cell–matrix adhesion

MINI-REVIEW 565J o u r n a l o fJ o u r n a l o f

CellularPhysiologyCellularPhysiology
Cell–Matrix Adhesion
ALLISON L. BERRIER1,2* AND KENNETH M. YAMADA2*1Katrina Visiting Faculty Program, National Center on Minority Health and Health Disparities,

National Institutes of Health, Bethesda, Maryland2Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research,

National Institutes of Health, Bethesda, Maryland

The complex interactions of cells with extracellular matrix (ECM) play crucial roles in mediating and regulating many processes, includingcell adhesion, migration, and signaling during morphogenesis, tissue homeostasis, wound healing, and tumorigenesis. Many of theseinteractions involve transmembrane integrin receptors. Integrins cluster in specific cell–matrix adhesions to provide dynamic linksbetween extracellular and intracellular environments by bi-directional signaling and by organizing the ECM and intracellular cytoskeletaland 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 reviewconcludes by speculating on the application of this knowledge of cell–matrix interactions in the formation of cell adhesions, assembly ofmatrix, migration, and tumorigenesis to potential future therapeutic approaches.J. Cell. Physiol. 213: 565–573, 2007. � 2007 Wiley-Liss, Inc.

Contract grant sponsor: Intramural Research Program, NIH.Contract grant sponsor: National Institute of Dental andCraniofacial Research.Contract grant sponsor: National Center on Minority Health andHealth 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: [email protected]; [email protected]

Received 28 June 2007; Accepted 29 June 2007

DOI: 10.1002/jcp.21237

The extracellular matrix (ECM) provides scaffolds for cellularsupport that are present in all tissues and organs. The ECM is acomplex mixture of matrix molecules, including theglycoproteins fibronectin, collagens, laminins, proteoglycans,and non-matrix proteins including growth factors. Celladhesion to the ECM induces discrete cell surface structurestightly associated with the matrix termed cell–matrixadhesions, which mediate direct interactions of the cell with itsextracellular environment. Cell–matrix adhesions are essentialfor cell migration, tissue organization, and differentiation, and asa result they play central roles in embryonic development,remodeling, and homeostasis of tissue and organ systems.Matrix adhesion signals cooperate with other pathways toregulate biological processes such as cell survival, cellproliferation, wound healing, and tumorigenesis. Thus,elucidating the structure and function of cell–matrix adhesionsprovides a critical vantage point for understanding theregulation of eukaryotic cellular phenotypes in vivo. For recentreviews see references Miranti and Brugge (2002); Danen andSonnenberg (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); Luoet al. (2007). Due to constraints on the length of the article, weapologize for not being able to cite all relevant references.

Integrins are the principle cell surface adhesion receptorsmediating cell–matrix adhesion. Integrins are heterodimericreceptors generated by selective pairing between 18 a and 8 bsubunits; there are 24 distinct integrin receptors that bindvarious ECM ligands with different affinities (Luo et al., 2007).Some integrin subunits are ubiquitously expressed, while othersubunits are expressed in a tissue- or stage-restricted manner(Humphries et al., 2006). For instance, integrin b1 isubiquitously expressed, whereas the b6 subunit is onlyexpressed in the adult during wound healing.

The extracellular domains of integrin receptors bind ECMligands and divalent cations, but they can also associate laterallywith other proteins at the cell surface, such as tetraspanins,growth factor receptors, matricellular proteins, and matrixproteases or their receptors (Miranti and Brugge, 2002).Integrins influence cell behavior not only by providing a dockingsite for the ECM at the cell surface, but also by actions of theintegrin intracellular domains. Integrin intracellular domains areshort regions of roughly 50 amino acids in length, except forintegrin 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 cellsignaling andwith adaptor proteins that provide a connection tothe cytoskeleton (Hynes, 2002).

