MINI-REVIEW

Journal of

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CellMatrix Adhesion
ALLISON L. BERRIER1,2*
1

Cellular Physiology

AND

KENNETH M. YAMADA2*

Katrina Visiting Faculty Program, National Center on Minority Health and Health Disparities, Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research,

National Institutes of Health, Bethesda, Maryland

2

National Institutes of Health, Bethesda, Maryland

The complex interactions of cells with extracellular matrix (ECM) play crucial roles in mediating and regulating many processes, including cell adhesion, migration, and signaling during morphogenesis, tissue homeostasis, wound healing, and tumorigenesis. Many of these interactions involve transmembrane integrin receptors. Integrins cluster in specic cellmatrix adhesions to provide dynamic links between extracellular and intracellular environments by bi-directional signaling and by organizing the ECM and intracellular cytoskeletal and signaling molecules. This mini review discusses these interconnections, including the roles of matrix properties such as composition, three-dimensionality, and porosity, the bi-directional functions of cellular contractility and matrix rigidity, and cell signaling. The review concludes by speculating on the application of this knowledge of cellmatrix interactions in the formation of cell adhesions, assembly of matrix, migration, and tumorigenesis to potential future therapeutic approaches. J. Cell. Physiol. 213: 565573, 2007. 2007 Wiley-Liss, Inc.

The extracellular matrix (ECM) provides scaffolds for cellular support that are present in all tissues and organs. The ECM is a complex mixture of matrix molecules, including the glycoproteins bronectin, collagens, laminins, proteoglycans, and non-matrix proteins including growth factors. Cell adhesion to the ECM induces discrete cell surface structures tightly associated with the matrix termed cellmatrix adhesions, which mediate direct interactions of the cell with its extracellular environment. Cellmatrix adhesions are essential for cell migration, tissue organization, and differentiation, and as a result they play central roles in embryonic development, remodeling, and homeostasis of tissue and organ systems. Matrix adhesion signals cooperate with other pathways to regulate biological processes such as cell survival, cell proliferation, wound healing, and tumorigenesis. Thus, elucidating the structure and function of cellmatrix adhesions provides a critical vantage point for understanding the regulation of eukaryotic cellular phenotypes in vivo. For recent reviews see references Miranti and Brugge (2002); Danen and Sonnenberg (2003); Guo and Giancotti (2004); Wozniak et al. (2004); Ginsberg et al. (2005); Li et al. (2005); Mitra et al. (2005); Vicente-Manzanares et al. (2005); Janes and Watt (2006); Luo et al. (2007). Due to constraints on the length of the article, we apologize for not being able to cite all relevant references. Integrins are the principle cell surface adhesion receptors mediating cellmatrix adhesion. Integrins are heterodimeric receptors generated by selective pairing between 18 a and 8 b subunits; there are 24 distinct integrin receptors that bind various ECM ligands with different afnities (Luo et al., 2007). Some integrin subunits are ubiquitously expressed, while other subunits are expressed in a tissue- or stage-restricted manner (Humphries et al., 2006). For instance, integrin b1 is ubiquitously expressed, whereas the b6 subunit is only expressed in the adult during wound healing. The extracellular domains of integrin receptors bind ECM ligands and divalent cations, but they can also associate laterally with other proteins at the cell surface, such as tetraspanins, growth factor receptors, matricellular proteins, and matrix proteases or their receptors (Miranti and Brugge, 2002). Integrins inuence cell behavior not only by providing a docking site for the ECM at the cell surface, but also by actions of the integrin intracellular domains. Integrin intracellular domains are short regions of roughly 50 amino acids in length, except for integrin b4 (1,000 amino acids). Integrin cytoplasmic domains
2 0 0 7 W I L E Y - L I S S , I N C .

form multi-molecular complexes with proteins involved in cell signaling and with adaptor proteins that provide a connection to the cytoskeleton (Hynes, 2002). Integrins provide a bi-directional conduit for mechanochemical information across the cell membrane, providing a major mechanism for connecting the intracellular and extracellular compartments. Cell adhesion to the ECM transmits information via integrin receptors that regulates intracellular signaling via outside-in signaling, which is important, for example, in cell spreading and cell migration. Conversely, intracellular signals can induce changes in integrin conformation and activation that alter its ligand-binding activity in a process termed inside-out signaling. Integrin engagement with matrix can also affect integrin activation, providing bidirectional crosstalk between inside-out and outside-in signaling (Ginsberg et al., 2005; Luo et al., 2007). Integrin clustering follows the engagement of integrins with the naturally multivalent ECM, and it promotes the localized intracellular concentration of signaling molecules. Clustering of integrin receptors, or particularly integrin b cytoplasmic domains, activates non-receptor tyrosine kinases such as focal adhesion kinase (FAK) leading to localized increases in the levels of tyrosine-phosphorylated proteins. Serine/threonine kinases including members of the protein kinase C family, lipid kinases such as PI 3-kinase, and phosphatases such as RPTPa are also regulated by integrin engagement and clustering. These kinase

