Z20 - Extracellular matrix stress relaxation regulates mammary branching morphogenesis

Introduction: : Branching morphogenesis is a fundamental process that drives the development of the mammary gland. Much of our current understanding of morphogenesis comes from the effects of chemical morphogens and transcriptional programs, though recent work has revealed that extracellular matrix (ECM) mechanics can regulate morphogenetic events. Conventionally, tissue development has been studied with in vivo and ex vivo systems where mechanical properties are either poorly controlled in space and time, lack tunability, or do not mimic the native ECM mechanics. For these reasons, it remains unknown how matrix stress relaxation rate, a time-dependent mechanical property shown to influence several cellular processes, regulates mammary branching. Our objective here is to determine the effect of ECM stress relaxation on mammary branching of human mammary epithelial cells using 3D collagen-rich matrices with tunable stress relaxation.

Materials and

Methods: : Collagen-alginate interpenetrating networks were fabricated to generate matrices with elastic moduli ~200 Pa , mimicking the stiffness of a mammary gland in vivo. The molecular weight of the alginate was varied to produce a range of stress-relaxation times (t1/2 = 100 s, 800 s, 1200 s, 4000 s) to span the range over which cells are mechanically responsive. To prepare MCF10A spheroids, 3000 cells were suspended in hanging drops and incubated for 24 hours. Spheroids were then encapsulated in matrices and imaged every 24 hours for 7 days. Spheroid morphology and branching was analyzed and quantified using Image J. Collagen fiber alignment was imaged via confocal reflectance microscopy and quantified with CurveAlign. Matrix displacement maps were generated using the PIV plugin in Image J and bead displacement was quantified using Imaris. Custom MATLAB scripts were used to generate the representative plots shown. For immunostaining analysis, gels were fixed, frozen, cryosectioned, and immunostained for β1 integrin 1:200 (Thermofisher, 14-0299-82) and FAK-P-Y397 1:500 (Thermofisher, 700255). Immunofluorescence intensity data was analyzed via a custom pipeline developed in CellProfiler. Pharmacological inhibitors used were: 50 μM NSC 23766 (Tocris Bioscience, Rac1 inhibitor), 50 μM Blebbistatin (Cayman Chemical, myosin II inhibitor), and 10 μM PF 573228 (Cayman Chemical, FAK inhibitor). Brightfield images were collected on day seven of culture and morphologically analyzed via Image J.

Results, Conclusions, and Discussions:: We fabricated engineered collagen-alginate interpenetrating networks (Fig. 1A) with a range of stress-relaxation times (t = 100 s, 800 s, 1200 s, 4000 s (Fig 1B) and an elastic modulus of 200 Pa (Fig. 1C). We were able to tune the mechanical properties of the hydrogel independently of the collagen microarchitecture of the substrates (Fig. 1D). Spheroids in slow stress-relaxing matrices grew rapidly in size and began to undergo branching morphogenesis as soon as 24-48 hours after encapsulation (Fig. 1E). This was characterized by a significant increase (586%) in the total number of branches (Fig. 1F) and a significant increase in branch length (390%) within slow stress relaxing matrices (t1/2 = 1200 s) compared to fast relaxing matrices (t1/2 = 100 s) after six days in culture (Fig. 1G). Confocal reflectance images and a density heat map demonstrated enhanced collagen fiber alignment in slow stress relaxing matrices along the branching axis (Fig. 2A). Upon binning and normalizing the orientation values, we observed significantly increased collagen fiber alignment (corresponding to bins between 60–90°) in front of the branching axis in slow stress relaxing matrices, but not in fast stress relaxing matrices (Fig. 2B–D). Upon performing live-cell imaging and particle image velocimetry, we found that larger, pulling displacements occurred in front of branch tips in slow stress relaxing matrices, compared to fast stress relaxing matrices where smaller, pushing displacements were applied to the ECM (Fig. 2E, F). Interestingly, spheroids in slow stress relaxing matrices exerted non-continuous, intermittent contractions to extend their branches (Fig. 2G). Mechanistically, this was driven by B1 integrin and pFAK which were localized to the tip cells of mammary branches in slow stress relaxing matrices (Fig. 3A-C). Furthermore, small molecule inhibition experiments revealed that Rac1 and non-muscle myosin II play key roles in contributing to mammary morphogenesis in slow stress relaxing matrices (Fig. 3D-G). This work provides us with an improved quantitative understanding of how the mechanical properties of the extracellular matrix regulate mammary morphogenesis, which will strengthen the foundational knowledge underlying tissue development.

Acknowledgements and/or References (Optional):: Funding for this project was provided by a National Science Foundation Graduate Research Fellowship (No: 2139319) to D.W., a National Science Foundation Data Driven Biology Grant (No. DGE-2125644) to D.W., a Crowe Family Fellowship to J.M., and a California Institute of Regenerative Medicine fellowship to A.S. (EDUC4-12821). The authors acknowledge the use of the Neuroscience Research Institute and the Department of Molecular, Cellular, and Developmental Biology Microscopy Facility supported by the University of California, Santa Barbara.