Integrins provide a bi-directional conduit formechanochemical information across the cell membrane,providing a major mechanism for connecting the intracellularand extracellular compartments. Cell adhesion to the ECMtransmits information via integrin receptors that regulatesintracellular signaling via outside-in signaling, which isimportant, for example, in cell spreading and cell migration.Conversely, intracellular signals can induce changes in integrinconformation and activation that alter its ligand-binding activityin a process termed inside-out signaling. Integrin engagementwith matrix can also affect integrin activation, providing bi-directional crosstalk between inside-out and outside-insignaling (Ginsberg et al., 2005; Luo et al., 2007).

Integrin clustering follows the engagement of integrins withthe naturally multivalent ECM, and it promotes the localizedintracellular concentration of signaling molecules. Clustering ofintegrin receptors, or particularly integrin b cytoplasmicdomains, activates non-receptor tyrosine kinases such as focaladhesion kinase (FAK) leading to localized increases in the levelsof tyrosine-phosphorylated proteins. Serine/threonine kinasesincluding members of the protein kinase C family, lipid kinasessuch as PI 3-kinase, and phosphatases such as RPTPa are alsoregulated by integrin engagement and clustering. These kinase

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and phosphatase signaling pathways induce post-translationalmodifications regulating the protein interactions and/orenzymatic activity of the substrates (Li et al., 2005). They specifyprotein recruitment to adhesion complexes and therebyselectively link matrix adhesions to various downstreamsignaling cascades that control cytoskeletal organization, generegulation, and diverse cellular processes and functions (Fig. 1)(Hervy et al., 2006).

More than 50 cytoplasmic proteins are present in cell–matrixadhesion structures (Lo, 2006). Because integrin receptors lackintrinsic enzymatic activity, they must recruit signaling proteinsto control adhesion-dependent processes (Liu et al., 2000;Mitra et al., 2005). Three basic categories of proteins arerecruited to cell–matrix adhesions: (1) integrin-bindingproteins, (2) adaptors and/or scaffolding proteins that lackintrinsic enzymatic activity, and (3) enzymes. Talin is an exampleof a protein that directly binds to integrin cytoplasmic domainsand is important for regulating integrin activation and signaling(Calderwood, 2004). Adaptors and/or scaffolding factors linkintegrin-associated proteins with actin or other proteins, andexamples include vinculin, paxillin, and a-actinin. Enzymes thatmodify integrin downstream effectors include the non-receptor tyrosine kinases FAK and Src. The profiles of proteinsrecruited to matrix adhesions specify the biochemical signalsand biophysical properties of matrix adhesions (Li et al., 2005).

The cytoskeleton contains three general classes offilamentous structure, F-actin, intermediate filaments, andmicrotubules. It has become clear that cell migration and tissueremodeling require coordinated crosstalk between the actin,intermediate filament, and microtubule cytoskeletal networks(e.g., see reference Even-Ram et al. (2007)). Actinpolymerization and proteins important for regulating actinorganization are essential regulators of membrane protrusionand cell migration. Rearrangements of the actin cytoskeletonare mediated by complex molecular pathways that promoteactin polymerization, actin depolymerization to renew theintracellular pool of monomeric actin, and modifications ofactin-crosslinking proteins (Vicente-Manzanares et al., 2005;

Fig. 1. General model of cell–matrix adhesions and their downstream reintegrins recruit cytoplasmic proteins, which in cooperation with other cephenotypes.

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

Pollard, 2007). Matrix adhesions associate with bundles of actinfilaments, and bi-directional interactions mediated by the actincytoskeleton involving actomyosin contraction and clusteringof integrins bound to matrix combine to increase cellcontractility, also referred to as endogenous tension. Thedynamic assembly and disassembly of adhesion structuresapplies different levels of force to the matrix that, in turn,regulates endogenous tension (Wozniak et al., 2004).Conversely, endogenous tension is transmitted throughintegrins to the ECM and can increase matrix rigidity, referredto as exogenous tension. Adhesion structures recruitcytoplasmic proteins that induce downstream effectorsinvolved in regulating matrix deposition or remodeling. Thus,integrin engagement with the ECM generates bi-directionalsignals that can alter endogenous tension, exogenous tension,and matrix composition (Fig. 2) (Katsumi et al., 2004; Petersonet al., 2004; Wang, 2007).