Contract grant sponsor: Intramural Research Program, NIH. Contract grant sponsor: National Institute of Dental and Craniofacial Research. Contract grant sponsor: National Center on Minority Health and Health Disparities. *Correspondence to: Allison L. Berrier or Kenneth M. Yamada, Building 30, Room 426, 30 Convent Drive, MSC 4370, Bethesda, MD 20892-4370. E-mail: berriera@mail.nih.gov; Kenneth.Yamada@nih.gov Received 28 June 2007; Accepted 29 June 2007 DOI: 10.1002/jcp.21237

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and phosphatase signaling pathways induce post-translational modications regulating the protein interactions and/or enzymatic activity of the substrates (Li et al., 2005). They specify protein recruitment to adhesion complexes and thereby selectively link matrix adhesions to various downstream signaling cascades that control cytoskeletal organization, gene regulation, and diverse cellular processes and functions (Fig. 1) (Hervy et al., 2006). More than 50 cytoplasmic proteins are present in cellmatrix adhesion structures (Lo, 2006). Because integrin receptors lack intrinsic enzymatic activity, they must recruit signaling proteins to control adhesion-dependent processes (Liu et al., 2000; Mitra et al., 2005). Three basic categories of proteins are recruited to cellmatrix adhesions: (1) integrin-binding proteins, (2) adaptors and/or scaffolding proteins that lack intrinsic enzymatic activity, and (3) enzymes. Talin is an example of a protein that directly binds to integrin cytoplasmic domains and is important for regulating integrin activation and signaling (Calderwood, 2004). Adaptors and/or scaffolding factors link integrin-associated proteins with actin or other proteins, and examples include vinculin, paxillin, and a-actinin. Enzymes that modify integrin downstream effectors include the nonreceptor tyrosine kinases FAK and Src. The proles of proteins recruited to matrix adhesions specify the biochemical signals and biophysical properties of matrix adhesions (Li et al., 2005). The cytoskeleton contains three general classes of lamentous structure, F-actin, intermediate laments, and microtubules. It has become clear that cell migration and tissue remodeling require coordinated crosstalk between the actin, intermediate lament, and microtubule cytoskeletal networks (e.g., see reference Even-Ram et al. (2007)). Actin polymerization and proteins important for regulating actin organization are essential regulators of membrane protrusion and cell migration. Rearrangements of the actin cytoskeleton are mediated by complex molecular pathways that promote actin polymerization, actin depolymerization to renew the intracellular pool of monomeric actin, and modications of actin-crosslinking proteins (Vicente-Manzanares et al., 2005;

Pollard, 2007). Matrix adhesions associate with bundles of actin laments, and bi-directional interactions mediated by the actin cytoskeleton involving actomyosin contraction and clustering of integrins bound to matrix combine to increase cell contractility, also referred to as endogenous tension. The dynamic assembly and disassembly of adhesion structures applies different levels of force to the matrix that, in turn, regulates endogenous tension (Wozniak et al., 2004). Conversely, endogenous tension is transmitted through integrins to the ECM and can increase matrix rigidity, referred to as exogenous tension. Adhesion structures recruit cytoplasmic proteins that induce downstream effectors involved in regulating matrix deposition or remodeling. Thus, integrin engagement with the ECM generates bi-directional signals that can alter endogenous tension, exogenous tension, and matrix composition (Fig. 2) (Katsumi et al., 2004; Peterson et al., 2004; Wang, 2007). Directional cell migration requires the establishment of cell polarity to create a leading edge and a trailing edge (Moissoglu and Schwartz, 2006). The leading edge undergoes membrane protrusive activities driven by actin polymerization that establish new matrix contacts, whereas at the trailing edge cell adhesions are disassembled to promote retraction of the cell rear and forward cell movement (Vicente-Manzanares et al., 2005). Local actin polymerization can induce membrane protrusions and favor formation of matrix contact by localizing integrin receptors in an active conformation at cell protrusions (Galbraith et al., 2007). The rate of cell migration can be limited by the rate of rear retraction, and thus the dynamic formation and disassembly of cellmatrix adhesions are critical to cell migration (Ridley et al., 2003). The Rho GTPase family of GTP-binding proteins including RhoA, Rac1, and Cdc42 are critical regulators of cell contractility, lamellipodial and lopodial formation, and cellular polarity. RhoA GTPase downstream signals such as activation of RhoA-kinase (ROCK) and inhibition of myosin phosphatase increase myosin light chain phosphorylation, leading to clustering of actin stress bers to regulate actomyosin

Fig. 1. General model of cellmatrix adhesions and their downstream regulation. Cell-extracellular matrix adhesions containing clusters of integrins recruit cytoplasmic proteins, which in cooperation with other cell surface receptors control diverse cellular processes, functions, and phenotypes.

