Breaching the basement membrane: who, when and how?

The basement membrane (BM), a specialized network of extracellular matrix macromolecules, surrounds epithelial, endothelial, muscle, fat and nerve cells. During development, immune surveillance and disease states ranging from cancer to fibrosis, host cells penetrate the BM by engaging tissue-invasive programs, the identity of which remain largely undefined. Although it is commonly assumed that all cells employ similar mechanisms to cross BM barriers, accumulating evidence indicates that cells might selectively mobilize protease-dependent or -independent invasion programs. New data indicate that protease-dependent transmigration is largely reliant on a group of membrane-anchored metalloenzymes, termed the membrane-type matrix metalloproteinases, which irreversibly remodel BM structure. By contrast, mechanisms that enable protease-independent transmigration remain undefined and potentially involve the reversible disassembly of the BM network. Further characterization of the molecular mechanisms underlying BM transmigration should provide important insights into pathophysiologic tissue remodeling events and also enable the development of novel therapeutics.     

The role of the cytoskeleton in cellular force generation in 2D and 3D environments

To adhere and migrate, cells generate forces through the cytoskeleton that are transmitted to the surrounding matrix. While cellular force generation has been studied on 2D substrates, less is known about cytoskeletal-mediated traction forces of cells embedded in more in vivo-like 3D matrices. Recent studies have revealed important differences between the cytoskeletal structure, adhesion, and migration of cells in 2D and 3D. Because the cytoskeleton mediates force, we sought to directly compare the role of the cytoskeleton in modulating cell force in 2D and 3D. MDA-MB-231 cells were treated with agents that perturbed actin, microtubules, or myosin, and analyzed for changes in cytoskeletal organization and force generation in both 2D and 3D. To quantify traction stresses in 2D, traction force microscopy was used; in 3D, force was assessed based on single cell-mediated collagen fibril reorganization imaged using confocal reflectance microscopy. Interestingly, even though previous studies have observed differences in cell behaviors like migration in 2D and 3D, our data indicate that forces generated on 2D substrates correlate with forces within 3D matrices. Disruption of actin, myosin or microtubules in either 2D or 3D microenvironments disrupts cell-generated force. These data suggest that despite differences in cytoskeletal organization in 2D and 3D, actin, microtubules and myosin contribute to contractility and matrix reorganization similarly in both microenvironments.     

Epidermal growth factor-induced enhancement of glioblastoma cell migration in 3D arises from an intrinsic increase in speed but an extrinsic matrix- and proteolysis-dependent increase in persistence

Epidermal growth factor (EGF) receptor-mediated cell migration plays a vital role in invasion of many tumor types. EGF receptor ligands increase invasiveness in vivo, but it remains unclear how consequent effects on intrinsic cell motility behavior versus effects on extrinsic matrix properties integrate to result in net increase of translational speed and/or directional persistence of migration in a 3D environment. Understanding this convolution is important for therapeutic targeting of tumor invasion, as key regulatory pathways for intrinsic versus extrinsic effects may not be coincident. Accordingly, we have undertaken a quantitative single-cell imaging study of glioblastoma cell movement in 3D matrices and on 2D substrata across a range of collagen densities with systematic variation of protease-mediated matrix degradation. In 3D, EGF induced a mild increase in cell speed and a strong increase in directional persistence, the latter depending heavily on matrix density and EGF-stimulated protease activity. In contrast, in 2D, EGF induced a similarly mild increase in speed but conversely a decrease in directional persistence (both independent of protease activity). Thus, the EGF-enhanced 3D tumor cell migration results only partially from cell-intrinsic effects, with override of cell-intrinsic persistence decrease by protease-mediated cell-extrinsic reduction of matrix steric hindrance.     

