In native states, animal cells of many types are supported by a fibrous network that forms the main structural component of the ECM. Mechanical interactions between cells and the 3D ECM critically regulate cell function, including growth and migration. However, the physical mechanism that governs the cell interaction with fibrous 3D ECM is still not known. In this article, we present single-cell traction force measurements using breast tumor cells embedded within 3D collagen matrices. We recreate the breast tumor mechanical environment by controlling the microstructure and density of type I collagen matrices. Our results reveal a positive mechanical feedback loop: cells pulling on collagen locally align and stiffen the matrix, and stiffer matrices, in return, promote greater cell force generation and a stiffer cell body. Furthermore, cell force transmission distance increases with the degree of strain-induced fiber alignment and stiffening of the collagen matrices. These findings highlight the importance of the nonlinear elasticity of fibrous matrices in regulating cell-ECM interactions within a 3D context, and the cell force regulation principle that we uncover may contribute to the rapid mechanical tissue stiffening occurring in many diseases, including cancer and fibrosis.cell traction force | 3D cell traction force microscopy | fibrous nonlinear elasticity | cell-ECM interaction | collagen A nimal cells of most cell types, including breast tumor cells, are supported structurally by a fibrous ECM within a 3D context (1, 2). Cells adhere to the fibers via the linkages between integrin receptors on the membrane surface and the adhesion molecules within the ECM. To migrate, cells pull/push along the fibers or squeeze through the pore structure of the network (3). The tensional balance between the cell and the ECM critically regulates many physiological and pathological processes, including immune response, tissue formation, and tumor progression (4-7). In the breast tumor, stiffening of the mechanical environment disrupts force balance between epithelial cells and the ECM, promoting a malignant phenotype (5, 8). Tumors stiffen as cells deposit more collagen than they digest (9-11), increasingly express cross-linking enzymes (8, 12), and exert traction forces to reorganize the ECM (13).The main structural component of the ECM is a network of cross-linked protein fibers. The fiber network aligns, stiffens, and sometimes, undergoes permanent changes when subjected to strain (14, 15). These adaptive mechanical properties of the fiber network provide cells entry points to modify their local microenvironment (16-18) and as such, perform physiologically realistic functions (1,3,(19)(20)(21)(22)(23). It has been reported that the nonlinear elasticity of fibrous matrices enables cells to transmit forces over distances of hundreds of micrometers, facilitating long-range communication between individual cells (24-26) and between tumor spheroids (27). Recent work has shown that individual cells are capable of stiffening the...

Physical changes in skin are among the most visible signs of aging. We found that young dermal fibroblasts secrete high levels of extracellular matrix (ECM) constituents, including proteoglycans, glycoproteins and cartilage-linking proteins. The most abundantly secreted was HAPLN1, a hyaluronic and proteoglycan link protein. HAPLN1 was lost in aged fibroblasts, resulting in a more aligned ECM that promoted metastasis of melanoma cells. Reconstituting HAPLN1 inhibited metastasis in an aged microenvironment, in 3D skin reconstruction models, and in vivo. Intriguingly, aged fibroblast-derived matrices had the opposite effects on the migration of T-cells, inhibiting their motility. HAPLN1 treatment of aged fibroblasts restored motility of mononuclear immune cells, while impeding that of polymorphonuclear immune cells, which in turn affected Treg recruitment. These data suggest while age-related physical changes in the ECM can promote tumor cell motility, they may adversely impact the motility of some immune cells, resulting in an overall change in the immune microenvironment. Understanding the physical changes in aging skin may provide avenues for more effective therapy for older melanoma patients.

Contractile cells can reorganize fibrous extracellular matrices and form dense tracts of fibers between neighboring cells. These tracts guide the development of tubular tissue structures and provide paths for the invasion of cancer cells. Here, we studied the mechanisms of the mechanical plasticity of collagen tracts formed by contractile premalignant acinar cells and fibroblasts. Using fluorescence microscopy and second harmonic generation, we quantified the collagen densification, fiber alignment, and strains that remain within the tracts after cellular forces are abolished. We explained these observations using a theoretical fiber network model that accounts for the stretch-dependent formation of weak cross-links between nearby fibers. We tested the predictions of our model using shear rheology experiments. Both our model and rheological experiments demonstrated that increasing collagen concentration leads to substantial increases in plasticity. We also considered the effect of permanent elongation of fibers on network plasticity and derived a phase diagram that classifies the dominant mechanisms of plasticity based on the rate and magnitude of deformation and the mechanical properties of individual fibers. Plasticity is caused by the formation of new cross-links if moderate strains are applied at small rates or due to permanent fiber elongation if large strains are applied over short periods. Finally, we developed a coarse-grained model for plastic deformation of collagen networks that can be employed to simulate multicellular interactions in processes such as morphogenesis, cancer invasion, and fibrosis.