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Fig. 2. Coordinate regulation of exogenous tension (matrix rigidity) and endogenous tension (contractility). A: Cells and matrix mutually interact to regulate tension. B: Tension in the cell microenvironment is thought to be distributed by integrin receptors that signal bi-directionally between extracellular and intracellular compartments. Tension levels may alter outside-in and inside-out integrin signaling. Integrin engagement with extracellular matrix (ECM) regulates endogenous cellular tension by triggering actin cytoskeletal organization and actomyosin contractility. Endogenous tension levels can indirectly or directly control exogenous tension (matrix rigidity) as indicated in the gure.

contractility and endogenous tension. Rho downstream signals regulate membrane retraction, thereby signicantly contributing to leading and trailing edge cell polarity in cell migration. The balance of Rac and Rho activation coordinates membrane protrusion, retraction, and numbers of protrusions during cell migration. Sites of high levels of active Rac1 will suppress RhoA and induce lamellipodia; in contrast, regions containing concentrated active RhoA will have low Rac1 activity and membrane retraction (Clark et al., 1998; Nimnual et al., 2003; Burridge and Wennerberg, 2004). Furthermore, integrin engagement with matrix regulates the activity of Rho GTPase family members and localization with downstream effectors. Thus, matrix adhesions establish feedback loops that control membrane protrusion and retraction during cell migration by coordinating and integrating the activities of individual Rho GTPase family members in the leading and trailing edges of the cell (Ridley et al., 2003). The remainder of our review will focus on the instructive role of cellmatrix adhesions on both cell phenotype and extracellular environment. First, we compare how cell morphology, cell migration, and cellmatrix adhesion structures respond to two-dimensional (2D) compared to three-dimensional (3D) matrices. Changes in cellmatrix adhesions associated with cancer are described. We then examine how alterations of the extracellular environment inuence the intracellular environment and vice versa. Finally, we speculate about ways in which our rapidly expanding knowledge about cellmatrix adhesion might be exploited for therapeutic purposes.
Matrix Dimensionality and Cell Behavior

have identied complex molecular and biochemical pathways activated or modied by integrin-mediated adhesion and have provided insights into mechanisms that regulate adhesiondependent cellular processes such as cell spreading, cell proliferation, cell differentiation, and cell survival. Fibroblasts cultured in 2D matrices interact with a rigid substratum at the ventral surface of the cell. The binding of integrin ligands and the differences in exogenous tension (matrix rigidity) between the dorsal and ventral cell surfaces can selectively trigger focal adhesion formation at the ventral surface, thereby inducing cell polarity (Giannone and Sheetz, 2006). Fibroblasts in 3D matrices are typically not exposed to these large differences in exogenous tension, and hence lack dorsalventral polarity. However, observations in 2D cultures suggest that regional variations in 3D-matrix exogenous tension (rigidity) may inuence the distribution of cellmatrix adhesions and cell behavior in vivo (Ingber, 2006). Intriguing new insights into the effects of 3D matrix on cell behavior elucidate the synergistic relationship of cell and ECM in vivo and the dynamic function of cellmatrix adhesions in a 3D environment.
Cell morphology

Eukaryotic cells adapted to grow in vitro are routinely cultured on a 2D substratum. Many studies have characterized cellular responses to growth on a 2D ECM-coated substratum. They
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

Three-dimensional ECMs can have striking effects on cell morphology, which differ depending on whether the cells are epithelial cells or broblasts. Mammary epithelial cells grown in 2D versus 3D matrices have dramatic differences in organization. In 3D, the mammary epithelial cells aggregate, form cellcell contacts, polarize, and establish spherical acini (Debnath and Brugge, 2005). In contrast, when cultured on a 2D ECM, these cells grow as a simple monolayer (Nelson and Bissell, 2006). Fibroblasts in vivo are normally embedded within a collagen-rich ECM. Fibroblasts adherent to a 2D matrix attach, spread out, and atten with large prominent lamellipodia. When placed back into a 3D matrix, the broblasts

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re-acquire an elongated spindle-shaped phenotype devoid of large, at lamellipodia. The differences between broblast and epithelial cell morphologies in 2D and 3D suggest that the physical conguration of the matrix itself provides spatial signals that control cell morphology (Larsen et al., 2006). In fact, mechanically attening a 3D matrix to a relatively 2D surface returns morphology to a 2D phenotype even though the same molecules and growth factors are present (Cukierman et al., 2001) yet sandwiching cells between two 2D surfaces can mimic a 3D environment (Beningo et al., 2004).
Cell migration