Hyaluronan matrices in pathobiological processes

Hyaluronan matrices are ubiquitous in normal and pathological biological processes. This remarkable diversity is related to their unique mechanism of synthesis by hyaluronan synthases. These enzymes are normally activated in the plasma membrane and utilize cytosolic substrates directly to form these large polyanionic glycosaminoglycans, which are extruded directly into the extracellular space. The extracellular matrices that are formed interact with cell surface receptors, notably CD44, that often dictate the biological processes, as described in the accompanying minireviews of this series. This article focuses on the discovery in recent studies that many cell stress responses initiate the synthesis of a monocyte-adhesive hyaluronan extracellular matrix, which forms a central focus for subsequent inflammatory processes that are modulated by the dialogue between the matrix and the inflammatory cells. The mechanisms involve active hyaluronan synthases at the cell membrane when cell stresses occur at physiological levels of glucose. However, dividing cells at hyperglycemic levels of glucose initiate the synthesis of hyaluronan in intracellular compartments, which induces endoplasmic reticulum stress and autophagy, processes that probably contribute greatly to diabetic pathologies.     

Contact guidance mediated three-dimensional cell migration is regulated by Rho/ROCK-dependent matrix reorganization

Cells generate mechanical force to organize the extracellular matrix (ECM) and drive important developmental and reparative processes. Likewise, tumor cells invading into three-dimensional (3D) matrices remodel the ECM microenvironment. Importantly, we previously reported a distinct radial reorganization of the collagen matrix surrounding tumors that facilitates local invasion. Here we describe a mechanism by which cells utilize contractility events to reorganize the ECM to provide contact guidance that facilitates 3D migration. Using novel assays to differentially organize the collagen matrix we show that alignment of collagen perpendicular to the tumor-explant boundary promotes local invasion of both human and mouse mammary epithelial cells. In contrast, organizing the collagen matrix to mimic the ECM organization associated with noninvading regions of tumors suppresses 3D migration/invasion. Moreover, we demonstrate that matrix reorganization is contractility-dependent and that the Rho/Rho kinase pathway is necessary for collagen alignment to provide contact guidance. Yet, if matrices are prealigned, inhibiting neither Rho nor Rho kinase inhibits 3D migration, which supports our conclusion that Rho-mediated matrix alignment is an early step in the invasion process, preceding and subsequently facilitating 3D migration.     

Contractile forces regulate cell division in three-dimensional environments

Physical forces direct the orientation of the cell division axis for cells cultured on rigid, two-dimensional (2D) substrates. The extent to which physical forces regulate cell division in three-dimensional (3D) environments is not known. Here, we combine live-cell imaging with digital volume correlation to map 3D matrix displacements and identify sites at which cells apply contractile force to the matrix as they divide. Dividing cells embedded in fibrous matrices remained anchored to the matrix by long, thin protrusions. During cell rounding, the cells released adhesive contacts near the cell body while applying tensile forces at the tips of the protrusions to direct the orientation of the cell division axis. After cytokinesis, the daughter cells respread into matrix voids and invaded the matrix while maintaining traction forces at the tips of persistent and newly formed protrusions. Mechanical interactions between cells and the extracellular matrix constitute an important mechanism for regulation of cell division in 3D environments.     

Cell jamming: Collective invasion of mesenchymal tumor cells imposed by tissue confinement

Background

Cancer invasion is a multi-step process which coordinates interactions between tumor cells with mechanotransduction towards the surrounding matrix, resulting in distinct cancer invasion strategies. Defined by context, mesenchymal tumors, including melanoma and fibrosarcoma, develop either single-cell or collective invasion modes, however, the mechanical and molecular programs underlying such plasticity of mesenchymal invasion programs remain unclear.

Methods

To test how tissue anatomy determines invasion mode, spheroids of MV3 melanoma and HT1080 fibrosarcoma cells were embedded into 3D collagen matrices of varying density and stiffness and analyzed for migration type and efficacy with matrix metalloproteinase (MMP)-dependent collagen degradation enabled or pharmacologically inhibited.

