinase (FAK), inhibition of which disrupted morphogenesis. Modulating mechanical stress by manipulating cellular contractility, matrix stiffness, intercellular cohesion and tissue geometry led to concomitant changes in both FAK activation and branching.

We found further that mammary epithelial branch extension in collagenous matrices was driven by tensile mechanical forces, which enhanced elongation by activating mechanosensitive signaling within the invading cells and conditioning the obstructing ECM. Specifically, cell-generated tension activated FAK and p130 Cas within the cells of the leading edge, and induced the nuclear translocation of myocardin-related transcription factor A (MRTF-A), which was required for branch extension. Blocking cellular tension led to loss of MRTF-A activation and prevented branch extension. Further, tensile forces at the leading edge facilitated invasion into the 3D matrix by remodeling and creating aligned tracks within the ECM. Although in vivo confirmation is pending, these data contribute to our understanding of the physical rules that guide the normal development of the mammary gland and other branching epithelia, and may help unlock general engineering strategies to build such organs ex vivo.