Migration of cells adherent to 2D matrices is based on cycles of lamellipodial extension, attachment, cell body translocation, and retraction of the cell rear. There are at least two major modes of cell migration through 3D matrices. In the mesenchymal mode of cell migration, just as on 2D surfaces, there are cycles of membrane protrusion at the leading edge, formation of matrix adhesions, dynamic cellular contractility, and retraction of the rear (Ridley et al., 2003; Li et al., 2005). For both 2D and 3D mesenchymal migration, cell traction forces are established by matrix-bound integrin receptors and transmitted by the actin cytoskeleton, and subsequent actomyosin contractility induces centripetal movements of actin and adhesion structures from the front and rear of cells. A second mode of 3D cell migration is termed amoeboid, similar to the process used by amoebae and leukocytes; this migration mode depends on non-adhesive conformational adaptation of cell shape to the local surrounding matrix. Cells adapt their shape to match the path of least resistance within the matrix, and migration is achieved by propulsive squeezing forward through gaps in the matrix (Friedl and Wolf, 2003). Fibroblasts in a 3D cell-derived matrix appear to migrate in a mesenchymal mode. Thus, the regulation of membrane protrusion, retraction, and endogenous tension by cellmatrix adhesions are likely to be important factors in broblast migration in 3D matrices. Levels of total Rac activity are lower in broblasts in a 3D matrix, resulting in fewer lamellae and more directional migration (Pankov et al., 2005). An additional mode of cellmatrix interaction has been dened for cells on a 2D substratum interacting with a collagen ber. In this mode, the cell translocates the collagen ber toward the cell body using cycles of membrane protrusion to establish ber contact and membrane retraction with the ber attached. The repetitive protrusion and retraction cycles elicit a hand-over-hand membrane dynamic associated with movement of the collagen ber (Meshel et al., 2005). It will be interesting to determine the role of hand-over-hand membrane dynamics in 3D matrices.
Molecular composition of cellmatrix adhesions

At least four different types of adhesion structures have been dened in broblasts, termed focal complexes, focal adhesions, brillar adhesions, and 3D-matrix adhesions. We will focus on matrixintegrinactin adhesion structures that are important contributors to regulating endogenous and exogenous tension (Katsumi et al., 2004). Focal complexes are small, transient matrix contact structures that provide early cell attachment at the leading edge. If stabilized, they will subsequently form focal adhesions, which can in turn transition to brillar adhesions. For cells on 2D matrices, the assembly and disassembly of matrix adhesions are regulated in a dynamic fashion in response to cell signals and the exogenous tension (matrix rigidity). The biological relevance of focal adhesions was initially questioned, since equivalent structures to these prominent 2D adhesion structures were not observed in most tissues. However, focal adhesions have been found at points of high uid shear stress in blood vessels (Romer et al., 2006). The fourth adhesion structure was identied in broblasts cultured within a cellJOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

derived 3D matrix and was also detected in tissues, indicating that adhesion structures containing integrins and actin do indeed form in 3D and in vivo, albeit with differing morphology and composition (Cukierman et al., 2001). The ability of integrins to localize to adhesion structures is not restricted to a particular integrin receptor. However, certain integrin receptors are preferentially concentrated at different cellmatrix adhesion structures. For example, broblasts adherent to a 2D bronectin matrix will form focal complexes and focal adhesions that are rich in avb3. While a5b1 is often excluded from the focal adhesion core, brillar adhesions contain a5b1. Three-dimensional-matrix adhesions contain primarily a5b1, but avb3 can be observed at the adhesion periphery. It seems likely that different integrin receptors will recruit different cytoplasmic factors and differentially control cell signaling and cellular tension (Cukierman et al., 2001). Subsets of proteins are recruited to different adhesion structures suggesting that adhesions may have signaling specicity. For instance, focal adhesions contain vinculin and numerous tyrosine-phosphorylated proteins including FAK and paxillin. Fibrillar adhesions contain abundant tensin, low levels of protein tyrosine phosphorylation, and a5b1 instead of avb3 integrins. Three-dimensional-matrix adhesions are similar to brillar adhesions regarding a5b1 localization; however, 3D-matrix adhesions contain high levels of vinculin, a-actinin, and phosphorylated paxillin. Proteins that are tyrosine phosphorylated localize to both 3D-matrix adhesions and focal adhesions, but levels of FAK Y397 phosphorylation are low in 3D-matrix adhesions, indicating that integrin signaling can differ substantially in 3D compared to 2D environments (Fig. 3) (Zamir and Geiger, 2001; Yamada et al., 2003). Levels of particular integrin receptors can be altered in various diseases, including cancer progression. Normally, integrin b6 is restricted in expression to epithelial cells during embryonic development and is not typically expressed in adult tissues. Integrin b6 is expressed at the cell surface in association with av as a receptor for bronectin, tenascin, and vitronectin. Expression of avb6 is induced on epithelial cells during wound healing and in carcinomas of the colon, lung, oral cavity, breast, and cervix (Janes and Watt, 2006). Increased expression of the integrin b1 subunit correlates with decreased breast cancer survival. Other integrin receptors such as avb3 and a6b4 are induced in highly metastatic melanoma cells and pancreatic adenocarcinoma progression, respectively. The shifting prole of integrin receptor expression may inuence endogenous (contractility) or exogenous (matrix rigidity) tension or facilitate tumor cell survival and migration in multiple tissues with different matrix compositions (Guo and Giancotti, 2004; Danen, 2005; Wilhelmsen et al., 2006). Changes in the expression of the integrin-associated proteins FAK, paxillin, a-actinin, and vinculin are also observed during tumor progression. Tumor cells appear to take advantage of the ability of FAK to regulate pathways important for cell proliferation, cell migration, gene expression, and survival (Mitra et al., 2005; Slack-Davis et al., 2007). For instance, FAK expression and activity are enhanced in metastatic tumors of the oral cavity, colon, rectum, thyroid, prostate, and cervix. In ovarian cancer, increased FAK expression correlates with decreased patient survival. In breast carcinoma, FAK activity is important for VEGF expression and tumor angiogenesis (McLean et al., 2005). Similar to FAK, increased expression of paxillin is observed in breast carcinoma, and a-actinin expression levels are increased in melanomas and in tumor cell lines with faster migration rates (Vadlamudi et al., 1999). In contrast, vinculin expression appears to have an inverse relationship with tumor metastasis. Vinculin expression is elevated in weakly metastatic melanoma cells. In highly metastatic melanoma cell lines, vinculin