We describe a multiscale model that incorporates force-dependent mechanical plasticity induced by interfiber cross-link breakage and stiffness-dependent cellular contractility to predict focal adhesion (FA) growth and mechanosensing in fibrous extracellular matrices (ECMs). The model predicts that FA size depends on both the stiffness of ECM and the density of ligands available to form adhesions. Although these two quantities are independent in commonly used hydrogels, contractile cells break cross-links in soft fibrous matrices leading to recruitment of fibers, which increases the ligand density in the vicinity of cells. Consequently, although the size of focal adhesions increases with ECM stiffness in nonfibrous and elastic hydrogels, plasticity of fibrous networks leads to a departure from the well-described positive correlation between stiffness and FA size. We predict a phase diagram that describes nonmonotonic behavior of FA in the space spanned by ECM stiffness and recruitment index, which describes the ability of cells to break cross-links and recruit fibers. The predicted decrease in FA size with increasing ECM stiffness is in excellent agreement with recent observations of cell spreading on electrospun fiber networks with tunable cross-link strengths and mechanics. Our model provides a framework to analyze cell mechanosensing in nonlinear and inelastic ECMs.focal adhesion | mechanosensing | cell contractility | matrix physical remodeling | Rho pathway F ocal adhesions (FAs) are large macromolecular assemblies through which mechanical force and regulatory signals are transmitted between the extracellular matrix (ECM) and cells. FAs play important roles in many cellular behaviors, including proliferation, differentiation, and locomotion, and pathological processes like tumorigenesis and wound healing (1-4). For this reason, intense efforts have been devoted to understanding how key signaling molecules and ECM characteristics influence the formation and growth of FAs. In particular, in vitro studies using elastic hydrogels have shown that forces generated by actomyosin contraction are essential for the stabilization of FAs (5, 6). Numerous observations have convincingly demonstrated that cells form larger FAs as well as develop higher intracellular traction forces on stiffer ECMs (7,8), evidencing the mechanosensitive nature of FAs which has been quantitatively modeled using different (continuum, coarse-grain, and molecular) approaches (9, 10).It must be pointed out that in all of the aforementioned investigations, the substrates considered were flat (2D) and linear elastic. However, in vivo, many cells reside within 3D fibrous scaffolds where the density and diameter of fibers can vary depending on the nature of the tissue (11-13). The local architecture of these fibrous networks may change significantly when cells exert forces on them, leading to phenomena such as nonlinear stiffening, reorientation, and physical remodeling of the ECM (14, 15). Our recent study on cells in synthetic fibrous matrices wit...

While cells within tissues generate and sense 3D states of strain, the current understanding of the mechanics of fibrous extracellular matrices (ECMs) stems mainly from uniaxial, biaxial, and shear tests. Here, we demonstrate that the multiaxial deformations of fiber networks in 3D cannot be inferred solely based on these tests. The interdependence of the three principal strains gives rise to anomalous ratios of biaxial to uniaxial stiffness between 8 and 9 and apparent Poisson's ratios larger than 1. These observations are explained using a microstructural network model and a coarsegrained constitutive framework that predicts the network Poisson effect and stress-strain responses in uniaxial, biaxial, and triaxial modes of deformation as a function of the microstructural properties of the network, including fiber mechanics and pore size of the network. Using this theoretical approach, we found that accounting for the Poisson effect leads to a 100-fold increase in the perceived elastic stiffness of thin collagen samples in extension tests, reconciling the seemingly disparate measurements of the stiffness of collagen networks using different methods. We applied our framework to study the formation of fiber tracts induced by cellular forces. In vitro experiments with low-density networks showed that the anomalous Poisson effect facilitates higher densification of fibrous tracts, associated with the invasion of cancerous acinar cells. The approach developed here can be used to model the evolving mechanics of ECM during cancer invasion and fibrosis.fibrous matrices | matrix realignment | 3D cell traction force microscopy | tissue swelling T he elastic modulus, strain-stiffening, and mechanical relaxation timescale of the extracellular matrix (ECM) regulate cellular behaviors such as differentiation and spreading (1-3), as well as the susceptibility of cells to infection by bacterial pathogens (4), and the response of cells to drugs. Mechanical cues and chemical signals are among the key factors that influence the migration of cells. Many cell types migrate toward stiffer regions of a substrate (5, 6) and follow the direction of fiber alignment (7,8). The characterization and modeling of the mechanics of collagen matrices are important for understanding the interaction between invading cancer cells and the tumor microenvironment, and the relation between mechanosensing and matrix biosynthesis in fibrosis, as well as tuning the cellular microenvironment in tissue engineering applications. Most previous microstructural studies and constitutive models of ECM mechanics have focused on either the uniaxial, biaxial, or shear loading of the fibrous ECMs, whereas fiber networks physiologically experience combinations of these modes of mechanical loading. For instance, large Poisson effects are involved in fiber densification at collagen tracts, which promote the invasion of cancerous acinar cells (9). Moreover, factors such as overexpression of hyaluronic acid in cancerous tissues cause swelling in the ECMs of tumors (10, 11)...