Results

With increasing collagen density and dependent on proteolytic collagen breakdown and track clearance, but independent of matrix stiffness, cells switched from single-cell to collective invasion modes. Conversion to collective invasion included gain of cell-to-cell junctions, supracellular polarization and joint guidance along migration tracks.

Conclusions

The density of the extracellulair matrix (ECM) determines the invasion mode of mesenchymal tumor cells. Whereas fibrillar, high porosity ECM enables single-cell dissemination, dense matrix induces cell–cell interaction, leader–follower cell behavior and collective migration as an obligate protease-dependent process.

General significance

These findings establish plasticity of cancer invasion programs in response to ECM porosity and confinement, thereby recapitulating invasion patterns of mesenchymal tumors in vivo. The conversion to collective invasion with increasing ECM confinement supports the concept of cell jamming as a guiding principle for melanoma and fibrosarcoma cells into dense tissue.This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

The effect of the extracellular matrix on the detachment of human endothelial cells

Human umbilical vein endothelial cells can be serially passaged by supplementing medium with a partially purified growth factor. Cell-substratum detachment of early and late passage endothelial cells was examined using trypsin, collagenase, or homocysteine. Late-passage cells detached more rapidly than early passage cells under all conditions tested. The rate of detachment was dependent upon the specific agent used. Protease-mediated detachment was most rapid, occurring over minutes, in contrast to homocysteine-induced detachment, which occurred over hours. When detached cells were collected and replated in the absence of the detaching agent, these cells reattached, spread, and continued to proliferate. No significant difference was observed in the rate of adhesion of either early or late passage cells to a gelatin matrix. When early or late-passage endothelial cells were plated and grown to confluence on a matrix synthesized by the opposite cell type, the rate of protease-mediated cell detachment resembled the cell type from which the matrix was derived. The ease of endothelial cell detachment was determined by the origin of the extracellular matrix. Examination of the extracellular matrices from early and late passage cells revealed significant differences in the amounts of glycosaminoglycans and sulfated proteins present. These studies demonstrate the importance of the endothelial cell extracellular matrix in protease-mediated cell detachment. The rate of cell detachment was controlled by the extracellular matrices are not altered by the endothelial cells.     

Traction Stresses and Translational Distortion of the Nucleus During Fibroblast Migration on a Physiologically Relevant ECM Mimic

Cellular traction forces, resulting in cell-substrate physical interactions, are generated by actin-myosin complexes and transmitted to the extracellular matrix through focal adhesions. These processes are highly dynamic under physiological conditions and modulate cell migration. To better understand the precise dynamics of cell migration, we measured the spatiotemporal redistribution of cellular traction stresses (force per area) during fibroblast migration at a submicron level and correlated it with nuclear translocation, an indicator of cell migration, on a physiologically relevant extracellular matrix mimic. We found that nuclear translocation occurred in pulses whose magnitude was larger on the low ligand density surfaces than on the high ligand density surfaces. Large nuclear translocations only occurred on low ligand density surfaces when the rear traction stresses completely relocated to a posterior nuclear location, whereas such relocation took much longer time on high ligand density surfaces, probably due to the greater magnitude of traction stresses. Nuclear distortion was also observed as the traction stresses redistributed. Our results suggest that the reinforcement of the traction stresses around the nucleus as well as the relaxation of nuclear deformation are critical steps during fibroblast migration, serving as a speed regulator, which must be considered in any dynamic molecular reconstruction model of tissue cell migration. A traction gradient foreshortening model was proposed to explain how the relocation of rear traction stresses leads to pulsed fibroblast migration.     