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Fig. 3. Comparison of focal adhesions, brillar adhesions, and 3D-matrix adhesions. These matrix adhesion structures recruit distinct cytoplasmic proteins and differ in signaling, for example, in the levels of protein tyrosine phosphorylation (pY) of adaptor and signaling proteins. For example, FAK pY 397(phosphorylation at tyrosine 397) levels are highin focal adhesionsbut substantially lower in brillar and 3D-matrix adhesions. Paxillin pY 31 and FAK pY 861 levels are high in both focal adhesions and 3D-matrix adhesions but are lower in brillar adhesions. Based on these differences, distinct protein complexes are likely to form at each type of matrix adhesion to trigger specic signaling pathways.

expression is reduced, and vinculin localizes to small punctate adhesion structures (Lifschitz-Mercer et al., 1997). It will be important to determine whether these changes in expression of integrin-associated proteins contribute directly to tumor progression or are secondary responses to altered tumor or tumor stromal microenvironments. Cellular interactions with the matrix are coordinated with local actin polymerization and F-actin organization. Fibroblasts adherent to a 2D matrix contain prominent bundles of actin laments or stress bers that insert into focal adhesions. The Arp2/3 complex that nucleates actin polymerization is localized in a broad band at the leading edge within prominent lamellipodia (Vicente-Manzanares et al., 2005). In a 3D matrix, thin stress bers are located at the periphery of membrane extensions. Focal adhesions are rare and appear as dot-like structures near the protrusive edge. The Arp2/3 complex is in foci at the tips of extensions and lamellipodia are smaller and narrow (Beningo et al., 2004). Actin and nucleators of actin polymerization such as the Arp 2/3 complex assume different spatial congurations in cells engaged with 2D and 3D matrices. Thus, dynamics in exogenous tension or rigidity within 3D matrices may inuence the cellular distribution of actin and the Arp2/3 complex.
The Inuence of CellMatrix Interaction on Extracellular and Intracellular Compartments Matrix control of cell phenotype

Matrix ligand density and exogenous tension levels. Conversion between motile and stationary phenotypes is critical for controlling developmental processes and tissue remodeling, for example, the recruitment and organization of
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epithelial cells and broblasts during wound healing. The density of matrix ligands regulates cell migration. There is a biphasic response of cell motility to increasing the matrix ligand density in both 2D and 3D matrices. Cells migrate poorly on substrates of relatively low or high matrix ligand density, and they preferentially migrate at an intermediate density (Li et al., 2005). The matrix rigidity or exogenous tension of collagen gels regulates broblast cell migration and cell signaling (Rhee et al., 2007). Fibroblasts migrate efciently on less rigid matrices with lower exogenous tension. The relative migration rate is impeded on rigid matrices with high exogenous tension. The sensitivity of cell migration to the matrix ligand density and matrix rigidity indicates that cellular mechanisms link exogenous tension (matrix rigidity) and ligand density to cell migration. Interestingly, if the migratory path of a cell includes differentials in either matrix ligand density or matrix rigidity, the cells will move toward the ligand-dense or more rigid regions and away from the low ligand density or less rigid matrix in phenomena termed haptotaxis and durotaxis, respectively (Lo et al., 2000). Mammary tumors and the adjacent tumor stroma have high exogenous tension compared to normal mammary gland. Metastatic breast tumors, unlike non-metastatic tumors, frequently express high levels of lysyl oxidase, a collagencrosslinking enzyme, and form rigid stroma in vivo. Overexpression of an isoform of lysyl oxidase in a nonmetastatic breast tumor cell line is sufcient to induce both tumor brosis and tumor invasion (Kirschmann et al., 2002). In breast tumor biopsies, invasive potential correlates with the presence of brotic foci associated with the tumor stroma (Hasebe et al., 2002).

