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Significance Extracellular matrix (ECM) stiffness is an influential factor in many biological processes. Temporal changes in ECM stiffness are observed in cancer, cardiovascular disease, and wound healing, and are likely involved in disease progression. However, no cell culture systems exist to appropriately model temporal changes in ECM stiffness to determine the biological relevance and mechanisms involved. Here, we present a 3D hydrogel cell culture system in which the matrix stiffness can be tuned by light. Our approach offers both spatial and temporal control of stiffness, is compatible with cell culture, and can be used transdermally for in vivo applications. This system is amenable to many applications to investigate the influence of matrix stiffness on cell behavior and fate. Hydrogels are widely used as in vitro culture models to mimic 3D cellular microenvironments. The stiffness of the extracellular matrix is known to influence cell phenotype, inspiring work toward unraveling the role of stiffness on cell behavior using hydrogels. However, in many biological processes such as embryonic development, wound healing, and tumorigenesis, the microenvironment is highly dynamic, leading to changes in matrix stiffness over a broad range of timescales. To recapitulate dynamic microenvironments, a hydrogel with temporally tunable stiffness is needed. Here, we present a system in which alginate gel stiffness can be temporally modulated by light-triggered release of calcium or a chelator from liposomes. Others have shown softening via photodegradation or stiffening via secondary cross-linking; however, our system is capable of both dynamic stiffening and softening. Dynamic modulation of stiffness can be induced at least 14 d after gelation and can be spatially controlled to produce gradients and patterns. We use this system to investigate the regulation of fibroblast morphology by stiffness in both nondegradable gels and gels with degradable elements. Interestingly, stiffening inhibits fibroblast spreading through either mesenchymal or amoeboid migration modes. We demonstrate this technology can be translated in vivo by using deeply penetrating near-infrared light for transdermal stiffness modulation, enabling external control of gel stiffness. Temporal modulation of hydrogel stiffness is a powerful tool that will enable investigation of the role that dynamic microenvironments play in biological processes both in vitro and in well-controlled in vivo experiments.

For mesenchymal stem cells (MSCs) cultured in three dimensional matrices, matrix remodeling is associated with enhanced osteogenic differentiation. However, the mechanism linking matrix remodeling in 3D to osteogenesis of MSCs remains unclear. Here, we find that MSCs in viscoelastic hydrogels exhibit volume expansion during cell spreading, and greater volume expansion is associated with enhanced osteogenesis. Restriction of expansion by either hydrogels with slow stress relaxation or increased osmotic pressure diminishes osteogenesis, independent of cell morphology. Conversely, induced expansion by hypoosmotic pressure accelerates osteogenesis. Volume expansion is mediated by activation of TRPV4 ion channels, and reciprocal feedback between TRPV4 activation and volume expansion controls nuclear localization of RUNX2, but not YAP, to promote osteogenesis. This work demonstrates the role of cell volume in regulating cell fate in 3D culture, and identifies TRPV4 as a molecular sensor of matrix viscoelasticity that regulates osteogenic differentiation. For mesenchymal stem cells (MSCs), matrix remodeling is associated with enhanced osteogenic differentiation. Here authors find that MSCs in viscoelastic hydrogels exhibit volume expansion during cell spreading, and greater volume expansion is associated with enhanced osteogenesis.

Neural progenitor cell (NPC) culture within three-dimensional (3D) hydrogels is an attractive strategy for expanding a therapeutically relevant number of stem cells. However, relatively little is known about how 3D material properties such as stiffness and degradability affect the maintenance of NPC stemness in the absence of differentiation factors. Over a physiologically relevant range of stiffness from ∼0.5 to 50 kPa, stemness maintenance did not correlate with initial hydrogel stiffness. In contrast, hydrogel degradation was both correlated with, and necessary for, maintenance of NPC stemness. This requirement for degradation was independent of cytoskeletal tension generation and presentation of engineered adhesive ligands, instead relying on matrix remodelling to facilitate cadherin-mediated cell–cell contact and promote β-catenin signalling. In two additional hydrogel systems, permitting NPC-mediated matrix remodelling proved to be a generalizable strategy for stemness maintenance in 3D. Our findings have identified matrix remodelling, in the absence of cytoskeletal tension generation, as a previously unknown strategy to maintain stemness in 3D. The physical properties of biomaterials affect cell behaviour. Here, the authors investigate how stiffness and degradation of hydrogels affect signalling pathways that modulate the maintenance of stemness of neural progenitor cells.

In breast cancer, the increased stiffness of the extracellular matrix is a key driver of malignancy. Yet little is known about the epigenomic changes that underlie the tumorigenic impact of extracellular matrix mechanics. Here, we show in a three-dimensional culture model of breast cancer that stiff extracellular matrix induces a tumorigenic phenotype through changes in chromatin state. We found that increased stiffness yielded cells with more wrinkled nuclei and with increased lamina-associated chromatin, that cells cultured in stiff matrices displayed more accessible chromatin sites, which exhibited footprints of Sp1 binding, and that this transcription factor acts along with the histone deacetylases 3 and 8 to regulate the induction of stiffness-mediated tumorigenicity. Just as cell culture on soft environments or in them rather than on tissue-culture plastic better recapitulates the acinar morphology observed in mammary epithelium in vivo, mammary epithelial cells cultured on soft microenvironments or in them also more closely replicate the in vivo chromatin state. Our results emphasize the importance of culture conditions for epigenomic studies, and reveal that chromatin state is a critical mediator of mechanotransduction. In a 3D model of breast cancer, a stiff extracellular matrix promotes a tumorigenic phenotype through broad changes in chromatin accessibility and in the activity of histone deacetylases and the transcription factor Sp1.

Structural and mechanical cues from the extracellular matrix (ECM) regulate tissue morphogenesis. Tissue development has conventionally been studied with ex vivo systems where the 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, the influence of matrix stress relaxation on mammary branching morphogenesis is systematically investigated using 3D alginate‐collagen matrices and spheroids of human mammary epithelial cells. Slow stress relaxing matrices enhanced branching, which is accompanied by local collagen fiber alignment, intermittent pulling contractions applied to the ECM, and focal adhesion signaling. On the contrary, it is 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 is 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.

Yes-associated protein (YAP) is a transcriptional regulator and mechanotransducer, relaying extracellular matrix (ECM) stiffness into proliferative gene expression in 2D culture. Previous studies show that YAP activation is dependent on F-actin stress fiber mediated nuclear pore opening, however the protein mediators of YAP translocation remain unclear. Here, we show that YAP co-localizes with F-actin during activating conditions, such as sparse plating and culturing on stiff 2D substrates. To identify proteins mediating YAP translocation, we performed co-immunoprecipitation followed by mass spectrometry (co-IP/MS) for proteins that differentially associated with YAP under activating conditions. Interestingly, YAP preferentially associates with β 1 integrin under activating conditions, and β 4 integrin under inactivating conditions. In activating conditions, CRISPR/Cas9 knockout (KO) of β 1 integrin (ΔITGB1) resulted in decreased cell area, which correlated with decreased YAP nuclear localization. ΔITGB1 did not significantly affect the slope of the correlation between YAP nuclear localization with area, but did decrease overall nuclear YAP independently of cell spreading. In contrast, β 4 integrin KO (ΔITGB4) cells showed no change in cell area and similarly decreased nuclear YAP. These results reveal proteins that differentially associate with YAP during activation, which may aid in regulating YAP nuclear translocation.

Tumors are much stiffer than healthy tissue, and progressively stiffen as the cancer develops. Tumor stiffening is largely the result of extracellular matrix (ECM) remodeling, for example, deposition and crosslinking of collagen I. Well established in vitro models have demonstrated the influence of the microenvironment in regulating tissue homeostasis, with matrix stiffness being a particularly influential mediator. Non-malignant MCF10A mammary epithelial cells (MECs) lose their epithelial characteristics and become invasive when cultured in stiff microenvironments, leading to the hypothesis that tumor stiffening could contribute directly to disease progression. However, previous studies demonstrating MCF10A invasion have been performed in gels with constant mechanical properties, unlike the dynamically stiffening tumor microenvironment. Here, we employ a temporally stiffening hydrogel platform to demonstrate that matrix stiffening induces invasion from and proliferation in MCF10A mammary acini. After allowing MCF10A acini to form in soft hydrogels for 14 days, the gels were stiffened to the level of a malignant tumor, giving rise to a proliferative and invasive phenotype. Cells were observed to collectively migrate away from mammary acini while maintaining cell–cell contacts. Small molecule inhibition of PI3K and Rac1 pathways was sufficient to significantly reduce the number and size of invasive acini after stiffening. Our results demonstrate that temporal matrix stiffening can induce invasion from mammary acini and supports the notion that tumor stiffening could be implicated in disease progression and metastasis.

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 (τ1 = 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 (τ1 = 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 β1- and β4-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.

The DNA methylation of the promoter region of the oncogenic Yes-associated protein is reversibly regulated by the stiffness of the extracellular matrix.

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.