Cell Traction Forces Direct Fibronectin Matrix Assembly

Interactions between cells and the surrounding matrix are critical to the development and engineering of tissues. We have investigated the role of cell-derived traction forces in the assembly of extracellular matrix using what we believe is a novel assay that allows for simultaneous measurement of traction forces and fibronectin fibril growth at discrete cell-matrix attachment sites. NIH3T3 cells were plated onto arrays of deformable cantilever posts for 2-24 h. Data indicate that developing fibril orientation is guided by the direction of the traction force applied to that fibril. In addition, cells initially establish a spatial distribution of traction forces that is largest at the cell edge and decreases toward the cell center. This distribution progressively shifts from a predominantly peripheral pattern to a more uniform pattern as compressive strain at the cell perimeter decreases with time. The impact of these changes on fibrillogenesis was tested by treating cells with blebbistatin or calyculin A to tonically block or augment, respectively, myosin II activity. Both treatments blocked the inward translation of traction forces, the dissipation of compressive strain, and fibronectin fibrillogenesis over time. These data indicate that dynamic spatial and temporal changes in traction force and local strain may contribute to successful matrix assembly.     

3D Traction forces in cancer cell invasion

Cell invasion through a dense three-dimensional (3D) matrix is believed to depend on the ability of cells to generate traction forces. To quantify the role of cell tractions during invasion in 3D, we present a technique to measure the elastic strain energy stored in the matrix due to traction-induced deformations. The matrix deformations around a cell were measured by tracking the 3D positions of fluorescent beads tightly embedded in the matrix. The bead positions served as nodes for a finite element tessellation. From the strain in each element and the known matrix elasticity, we computed the local strain energy in the matrix surrounding the cell. We applied the technique to measure the strain energy of highly invasive MDA-MB-231 breast carcinoma and A-125 lung carcinoma cells in collagen gels. The results were compared to the strain energy generated by non-invasive MCF-7 breast and A-549 lung carcinoma cells. In all cases, cells locally contracted the matrix. Invasive breast and lung carcinoma cells showed a significantly higher contractility compared to non-invasive cells. Higher contractility, however, was not universally associated with higher invasiveness. For instance, non-invasive A-431 vulva carcinoma cells were the most contractile cells among all cell lines tested. As a universal feature, however, we found that invasive cells assumed an elongated spindle-like morphology as opposed to a more spherical shape of non-invasive cells. Accordingly, the distribution of strain energy density around invasive cells followed patterns of increased complexity and anisotropy. These results suggest that not so much the magnitude of traction generation but their directionality is important for cancer cell invasion.     

Cell-cell mechanical communication through compliant substrates

The role of matrix mechanics on cell behavior is under intense investigation. Cells exert contractile forces on their matrix and the matrix elasticity can alter these forces and cell migratory behavior. However, little is known about the contribution of matrix mechanics and cell-generated forces to stable cell-cell contact and tissue formation. Using matrices of varying stiffness and measurements of endothelial cell migration and traction stresses, we find that cells can detect and respond to substrate strains created by the traction stresses of a neighboring cell, and that this response is dependent on matrix stiffness. Specifically, pairs of endothelial cells display hindered migration on gels with elasticity below 5500 Pa in comparison to individual cells, suggesting these cells sense each other through the matrix. We believe that these results show for the first time that matrix mechanics can foster tissue formation by altering the relative motion between cells, promoting the formation of cell-cell contacts. Moreover, our data indicate that cells have the ability to communicate mechanically through their matrix. These findings are critical for the understanding of cell-cell adhesion during tissue formation and disease progression, and for the design of biomaterials intended to support both cell-matrix and cell-cell adhesion.     

Effects of extracellular fiber architecture on cell membrane shear stress in a 3D fibrous matrix

Interstitial fluid flow has been shown to affect the organization and behavior of cells in 3D environments in vivo and in vitro, yet the forces driving such responses are not clear. Due to the complex architecture of the extracellular matrix (ECM) and the difficulty of measuring fluid flow near cells embedded in it, the levels of shear stress experienced by cells in this environment are typically estimated using bulk-averaged matrix parameters such as hydraulic permeability. While this is useful for estimating average stresses, it cannot yield insight into how local matrix fiber architecture-which is cell-controlled in the immediate pericellular environment-affects the local stresses imposed on the cell surface. To address this, we used computational fluid dynamics to study flow through an idealized mesh constructed of a cubic lattice of fibers simulating a typical in vitro collagen gel. We found that, in such high porosity matrices, the fibers strongly affect the flow fields near the cell, with peak shear stresses up to five times higher than those predicted by the Brinkman equation. We also found that minor remodeling of the fibers near the cell surface had major effects on the shear stress profile on the cell. These findings demonstrate the importance of fiber architecture to the fluid forces on a cell embedded in a 3D matrix, and also show how small modifications in the local ECM can lead to large changes in the mechanical environment of the cell.     