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The inuence of tumor stroma tension on cell phenotype was recapitulated in 3D cultures using the basement membrane extract Matrigel crosslinked to polyacrylamide gels of variable tension. Exogenous tension (rigidity) was adjusted to the levels observed in normal and tumor-containing tissue by changing the polyacrylamide gel composition. Elevation of exogenous tension enhances Rho activity, induces cytoskeletal tension, increases focal adhesions, decreases cellcell contact, perturbs tissue polarity, and increases growth. Exogenous tension (rigidity) at levels comparable to the tumor stroma induces integrin clustering and activation of intracellular signaling, such as ERK phosphorylation and ROCK-dependent contractility. Inhibiting Rho and ERK signaling was sufcient to decrease endogenous tension in cells adhering to a rigid matrix, and this resulted in decreased formation of focal adhesions and increased cellcell contacts (Fig. 4) (Paszek et al., 2005). Thus, the high exogenous tension of the tumor stroma can inuence the endogenous tension and cellmatrix adhesions that form in tumor cells (Bershadsky et al., 2006). The levels of exogenous tension or rigidity also regulate preosteoblast proliferation, differentiation, and focal adhesion dynamics (Kong et al., 2005). It is interesting to speculate that due to durotaxis, the gradient in stroma exogenous tension at the tumor boundaryhigh at the tumor and low in surrounding tissue may induce a migratory response of cells in the surrounding tissue, leading to preferential recruitment of cells to the tumor and its stroma to continually increase tumor size. The idea of cells responding to the presence of a rigid body in a matrix is not novel. Chicken heart broblasts are known to align and induce membrane protrusions toward an embedded rigid body from adjacent compliant areas (Boocock, 1989). Composition of the ECM. Rarely will cells encounter only a single isolated ECM protein in vivo. The basement

membrane and connective tissue are specialized matrices containing networks of multiple proteins including several collagen isoforms, laminin isoforms, and bronectin. Migration from a collagen plug and collagen gel contractility by broblasts adherent to collagen or mixtures of collagen and bronectin indicate that the matrix mixture elicits a cell phenotype that is distinct from cells adherent to only collagen (Greiling and Clark, 1997; Liu et al., 2006). Consequently, this and many other studies indicate that the composition of the ECM, rather than simply the presence of an extracellular scaffold, is critical for regulating cell phenotype.
Cell control of the matrix

Fig. 4. Increasing the exogenous tension (matrix rigidity) has dramatic effects on intracellular signaling, matrix adhesions, and endogenous tension (contractility). An increase in exogenous tension alters RhoA activity, the actin cytoskeleton, focal adhesions, cellcell contacts, tissue polarity, and importantly the growth rate. The gure shows a comparison of cellular phenotypes on matrices of different rigidity (" indicates relative increase and # relative decrease). Thus, dense desmoplastic tumor stroma will inuence the endogenous tension, matrix adhesion structures, and cell signaling pathways. If some mechanism could be developed to reduce both the exogenous tension (rigidity) and endogenous tension (contractility) of rigid tumors, it might be possible to inhibit tumor growth or progression. [Color gure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Matrix porosity. The matrix is a meshwork of brillar and non-brillar proteins containing pores. At a constant matrix concentration, crosslinking of the matrix will reduce pore size and enhance the barrier function of the matrix (McKegney et al., 2001). Lysyl oxidase is a cell-derived collagen-crosslinking enzyme that associates with bronectin to form a complex that retains crosslinking activity (Fogelgren et al., 2005). Thus, cellderived lysyl oxidase may utilize bronectin as a scaffold to locally increase collagen crosslinking and decrease matrix porosity. Matrix cleavage. Cells use matrix proteases to remodel the matrix. ECM remodeling is required for normal physiological processes such as embryonic development, morphogenesis, and wound repair. Tissues normally harbor low levels of matrix protease activity that are controlled by inhibitors such as tissue inhibitors of metalloproteinases (TIMPs) (recently reviewed in references Nagase et al. (2006); Page-McCaw et al. (2007)). In many disease states, or following tissue damage, there is a shift favoring matrix protease activity, and matrix remodeling ensues. The triple-helical structure of brillar collagen confers resistance to many proteases except collagenase. Collagenase promotes uncoiling of the triple helix and exposes additional sites that become susceptible to proteolysis. Thus, the activity of collagenases can disrupt the brous meshwork of the ECM and increase matrix porosity. Endothelial cells provide an excellent example of inherent differences between 2D and 3D matrix adhesions and associated downstream pathways that regulate matrix protease activity and cell migration. Endothelial cell migration occurs independent of matrix protease activity in a 2D matrix. In contrast, in a 3D matrix environment, endothelial cell migration is dependent on protease activity. Interestingly, the activation of matrix metalloproteinase-2 also occurs selectively for endothelial cells cultured in a 3D matrix. Thus, 2D and 3D matrix adhesions induce different cell signaling pathways that control matrix protease activity and the mode of cell migration (Koike et al., 2002; Fisher et al., 2006). Invasive tumor cells shift the proteolytic balance and display enhanced matrix protease activities. Tumor-associated matrix proteases can promote cell invasion, in part, by increasing matrix porosity or generating pro-migratory matrix peptides (Giannelli et al., 1997; Hotary et al., 2006). However, the requirement for matrix remodeling during tumor cell migration in vivo is controversial, since cells can reportedly shift between protease-dependent and independent migration modes (Friedl and Wolf, 2003; Wolf et al., 2003). Nonetheless, carcinoma cells acquire the ability to invade through the basement membrane and connective tissues, potentially utilizing both matrix protease-dependent and independent modes of cell migration. Invadopodia are membrane protrusions on the cell surface of tumor cells that mediate matrix cleavage. Many of the cytoplasmic proteins that are recruited to matrix adhesions in primary cells are also recruited to invadopodia in tumor cells.

