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.

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.

Nanoscale Tracking Combined with Cell-Scale Microrheology Reveals Stepwise Increases in Force Generated by Cancer Cell Protrusions.

In early breast cancer progression, cancer cells invade through a nanoporous basement membrane (BM) as a first key step toward metastasis. This invasion is thought to be mediated by a combination of proteases, which biochemically degrade BM matrix, and physical forces, which mechanically open up holes in the matrix. To date, techniques that quantify cellular forces of BM invasion in 3D culture have been unavailable. Here, we developed cellular-force measurements for breast cancer cell invasion in 3D culture that combine multiple-particle tracking of force-induced BM-matrix displacements at the nanoscale, and magnetic microrheometry of localized matrix mechanics. We find that cancer-cell protrusions exert forces from picoNewtons up to nanoNewtons during invasion. Strikingly, the protrusions extension involves stepwise increases in force, in steps of 0.2 to 0.5 nN exerted from every 30 s to 6 min. Thus, this technique reveals previously unreported dynamics of force generation by invasive protrusions in cancer cells.

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.

Effect of substrate stiffness on friction in collective cell migration.

In collective cell migration, the motion results from forces produced by each cell and transmitted to the neighboring cells and to the substrate. Because inertia is negligible and the migration occurs over long time scales, the cell layer exhibits viscous behavior, where force and motion are connected by an apparent friction that results from the breaking and forming of adhesive bonds at the cell-cell and cell-substrate interfaces. Most theoretical models for collective migration include an apparent friction to connect force and motion, with many models making predictions that depend on the ratio of cell-cell and cell-substrate friction. However, little is known about factors that affect friction, leaving predictions of many theoretical models untested. Here, we considered how substrate stiffness and the number of adhesions affected friction at the cell-substrate interface. The experimental data were interpreted through prior theoretical models, which led to the same conclusion, that increased substrate stiffness increased the number of cell-substrate adhesions and caused increased cell-substrate friction. In turn, the friction affected the collective migration by altering the curvature at the edge of the cell layer. By revealing underlying factors affecting friction and demonstrating how friction perturbs the collective migration, this work provides experimental evidence supporting prior theoretical models and motivates the study of other ways to alter the collective migration by changing friction.

Coordination of contractile tension and cell area changes in an epithelial cell monolayer.

During tissue development and repair, cells contract and expand in coordination with their neighbors, giving rise to tissue deformations that occur on length scales far larger than that of a single cell. The biophysical mechanisms by which the contractile forces of each cell cause deformations on multicellular length scales are not fully clear. To investigate this question, we began with the principle of force equilibrium, which dictates a balance of tensile forces between neighboring cells. Based on this principle, we hypothesized that coordinated changes in cell area result from tension transmitted across the cell layer. To test this hypothesis, spatial correlations of both contractile tension and the divergence of cell velocities were measured as readouts of coordinated contractility and collective area changes, respectively. Experiments were designed to alter the spatial correlation of contractile tension using three different methods, including disrupting cell-cell adhesions, modulating the alignment of actomyosin stress fibers between neighboring cells, and changing the size of the cell monolayer. In all experiments, the spatial correlations of both tension and divergence increased or decreased together, in agreement with our hypothesis. To relate our findings to the intracellular mechanism connecting changes in cell area to contractile tension, we disrupted activation of extracellular signal-regulated kinase (ERK), which is known to mediate the intracellular relationship between cell area and contraction. Consistent with prior knowledge, a temporal cross-correlation between cell area and tension revealed that ERK was responsible for a proportional relationship between cell area and contraction. Inhibition of ERK activation reduced the spatial correlations of the divergence of cell velocity but not of tension. Together, our findings suggest that coordination of cell contraction and expansion requires transfer of cell tension over space and ERK-mediated coordination between cell area and contraction in time.

Spatiotemporal force and motion in collective cell migration.

Cells move in collective groups in biological processes such as wound healing, morphogenesis, and cancer metastasis. How active cell forces produce the motion in collective cell migration is still unclear. Many theoretical models have been introduced to elucidate the relationship between the cell's active forces and different observations about the collective motion such as collective swirls, oscillations, and rearrangements. Though many models share the common feature of balancing forces in the cell layer, the specific relationships between force and motion vary among the different models, which can lead to different conclusions. Simultaneous experimental measurements of force and motion can aid in testing assumptions and predictions of the theoretical models. Here, we provide time-lapse images of cells in 1 mm circular islands, which are used to compute cell velocities, cell-substrate tractions, and monolayer stresses. Additional data are included from experiments that perturbed cell number density and actomyosin contractility. We expect this data set to be useful to researchers interested in force and motion in collective cell migration.