Assistant Professor University of California, Santa Barbara, United States
Introduction:: Branching morphogenesis is a fundamental process that occurs during the development of mammary tissue, and several signaling pathways that regulate mammary branching are disrupted during the development and progression of breast cancer. Much of our current understanding of morphogenesis comes from the effects of chemical morphogens, though recent work has revealed that the mechanics of the extracellular matrix can regulate morphogenetic events. Conventionally, tissue development has been studied with in vivo or ex vivo/in vitro 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 functions and processes, regulates tissue branching in mammary development. Our objective here is to determine the effect of ECM viscoelasticity on mammary branching using 3D collagen-rich matrix with tunable stress relaxation and spheroids of human mammary epithelial cells.
Materials and Methods:: Collagen-alginate interpenetrating networks were fabricated to generate matrices with elastic moduli ~100 Pa, mimicking the stiffness of a mammary gland. The molecular weight of the alginate was varied to produce slow relaxing (t1/2~1000 s) and fast relaxing (t1/2 ~100 s) matrices. To prepare MCF10A spheroids, 3000 cells suspended in hanging drop culture per spheroid and incubated for 48 hours. Spheroids were then encapsulated in matrices, and imaged every 24 hours for 5 days before fixation, immunostaining, and confocal microscopy. Spheroid morphology and branching was analyzed and quantified using Image J.
Results, Conclusions, and Discussions:: Spheroids in slow stress-relaxing matrices grew rapidly in size and began to undergo branching morphogenesis as soon as 24-48 hours after encapsulation, forming several primary, secondary, and tertiary branches into the surrounding matrix (Fig. 1A). Interestingly, spheroids in fast stress-relaxing matrices had significantly fewer branches which were also shorter in length. We found that the primary branch length of MCF10A spheroids in both fast stress-relaxing matrices and slow stress-relaxing matrices increased linearly overtime, though the branch length was much statistically significantly greater for spheroids in slow stress-relaxing matrices (Fig. 1B). Additionally, the total number of branches grew overtime in slow stress-relaxing matrices, yet maintained a constant linear trajectory in fast stress-relaxing matrices and began to taper after 144 hours in culture (Fig 1C). MCF10A spheroid area in slow stress-relaxing matrices was significantly larger after 120 hours compared to 24 hours in culture, whereas the spheroid area in fast stress-relaxing matrices between these time scales were not significantly different (Fig. 1D). To conclude, we show that extracellular matrix viscoelasticity regulates spatiotemporal epithelial tissue organization within human mammary epithelial cells. This work provides us with an improved quantitative understanding of the mechanical cues regulating mammary morphogenesis, which will strengthen the foundational knowledge underlying tissue development and breast cancer development and progression.