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Invasive cancer cells can extend multiple invadopodia that induce matrix cleavage, leading to increases in matrix porosity and liberation of pro-migratory matrix peptides. Because of their matrix remodeling properties, invadopodia are suggested to promote tumor cell invasion (Artym et al., 2006; Weaver, 2006; Linder, 2007). Cellular contractility or endogenous tension. Cell matrix interactions appear to utilize a molecular clutch mechanism that provides a bi-directional conduit for controlling mechanical tension across the cell membrane (Evans and Calderwood, 2007). Endogenous intracellular tension uctuates in response to disruption or stabilization of the linkages between matrix-engaged integrins, the F-actin cytoskeleton, and myosin. Actomyosin contractility increases endogenous cell tension through sliding of actin laments and subsequent force applied to matrix adhesions. Increasing the cytoplasmic tension of matrix-engaged integrins can transfer tension to the tethered matrix and increase exogenous tension (matrix rigidity) (Smilenov et al., 1999; Brown et al., 2006; Hu et al., 2007). As an example, during broblast migration, cellular force applied through matrix adhesions in protrusions at the leading edge will displace the matrix centripetally, and this local matrix stretch increases the exogenous tension. Fibronectin is comprised of a series of modular domains that fold into tertiary structures that can undergo conformational rearrangements in response to tension. Elevating actomyosin contractility or endogenous tension induces cell-associated bronectin to undergo a conformational change that reveals previously concealed bronectin-binding sites and triggers bronectin matrix assembly (Pankov and Yamada, 2002; Mao and Schwarzbauer, 2005). Adjusting either the endogenous (contractility) or exogenous tension (matrix rigidity) can potentially regulate bronectin matrix assembly and matrix structure.
Potential Future Therapeutic Applications

future research. Although highly speculative, they provide examples of novel possible approaches based on new ndings in matrix biology.
Potential therapeutic approaches based on altering cell matrix adhesion in cancer

The major advances in knowledge of the mechanisms and sequelae of cellmatrix interactions that we have summarized in this mini review should lead to new therapeutic approaches in the future. Principles such as the roles of matrix composition, three-dimensionality, and rigidity, as well as the existence of distinct types of cellmatrix adhesions and bi-directional signaling responses provide a rational foundation for the development of novel approaches to tissue repair and intervention in disease processes. For example, there are already many applications of matrix molecules and synthetic biomaterials to tissue engineering that speed wound repair and potentially replace failed organs (Lutolf and Hubbell, 2005; Maskarinec and Tirrell, 2005; Clark et al., 2007; Kong and Mooney, 2007; Metcalfe and Ferguson, 2007). Applying new knowledge of the principles of the 3D organization and rigidity of the microenvironment to controlling the cell signaling response should accelerate research in engineering tissues. Similarly, increasing the understanding of the roles of local tension and feedback mechanisms may lead to the development of approaches to facilitate wound repair and prevent scarring and brosis (Xiao et al., 2004; Metcalfe and Ferguson, 2007). Since integrin-mediated adhesion and signaling are crucial for thrombosis and inammation, there are major efforts already underway to develop novel therapies by manipulating integrin activation and functions (Rose et al., 2000; Coller, 2001; Meadows and Bhatt, 2007). Complex diseases with an inammatory and mechanical component, such as atherosclerosis, may eventually include management of cell matrix interactions, since they play crucial roles in cell recruitment, adhesion, and tissue remodeling (Hamm, 2003). A less-explored area of future application of cellmatrix biology involves cancer, and we propose below a few possible areas of
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

