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Abstract Breast cancer progression is marked by extracellular matrix (ECM) remodeling, including increased stiffness, faster stress relaxation, and elevated collagen levels. In vitro experiments have revealed a role for each of these factors to individually promote malignant behavior, but their combined effects remain unclear. To address this, we developed alginate-collagen hydrogels with independently tunable stiffness, stress relaxation, and collagen density. We show that these combined tumor-mimicking ECM cues reinforced invasive morphologies and promoted spheroid invasion in breast cancer and mammary epithelial cells. High stiffness and low collagen density in slow-relaxing matrices led to the greatest cell migration speed and displacement. RNA-seq revealed Sp1 target gene enrichment in response to both individual and combined ECM cues, with a greater enrichment observed under multiple cues. Notably, high expression of Sp1 target genes upregulated by fast stress relaxation correlated with poor patient survival. Mechanistically, we found that phosphorylated-Sp1 (T453) was increasingly located in the nucleus in stiff and/or fast relaxing matrices, which was regulated by PI3K and ERK1/2 signaling, as well as actomyosin contractility. This study emphasizes how multiple ECM cues in complex microenvironments reinforce malignant traits and supports an emerging role for Sp1 as a mechanoresponsive transcription factor.
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This content will become publicly available on May 20, 2026
Extracellular matrix viscoelasticity regulates mammary branching morphogenesis
Abstract Structural and mechanical cues from the extracellular matrix (ECM) regulate tissue morphogenesis. Tissue development has conventionally been studied withex vivosystems where mechanical properties of the extracellular environment are either poorly controlled in space and time, lack tunability, or do not mimic ECM mechanics. For these reasons, it remains unknown how matrix stress relaxation rate, a time-dependent mechanical property that influences several cellular processes, regulates mammary branching morphogenesis. Here, we systematically investigated the influence of matrix stress relaxation on mammary branching morphogenesis using 3D alginate-collagen matrices and spheroids of human mammary epithelial cells. Slow stress relaxing matrices promoted significantly greater branch formation compared to fast stress relaxing matrices. Branching in slow stress relaxing matrices was accompanied by local collagen fiber alignment, while collagen fibers remained randomly oriented in fast stress relaxing matrices. In slow stress relaxing matrices, branch formation was driven by intermittent pulling contractions applied to the local ECM at the tips of elongating branches, which was accompanied by an abundance of phosphorylated focal adhesion kinase (phospho-FAK) and β1 integrin at the tips of branches. On the contrary, we observed that growing spheroids in fast stress relaxing matrices applied isotropic pushing forces to the ECM. Pharmacological inhibition of both Rac1 and non-muscle myosin II prevented epithelial branch formation, regardless of matrix stress relaxation rate. Interestingly, restricting cellular expansion via increased osmotic pressure was sufficient to impede epithelial branching in slow stress relaxing matrices. This work highlights the importance of stress relaxation in regulating and directing mammary branch elongation.
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- Award ID(s):
- 2125644
- PAR ID:
- 10626397
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Institution:
- bioRxiv
- Sponsoring Org:
- National Science Foundation
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