Passive and active contributions to generated force and retraction in heart valve tissue engineering

In tissue engineered heart valves, cell-mediated stress development during culture results in leaflet retraction at time of implantation. This tissue retraction is partly active due to traction forces exerted by the cells and partly passive due to release of residual stress in the extracellular matrix and the cells. Within this study, we unraveled the passive and active contributions of cells and matrix to generated force and retraction in engineered heart valve tissues. Tissue engineered rectangular strips, fabricated from PGA/P4HB scaffolds and seeded with human myofibroblasts, were cultured for 4 weeks, after which the cellular contribution was changed at different levels. Elimination of the active cellular traction forces was achieved with Cytochalasin D and inhibition of the Rho-associated kinase pathway. Both active and passive cellular contributions were eliminated by lysation and/or decellularization of the tissue. Maximum cell activity was reached by increasing the fetal bovine serum concentration to 50%. The generated force decreased ~20% after elimination of the active cellular component, ~25% when the passive cellular component was removed as well and remained unaffected by increased serum concentrations. Passive retraction accounted for ~60% of total retraction, of which ~15% was residual stress in the matrix and ~45% was passive cell retraction. Cell traction forces accounted for the remainder ~40% of the retraction. Full activation of the cells increased retraction by ~45%. These results illustrate the importance of the cells in the process of tissue retraction, not only actively retracting the tissue, but also in a passive manner to a large extent.     

Integrins and the activation of latent transforming growth factor beta1 - an intimate relationship

Integrins are crucial for the ability of cells to sense mechanical perturbations and to transmit intracellular stress to their environment. We here review the more recently discovered role of integrins in activating the pleiotrophic cytokine transforming growth factor beta 1 (TGF-beta1). TGF-beta1 controls tissue homeostasis in embryonic and normal adult tissues and contributes to the development of fibrosis, cancer, autoimmune and vascular diseases when being mis-regulated. In most of these conditions, active TGF-beta1 is generated by dissociation from a large latent protein complex that sequesters latent TGF-beta1 in the extracellular matrix (ECM). Two main models are proposed how integrins contribute to latent TGF-beta1 activation: (1) In a protease-dependent mechanism, integrins alphavbeta8 and alphavbeta3 are suggested to simultaneously bind the latent TGF-beta1 complex and proteinases. This close vicinity at the cell surface improves enzymatic cleavage of the latent complex to release active TGF-beta1. (2) Integrins alphavbeta3, alphavbeta5, alphavbeta6, and alphavbeta8 appear to change the conformation of the latent TGF-beta1 complex by transmitting cell traction forces. This action requires association of the latent complex with a mechanically resistant ECM and is independent from proteolysis. Understanding that different integrins use different mechanisms to activate latent TGF-beta1 opens new possibilities to develop cell-specific therapeutic strategies for TGF-beta1-induced pathologies.     

Microtubules regulate GEF-H1 in response to extracellular matrix stiffness

Breast epithelial cells sense the stiffness of the extracellular matrix through Rho-mediated contractility. In turn, matrix stiffness regulates RhoA activity. However, the upstream signaling mechanisms are poorly defined. Here we demonstrate that the Rho exchange factor GEF-H1 mediates RhoA activation in response to extracellular matrix stiffness. We demonstrate the novel finding that microtubule stability is diminished by a stiff three-dimensional (3D) extracellular matrix, which leads to the activation of GEF-H1. Surprisingly, activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway did not contribute to stiffness-induced GEF-H1 activation. Loss of GEF-H1 decreases cell contraction of and invasion through 3D matrices. These data support a model in which matrix stiffness regulates RhoA through microtubule destabilization and the subsequent release and activation of GEF-H1.     