Selective expression of integrin receptors on tumor cells presents an opportunity for targeting drugs, peptides, or radioisotopes using anti-integrin antibody conjugates. In particular, integrin b6 expression is induced on several carcinomas, whereas b6 subunit expression is normally only detectable in the adult during wound healing (Jones et al., 1997). Tumor cells retain expression of the b6 subunit at metastatic sites, such as in lymph nodes (Hazelbag et al., 2007). Thus, antiavb6 antibody conjugates may provide a tool for delivering cytotoxic agents to the primary tumor and metastatic sites for potential therapy at both early and late tumor stages (Weinreb et al., 2004; Sheppard, 2005; Hehlgans et al., 2007). Clearly, this approach is limited to tumors that express the integrin b6 subunit. The localized expression of lysyl oxidase in metastatic tumor cells provides a unique opportunity to target proteins to invasive tumors or the associated stroma (Kirschmann et al., 2002; Erler and Giaccia, 2006), but the challenge will be how to obtain drug targeting. Invasive tumor cells expressing lysyl oxidase might be targeted with a fusion protein containing as a fusion partner the collagen substrate domain for lysyl oxidase. The substrate domain could crosslink the fusion protein to the tumor matrix (Lucero and Kagan, 2006). Once a fusion protein or antibody is targeted to the tumor by taking advantage of selective tumor cell expression of an integrin or lysyl oxidase, a number of therapeutic approaches are possible (Fig. 5); some can take further advantage of knowledge of matrix biology. The fusion protein could contain a toxin that is specically released in active form by engineering sites specic for matrix metalloprotease(s) known to be expressed by the particular tumor or its stroma. Alternatively, the fusion protein could carry a selective matrix protease inhibitor or non-cleavable substrate to inhibit local matrix remodeling and invasion. The strategy in this case would be to restrain a potentially metastatic tumor within a molecular cage designed to prevent cell invasion and tumor cell matrix remodeling. Engineered fusion proteins could be injected into sites of tumor excision after surgery to target any remaining tumor cells. Breast and other tumors can associate with dense, collagenrich desmoplastic stroma. The high exogenous tension or rigidity associated with breast tumor stroma is known to induce signaling and cytoskeletal changes, disrupt tissue polarity, and stimulate cell growth (Paszek et al., 2005). Therefore, strategies to reduce the exogenous and endogenous tension of breast and other tumors characterized by high exogenous tension may provide a novel therapeutic opportunity to control tumor progression or recurrence (Fig. 4). Developing approaches to decrease exogenous and/or endogenous tension of tumors in vivo will require creative new technologies.
Conclusions

Over the past few decades, there has been exciting progress in understanding the molecular mechanisms that regulate the formation and function of cellmatrix adhesions. Many cellular processes are now known to be regulated by signals from cell matrix adhesion structures that are transmitted bi-directionally across the cell membrane and dynamically link the intracellular and extracellular microenvironments. Future studies will clarify further the roles of cellmatrix interactions in determining cellular fate in vivo. This knowledge should provide a foundation for developing molecular tools to specically modify the cell

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Fig. 5. Speculative applications of matrix biology: lysyl oxidase (LOX)-targeted inhibitors and a molecular cage for tumors. Certain metastatic tumors frequently express elevated lysyl oxidase, which leads to a rigid stroma with high exogenous tension. This selective expression of the collagen-crosslinking enzyme lysyl oxidase in metastatic tumors could potentially provide a unique opportunity to target and crosslink proteins to the tumor stroma. For example, modular proteins for targeting could contain the lysyl oxidase collagen substrate domain (LOX substrate domain) fused to effector domains such as inhibitors of matrix remodeling or toxins to be crosslinked to tumor stroma. The modular protein could be engineered to contain a protease-resistant matrix protein to be crosslinked to the tumor stroma, which together with targeted matrix protease inhibitors could trap tumor cells within a molecular cage designed to resist invasion and matrix remodeling. In addition, a targeted toxin could also be crosslinked to tumor stroma that is tailored to the matrix protease activity signature of the tumor for local activation and release. [Color gure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

matrix interface in order to control particular cellular functions such as gene expression, cell migration, and differentiation. The ability to control cellular phenotype in vivo could be translated to therapeutic technologies to prevent progression of cancer and other diseases.
Acknowledgments

Health Disparities. We would like to dedicate this review to the memory of Dr. Suzanne Bernier.
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We appreciate the insightful comments of Marinilce dos Santos and Andrew Doyle in the preparation of this article. This research was supported by the Intramural Research Program of the NIH, the National Institute of Dental and Craniofacial Research, and the National Center on Minority Health and
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