Lumen expansion is initially driven by apical actin polymerization followed by osmotic pressure in a human epiblast model.

Post-implantation, the pluripotent epiblast in a human embryo forms a central lumen, paving the way for gastrulation. Osmotic pressure gradients are considered the drivers of lumen expansion across development, but their role in human epiblasts is unknown. Here, we study lumenogenesis in a pluripotent-stem-cell-based epiblast model using engineered hydrogels. We find that leaky junctions prevent osmotic pressure gradients in early epiblasts and, instead, forces from apical actin polymerization drive lumen expansion. Once the lumen reaches a radius of ∼12 µm, tight junctions mature, and osmotic pressure gradients develop to drive further growth. Computational modeling indicates that apical actin polymerization into a stiff network mediates initial lumen expansion and predicts a transition to pressure-driven growth in larger epiblasts to avoid buckling. Human epiblasts show transcriptional signatures consistent with these mechanisms. Thus, actin polymerization drives lumen expansion in the human epiblast and may serve as a general mechanism of early lumenogenesis.

Cell volume expansion and local contractility drive collective invasion of the basement membrane in breast cancer.

Breast cancer becomes invasive when carcinoma cells invade through the basement membrane (BM)-a nanoporous layer of matrix that physically separates the primary tumour from the stroma. Single cells can invade through nanoporous three-dimensional matrices due to protease-mediated degradation or force-mediated widening of pores via invadopodial protrusions. However, how multiple cells collectively invade through the physiological BM, as they do during breast cancer progression, remains unclear. Here we developed a three-dimensional in vitro model of collective invasion of the BM during breast cancer. We show that cells utilize both proteases and forces-but not invadopodia-to breach the BM. Forces are generated from a combination of global cell volume expansion, which stretches the BM, and local contractile forces that act in the plane of the BM to breach it, allowing invasion. These results uncover a mechanism by which cells collectively interact to overcome a critical barrier to metastasis.

Monocytes use protrusive forces to generate migration paths in viscoelastic collagen-based extracellular matrices.

Circulating monocytes are recruited to the tumor microenvironment, where they can differentiate into macrophages that mediate tumor progression. To reach the tumor microenvironment, monocytes must first extravasate and migrate through the type-1 collagen rich stromal matrix. The viscoelastic stromal matrix around tumors not only stiffens relative to normal stromal matrix, but often exhibits enhanced viscous characteristics, as indicated by a higher loss tangent or faster stress relaxation rate. Here, we studied how changes in matrix stiffness and viscoelasticity, impact the three-dimensional migration of monocytes through stromal-like matrices. Interpenetrating networks of type-1 collagen and alginate, which enable independent tunability of stiffness and stress relaxation over physiologically relevant ranges, were used as confining matrices for three-dimensional culture of monocytes. Increased stiffness and faster stress relaxation independently enhanced the 3D migration of monocytes. Migrating monocytes have an ellipsoidal or rounded wedge-like morphology, reminiscent of amoeboid migration, with accumulation of actin at the trailing edge. Matrix adhesions and Rho-mediated contractility were dispensable for monocyte migration in 3D, but migration did require actin polymerization and myosin contractility. Mechanistic studies indicate that actin polymerization at the leading edge generates protrusive forces that open a path for the monocytes to migrate through in the confining viscoelastic matrices. Taken together, our findings implicate matrix stiffness and stress relaxation as key mediators of monocyte migration and reveal how monocytes use pushing forces at the leading edge mediated by actin polymerization to generate migration paths in confining viscoelastic matrices. Significance Statement: Cell migration is essential for numerous biological processes in health and disease, including for immune cell trafficking. Monocyte immune cells migrate through extracellular matrix to the tumor microenvironment where they can play a role in regulating cancer progression. Increased extracellular matrix (ECM) stiffness and viscoelasticity have been implicated in cancer progression, but the impact of these changes in the ECM on monocyte migration remains unknown. Here, we find that increased ECM stiffness and viscoelasticity promote monocyte migration. Interestingly, we reveal a previously undescribed adhesion-independent mode of migration whereby monocytes generate a path to migrate through pushing forces at the leading edge. These findings help elucidate how changes in the tumor microenvironment impact monocyte trafficking and thereby disease progression.

Cell-extracellular matrix mechanotransduction in 3D.

Mechanical properties of extracellular matrices (ECMs) regulate essential cell behaviours, including differentiation, migration and proliferation, through mechanotransduction. Studies of cell-ECM mechanotransduction have largely focused on cells cultured in 2D, on top of elastic substrates with a range of stiffnesses. However, cells often interact with ECMs in vivo in a 3D context, and cell-ECM interactions and mechanisms of mechanotransduction in 3D can differ from those in 2D. The ECM exhibits various structural features as well as complex mechanical properties. In 3D, mechanical confinement by the surrounding ECM restricts changes in cell volume and cell shape but allows cells to generate force on the matrix by extending protrusions and regulating cell volume as well as through actomyosin-based contractility. Furthermore, cell-matrix interactions are dynamic owing to matrix remodelling. Accordingly, ECM stiffness, viscoelasticity and degradability often play a critical role in regulating cell behaviours in 3D. Mechanisms of 3D mechanotransduction include traditional integrin-mediated pathways that sense mechanical properties and more recently described mechanosensitive ion channel-mediated pathways that sense 3D confinement, with both converging on the nucleus for downstream control of transcription and phenotype. Mechanotransduction is involved in tissues from development to cancer and is being increasingly harnessed towards mechanotherapy. Here we discuss recent progress in our understanding of cell-ECM mechanotransduction in 3D.

Viscoelasticity and Adhesion Signaling in Biomaterials Control Human Pluripotent Stem Cell Morphogenesis in 3D Culture.

Organoids are lumen-containing multicellular structures that recapitulate key features of the organs, and are increasingly used in models of disease, drug testing, and regenerative medicine. Recent work has used 3D culture models to form organoids from human induced pluripotent stem cells (hiPSCs) in reconstituted basement membrane (rBM) matrices. However, rBM matrices offer little control over the microenvironment. More generally, the role of matrix viscoelasticity in directing lumen formation remains unknown. Here, viscoelastic alginate hydrogels with independently tunable stress relaxation (viscoelasticity), stiffness, and arginine-glycine-aspartate (RGD) ligand density are used to study hiPSC morphogenesis in 3D culture. A phase diagram that shows how these properties control hiPSC morphogenesis is reported. Higher RGD density and fast stress relaxation promote hiPSC viability, proliferation, apicobasal polarization, and lumen formation, while slow stress relaxation at low RGD densities triggers hiPSC apoptosis. Notably, hiPSCs maintain pluripotency in alginate hydrogels for much longer times than is reported in rBM matrices. Lumen formation is regulated by actomyosin contractility and is accompanied by translocation of Yes-associated protein (YAP) from the nucleus to the cytoplasm. The results reveal matrix viscoelasticity as a potent factor regulating stem cell morphogenesis and provide new insights into how engineered biomaterials may be leveraged to build organoids.

Transient mechanical interactions between cells and viscoelastic extracellular matrix.

During various physiological processes, such as wound healing and cell migration, cells continuously interact mechanically with a surrounding extracellular matrix (ECM). Contractile forces generated by the actin cytoskeleton are transmitted to a surrounding ECM, resulting in structural remodeling of the ECM. To better understand how matrix remodeling takes place, a myriad of in vitro experiments and simulations have been performed during recent decades. However, physiological ECMs are viscoelastic, exhibiting stress relaxation or creep over time. The time-dependent nature of matrix remodeling induced by cells remains poorly understood. Here, we employed a discrete model to investigate how the viscoelastic nature of ECMs affects matrix remodeling and stress profiles. In particular, we used explicit transient cross-linkers with varied density and unbinding kinetics to capture viscoelasticity unlike most of the previous models. Using this model, we quantified the time evolution of generation, propagation, and relaxation of stresses induced by a contracting cell in an ECM. It was found that matrix connectivity, regulated by fiber concentration and cross-linking density, significantly affects the magnitude and propagation of stress and subsequent matrix remodeling, as characterized by fiber displacements and local net deformation. In addition, we demonstrated how the base rate and force sensitivity of cross-linker unbinding regulate stress profiles and matrix remodeling. We verified simulation results using in vitro experiments performed with fibroblasts encapsulated in a three-dimensional collagen matrix. Our study provides key insights into the dynamics of physiologically relevant mechanical interactions between cells and a viscoelastic ECM.

Tuning Viscoelasticity in Alginate Hydrogels for 3D Cell Culture Studies.

Physical properties of the extracellular matrix (ECM) affect cell behaviors ranging from cell adhesion and migration to differentiation and gene expression, a process known as mechanotransduction. While most studies have focused on the impact of ECM stiffness, using linearly elastic materials such as polyacrylamide gels as cell culture substrates, biological tissues and ECMs are viscoelastic, which means they exhibit time-dependent mechanical responses and dissipate mechanical energy. Recent studies have revealed ECM viscoelasticity, independent of stiffness, as a critical physical parameter regulating cellular processes. These studies have used biomaterials with tunable viscoelasticity as cell-culture substrates, with alginate hydrogels being one of the most commonly used systems. Here, we detail the protocols for three approaches to modulating viscoelasticity in alginate hydrogels for 2D and 3D cell culture studies, as well as the testing of their mechanical properties. Viscoelasticity in alginate hydrogels can be tuned by varying the molecular weight of the alginate polymer, changing the type of crosslinker-ionic versus covalent-or by grafting short poly(ethylene-glycol) (PEG) chains to the alginate polymer. As these approaches are based on commercially available products and simple chemistries, these protocols should be accessible for scientists in the cell biology and bioengineering communities. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Tuning viscoelasticity by varying alginate molecular weight Basic Protocol 2: Tuning viscoelasticity with ionic versus covalent crosslinking Basic Protocol 3: Tuning viscoelasticity by adding PEG spacers to alginate chains Support Protocol 1: Testing mechanical properties of alginate hydrogels Support Protocol 2: Conjugating cell-adhesion peptide RGD to alginate.