Two-photon laser-generated microtracks in 3D collagen lattices: principles of MMP-dependent and -independent collective cancer cell invasion

Cancer invasion into an extracellular matrix (ECM) results from a biophysical reciprocal interplay between the expanding cancer lesion and tissue barriers imposed by the adjacent microenvironment. In vivo, connective tissue provides both densely packed ECM barriers adjacent to channel/track-like spaces and loosely organized zones, both of which may impact cancer invasion mode and efficiency; however little is known about how three-dimensional (3D) spaces and aligned tracks present in interstitial tissue guide cell invasion. We here describe a two-photon laser ablation procedure to generate 3D microtracks in dense 3D collagen matrices that support and guide collective cancer cell invasion. Whereas collective invasion of mammary tumor (MMT) breast cancer cells into randomly organized collagen networks required matrix metalloproteinase (MMP) activity for cell-derived collagen breakdown, re-alignment and track generation, preformed tracks supported MMP-independent collective invasion down to a track caliber of 3 μm. Besides contact guidance along the track of least resistance and initial cell deformation (squeezing), MMP-independent collective cell strands led to secondary track expansion by a pushing mechanism. Thus, two-photon laser ablation is useful to generate barrier-free microtracks in a 3D ECM which guide collective invasion independently of pericellular proteolysis.     

Rab25 associates with alpha5beta1 integrin to promote invasive migration in 3D microenvironments

Here, we report a direct interaction between the beta1 integrin cytoplasmic tail and Rab25, a GTPase that has been linked to tumor aggressiveness and metastasis. Rab25 promotes a mode of migration on 3D matrices that is characterized by the extension of long pseudopodia, and the association of the GTPase with alpha5beta1 promotes localization of vesicles that deliver integrin to the plasma membrane at pseudopodial tips as well as the retention of a pool of cycling alpha5beta1 at the cell front. Furthermore, Rab25-driven tumor-cell invasion into a 3D extracellular matrix environment is strongly dependent on ligation of fibronectin by alpha5beta1 integrin and the capacity of Rab25 to interact with beta1 integrin. These data indicate that Rab25 contributes to tumor progression by directing the localization of integrin-recycling vesicles and thereby enhancing the ability of tumor cells to invade the extracellular matrix.     

Computational model for cell migration in three-dimensional matrices

Although computational models for cell migration on two-dimensional (2D) substrata have described how various molecular and cellular properties and physiochemical processes are integrated to accomplish cell locomotion, the same issues, along with certain new ones, might contribute differently to a model for migration within three-dimensional (3D) matrices. To address this more complicated situation, we have developed a computational model for cell migration in 3D matrices using a force-based dynamics approach. This model determines an overall locomotion velocity vector, comprising speed and direction, for individual cells based on internally generated forces transmitted into external traction forces and considering a timescale during which multiple attachment and detachment events are integrated. Key parameters characterize cell and matrix properties, including cell/matrix adhesion and mechanical and steric properties of the matrix; critical underlying molecular properties are incorporated explicitly or implicitly. Model predictions agree well with experimental results for the limiting case of migration on 2D substrata as well as with recent experiments in 3D natural tissues and synthetic gels. Certain predicted features such as biphasic behavior of speed with density of matrix ligands for 3D migration are qualitatively similar to their 2D counterparts, but new effects generally absent in 2D systems, such as effects due to matrix sterics and mechanics, are now predicted to arise in many 3D situations. As one particular sample manifestation of these effects, the optimal levels of cell receptor expression and matrix ligand density yielding maximal migration are dependent on matrix mechanical compliance.     

Toward single cell traction microscopy within 3D collagen matrices

Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels.