Effect of matrix heterogeneity on cell mechanosensing.

Cells sense mechanical signals within the extracellular matrix, the most familiar being stiffness, but matrix stiffness cannot be simply described by a single value. Randomness in matrix structure causes stiffness at the scale of a cell to vary by more than an order of magnitude. Additionally, the extracellular matrix contains ducts, blood vessels, and, in cancer or fibrosis, regions with abnormally high stiffness. These different features could alter the stiffness sensed by a cell, but it is unclear whether the change in stiffness is large enough to overcome the noise caused by heterogeneity due to the random fibrous structure. Here we used a combination of experiments and modeling to determine the extent to which matrix heterogeneity disrupts the potential for cell sensing of a locally stiff feature in the matrix. Results showed that, at the scale of a single cell, spatial heterogeneity in local stiffness was larger than the increase in stiffness due to a stiff feature. The heterogeneity was reduced only for large length scales compared to the fiber length. Experiments verified this conclusion, showing spheroids of cells, which were large compared to the average fiber length, spreading preferentially toward stiff inclusions. Hence, the propagation of mechanical cues through the matrix depends on length scale, with single cells being able to sense only the stiffness of the nearby fibers and multicellular structures, such as tumors, also sensing the stiffness of distant matrix features.

Effect of hyaluronic acid on microscale deformations of collagen gels.

As fibrous collagen is the most abundant protein in mammalian tissues, gels of collagen fibers have been extensively used as an extracellular matrix scaffold to study how cells sense and respond to cues from their microenvironment. Other components of native tissues, such as glycosaminoglycans like hyaluronic acid, can affect cell behavior in part by changing the mechanical properties of the collagen gel. Prior studies have quantified the effects of hyaluronic acid on the mechanical properties of collagen gels in experiments of uniform shear or compression at the macroscale. However, there remains a lack of experimental studies of how hyaluronic acid changes the mechanical properties of collagen gels at the scale of a cell. Here, we studied how addition of hyaluronic acid to gels of collagen fibers affects the local field of displacements in response to contractile loads applied on length scales similar to those of a contracting cell. Using spherical poly(N-isopropylacrylamide) particles, which contract when heated, we induced displacement in gels of collagen and collagen with hyaluronic acid. Displacement fields were quantified using a combination of confocal microscopy and digital image correlation. Results showed that hyaluronic acid suppressed the distance over which displacements propagated, suggesting that it caused the network to become more linear. Additionally, hyaluronic acid had no statistical effect on heterogeneity of the displacement fields, but it did make the gels more elastic by substantially reducing the magnitude of permanent deformations. Lastly, we examined the effect of hyaluronic acid on fiber remodeling due to localized forces and found that hyaluronic acid partially - but not fully - inhibited remodeling. This result is consistent with prior studies suggesting that fiber remodeling is associated with a phase transition resulting from an instability caused by nonlinearity of the collagen gel.

Cells exploit a phase transition to mechanically remodel the fibrous extracellular matrix.

Through mechanical forces, biological cells remodel the surrounding collagen network, generating striking deformation patterns. Tethers-tracts of high densification and fibre alignment-form between cells, thinner bands emanate from cell clusters. While tethers facilitate cell migration and communication, how they form is unclear. Combining modelling, simulation and experiment, we show that tether formation is a densification phase transition of the extracellular matrix, caused by buckling instability of network fibres under cell-induced compression, featuring unexpected similarities with martensitic microstructures. Multiscale averaging yields a two-phase, bistable continuum energy landscape for fibrous collagen, with a densified/aligned second phase. Simulations predict strain discontinuities between the undensified and densified phase, which localizes within tethers as experimentally observed. In our experiments, active particles induce similar localized patterns as cells. This shows how cells exploit an instability to mechanically remodel the extracellular matrix simply by contracting, thereby facilitating mechanosensing, invasion and metastasis.

Directional cues in the tumor microenvironment due to cell contraction against aligned collagen fibers.

It is well established that collagen alignment in the breast tumor microenvironment provides biophysical cues to drive disease progression. Numerous mechanistic studies have demonstrated that tumor cell behavior is driven by the architecture and stiffness of the collagen matrix. However, the mechanical properties within a 3D collagen microenvironment, particularly at the scale of the cell, remain poorly defined. To investigate cell-scale mechanical cues with respect to local collagen architecture, we employed a combination of intravital imaging of the mammary tumor microenvironment and a 3D collagen gel system with both acellular pNIPAAm microspheres and MDA-MB-231 breast carcinoma cells. Within the in vivo tumor microenvironment, the displacement of collagen fiber was identified in response to tumor cells migrating through the stromal matrix. To further investigate cell-scale stiffness in aligned fiber architectures and the propagation of cell-induced fiber deformations, precise control of collagen architecture was coupled with innovative methodology to measure mechanical properties of the collagen fiber network. This method revealed up to a 35-fold difference in directional cell-scale stiffness resulting from contraction against aligned fibers. Furthermore, the local anisotropy of the matrix dramatically altered the rate at which contractility-induced fiber displacements decayed over distance. Together, our results reveal mechanical properties in aligned matrices that provide dramatically different cues to the cell in perpendicular directions. These findings are supported by the mechanosensing behavior of tumor cells and have important implications for cell-cell communication within the tissue microenvironment. STATEMENT OF SIGNIFICANCE: It is widely appreciated that the architecture of the extracellular matrix impacts cellular behavior in normal and disease states. Numerous studies have determined the fundamental role of collagen matrix architecture on cellular mechanosensing, but effectively quantifying anisotropic mechanical properties of the collagen matrix at the cell-scale remains challenging. Here, we developed innovative methodology to discover that collagen alignment results in a 35-fold difference in cell-scale stiffness and alters contractile force transmission through the fiber network. Furthermore, we identified bias in cell response along the axis of alignment, where local stiffness is highest. Overall, our results define cell-scale stiffness and fiber deformations due to collagen architecture that may instruct cell communication within a broad range of tissue microenvironments.

Heterogeneity and nonaffinity of cell-induced matrix displacements.

Cell contractile forces deform and reorganize the surrounding matrix, but the relationship between the forces and the resulting displacements is complicated by the fact that the fibrous structure brings about a complex set of mechanical properties. Many studies have quantified nonlinear and time-dependent properties at macroscopic scales, but it is unclear whether macroscopic properties apply to the scale of a cell, where the matrix is composed of a heterogeneous network of fibers. To address this question, we mimicked the contraction of a cell embedded within a fibrous collagen matrix and quantified the resulting displacements. The data revealed displacements that were heterogeneous and nonaffine. The heterogeneity was reproducible during cyclic loading, and it decreased with decreasing fiber length. Both the experiments and a fiber network model showed that the heterogeneous displacements decayed over distance at a rate no faster than the average displacement field, indicating no transition to homogeneous continuum behavior. Experiments with cells fully embedded in collagen matrices revealed the presence of heterogeneous displacements as well, exposing the dramatic heterogeneity in matrix reorganization that is induced by cells at different positions within the same fibrous matrix.