Biology subjects

Stowers, R.

Publications and source records attributed to Stowers, R..

3 recordsLinked to original sources

bioRxiv2025

Extracellular matrix viscoelasticity regulates mammary branching morphogenesis

Structural and mechanical cues from the extracellular matrix (ECM) regulate tissue morphogenesis. Tissue development has conventionally been studied with ex vivo systems 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 {beta}1 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.

bioengineering↗

bioRxiv2025

Engineered basement membrane mimetic hydrogels to study mammary epithelial morphogenesis and invasion

Reconstituted basement membrane (rBM) products like Matrigel are widely used in 3D culture models of epithelial tissues and cancer. However, their utility is hindered by key limitations, including batch variability, xenogenic contaminants, and a lack of tunability. To address these challenges, we engineered a 3D basement membrane (eBM) matrix by conjugating defined extracellular matrix (ECM) adhesion peptides (IKVAV, YIGSR, RGD) to an alginate hydrogel network with precisely tunable stiffness and viscoelasticity. We optimized the mechanical and biochemical properties of the engineered basement membranes (eBMs) to support mammary acinar morphogenesis in MCF10A cells, similar to rBM. We found that IKVAV-modified, fast-relaxing ({tau}1/2 = 30-150 s), and soft (E = 200 Pa) eBMs best promoted polarized acinar structures. Clusters became invasive and lost polarity only when the IKVAV-modified eBM exhibited both similar stiffness to a malignant breast tumor (E = 4000 Pa) and slow stress relaxation ({tau}1/2 = 600-1100 s). Notably, tumor-like stiffness alone was not sufficient to drive invasion in fast stress relaxing matrices modified with IKVAV. In contrast, RGD-modified matrices promoted a malignant phenotype regardless of mechanical properties. We also utilized this system to interrogate the mechanism driving acinar and tumorigenic phenotypes in response to microenvironmental parameters. A balance in activity between {beta}1- and {beta}4-integrins was observed in the context of IKVAV-modified eBMs, prompting further investigation into the downstream mechanisms. We found differences in hemidesmosome formation and production of endogenous laminin in response to peptide type, stress relaxation, and stiffness. We also saw that inhibiting either focal adhesion kinase or hemidesmosome signaling in IKVAV eBMs prevented acinus formation. This eBM matrix is a powerful, reductionist, xenogenic-free system, offering a robust platform for both fundamental research and translational applications in tissue engineering and disease modeling.

bioengineering↗

bioRxiv2023

Spatial and temporal control of 3D hydrogel viscoelasticity through phototuning

The mechanical properties of the extracellular environment can regulate a variety of cellular functions, such as spreading, migration, proliferation, and even differentiation and phenotypic determination. Much effort has been directed at understanding the effects of the extracellular matrix (ECM) elastic modulus and more recently, stress relaxation, on cellular processes. In physiological contexts like development, wound healing, and fibrotic disease progression, ECM mechanical properties change substantially over time or space. Dynamically tunable hydrogel platforms have been developed to spatiotemporally modulate a gels elastic modulus. However, dynamically altering the stress relaxation rate of a hydrogel remains a challenge. Here, we present a strategy to tune hydrogel stress relaxation rates in time or space using a light-triggered tethering of poly(ethylene glycol) (PEG) to alginate. We show that stress relaxation rate can be tuned without altering the elastic modulus of the hydrogel. We found that cells are capable of sensing and responding to dynamic stress relaxation rate changes, both morphologically and through differences in proliferation rates. We also exploited the light-based technique to generate spatial patterns of stress relaxation rates in 3D hydrogels. We anticipate that user-directed control of 3D hydrogel stress relaxation rate will be a powerful tool that enables studies that mimic dynamic ECM contexts, or as a means to guide cell fate in space and time for tissue engineering applications.

bioengineering↗