Cells under pressure.

A new method for applying solid stress to aggregates of cells is shedding light on the impact of mechanical forces on cancer cells.

Magnetic probe-based microrheology reveals local softening and stiffening of 3D collagen matrices by fibroblasts.

Changes in extracellular matrix stiffness impact a variety of biological processes including cancer progression. However, cells also actively remodel the matrices they interact with, dynamically altering the matrix mechanics they respond to. Further, cells not only react to matrix stiffness, but also have a distinct reaction to matrix viscoelasticity. The impact of cell-driven matrix remodeling on matrix stiffness and viscoelasticity at the microscale remains unclear, as existing methods to measure mechanics are largely at the bulk scale or probe only the surface of matrices, and focus on stiffness. Yet, establishing the impact of the matrix remodeling at the microscale is crucial to obtaining an understanding of mechanotransduction in biological matrices, and biological matrices are not just elastic, but are viscoelastic. Here, we advanced magnetic probe-based microrheology to overcome its previous limitations in measuring viscoelasticity at the cell-size-scale spatial resolution within 3D cell cultures that have tissue-relevant stiffness levels up to a Young's modulus of 0.5 kPa. Our magnetic microrheometers exert controlled magnetic forces on magnetic microprobes within reconstituted extracellular matrices and detect microprobe displacement responses to measure matrix viscoelasticity and determine the frequency-dependent shear modulus (stiffness), the loss tangent, and spatial heterogeneity. We applied these tools to investigate how microscale viscoelasticity of collagen matrices is altered by fibroblast cells as they contract collagen gels, a process studied extensively at the macroscale. Interestingly, we found that fibroblasts first soften the matrix locally over the first 32 hours of culture, and then progressively stiffen the matrix thereafter. Fibroblast activity also progressively increased the matrix loss tangent. We confirmed that the softening is caused by matrix-metalloproteinase-mediated collagen degradation, whereas stiffening is associated with local alignment and densification of collagen fibers around the fibroblasts. This work paves the way for the use of measurement systems that quantify microscale viscoelasticity within 3D cell cultures for studies of cell-matrix interactions in cancer progression and other areas.

Are college campuses superspreaders? A data-driven modeling study.

The COVID-19 pandemic continues to present enormous challenges for colleges and universities and strategies for save reopening remain a topic of ongoing debate. Many institutions that reopened cautiously in the fall experienced a massive wave of infections and colleges were soon declared as the new hotspots of the pandemic. However, the precise effects of college outbreaks on their immediate neighborhood remain largely unknown. Here we show that the first two weeks of instruction present a high-risk period for campus outbreaks and that these outbreaks tend to spread into the neighboring communities. By integrating a classical mathematical epidemiology model and Bayesian learning, we learned the dynamic reproduction number for 30 colleges from their daily case reports. Of these 30 institutions, 14 displayed a spike of infections within the first two weeks of class, with peak seven-day incidences well above 1,000 per 100,000, an order of magnitude larger than the nation-wide peaks of 70 and 150 during the first and second waves of the pandemic. While most colleges were able to rapidly reduce the number of new infections, many failed to control the spread of the virus beyond their own campus: Within only two weeks, 17 campus outbreaks translated directly into peaks of infection within their home counties. These findings suggests that college campuses are at risk to develop an extreme incidence of COVID-19 and become superspreaders for neighboring communities. We anticipate that tight test-trace-quarantine strategies, flexible transition to online instruction, and-most importantly-compliance with local regulations will be critical to ensure a safe campus reopening after the winter break.

Covalent cross-linking of basement membrane-like matrices physically restricts invasive protrusions in breast cancer cells.

The basement membrane (BM) provides a physical barrier to invasion in epithelial tumors, and alterations in the molecular makeup and structural integrity of the BM have been implicated in cancer progression. Invadopodia are the invasive protrusions that enable cancer cells to breach the nanoporous basement membrane, through matrix degradation and generation of force. However, the impact of covalent cross-linking on invadopodia extension into the BM remains unclear. Here, we examine the impact of covalent cross-linking of extracellular matrix on invasive protrusions using biomaterials that present ligands relevant to the basement membrane and provide a nanoporous, confining microenvironment. We find that increased covalent cross-linking of reconstituted basement membrane (rBM) matrix diminishes matrix mechanical plasticity, or the ability of the matrix to permanently retain deformation due to force. Covalently cross-linked rBM matrices, and rBM-alginate interpenetrating networks (IPNs) with covalent cross-links and low plasticity, restrict cell spreading and protrusivity. The reduced spreading and reduced protrusivity in response to low mechanical plasticity occurred independent of proteases. Mechanistically, our computational model reveals that the reduction in mechanical plasticity due to covalent cross-linking is sufficient to mechanically prevent cell protrusions from extending, independent of the impact of covalent cross-linking or matrix mechanical plasticity on cell signaling pathways. These findings highlight the biophysical role of covalent cross-linking in regulating basement membrane plasticity, as well as cancer cell invasion of this confining tissue layer.