Directional cell migration requires the establishment of cellpolarity to create a leading edge and a trailing edge (Moissogluand Schwartz, 2006). The leading edge undergoes membraneprotrusive activities driven by actin polymerization thatestablish new matrix contacts, whereas at the trailing edge celladhesions are disassembled to promote retraction of the cellrear and forward cell movement (Vicente-Manzanares et al.,2005). Local actin polymerization can induce membraneprotrusions and favor formation of matrix contact by localizingintegrin receptors in an active conformation at cell protrusions(Galbraith et al., 2007). The rate of cell migration can be limitedby the rate of rear retraction, and thus the dynamic formationand disassembly of cell–matrix adhesions are critical to cellmigration (Ridley et al., 2003).

The Rho GTPase family of GTP-binding proteins includingRhoA, Rac1, and Cdc42 are critical regulators of cellcontractility, lamellipodial and filopodial formation, and cellularpolarity. RhoAGTPase downstream signals such as activation ofRhoA-kinase (ROCK) and inhibition of myosin phosphataseincrease myosin light chain phosphorylation, leading toclustering of actin stress fibers to regulate actomyosin

gulation. Cell-extracellular matrix adhesions containing clusters ofll surface receptors control diverse cellular processes, functions, and

Fig. 2. Coordinate regulation of exogenous tension (matrix rigidity) and endogenous tension (contractility). A: Cells and matrix mutuallyinteract to regulate tension. B: Tension in the cell microenvironment is thought to be distributed by integrin receptors that signal bi-directionallybetween extracellular and intracellular compartments. Tension levels mayalter outside-inand inside-out integrin signaling. Integrin engagementwith 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 figure.

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contractility and endogenous tension. Rho downstream signalsregulate membrane retraction, thereby significantlycontributing to leading and trailing edge cell polarity in cellmigration. The balance of Rac and Rho activation coordinatesmembrane protrusion, retraction, and numbers of protrusionsduring cell migration. Sites of high levels of active Rac1 willsuppress RhoA and induce lamellipodia; in contrast, regionscontaining concentrated active RhoAwill have lowRac1 activityand membrane retraction (Clark et al., 1998; Nimnual et al.,2003; Burridge andWennerberg, 2004). Furthermore, integrinengagement with matrix regulates the activity of Rho GTPasefamily members and localization with downstream effectors.Thus, matrix adhesions establish feedback loops that controlmembrane protrusion and retraction during cell migration bycoordinating and integrating the activities of individual RhoGTPase family members in the leading and trailing edges of thecell (Ridley et al., 2003).

The remainder of our review will focus on the instructiverole of cell–matrix adhesions on both cell phenotype andextracellular environment. First, we compare how cellmorphology, cell migration, and cell–matrix adhesionstructures respond to two-dimensional (2D) compared tothree-dimensional (3D) matrices. Changes in cell–matrixadhesions associated with cancer are described. We thenexamine how alterations of the extracellular environmentinfluence the intracellular environment and vice versa. Finally,we speculate about ways in which our rapidly expandingknowledge about cell–matrix adhesion might be exploited fortherapeutic purposes.

Matrix Dimensionality and Cell Behavior

Eukaryotic cells adapted to grow in vitro are routinely culturedon a 2D substratum. Many studies have characterized cellularresponses to growth on a 2D ECM-coated substratum. They


have identified complex molecular and biochemical pathwaysactivated or modified by integrin-mediated adhesion and haveprovided insights into mechanisms that regulate adhesion-dependent cellular processes such as cell spreading, cellproliferation, cell differentiation, and cell survival. Fibroblastscultured in 2D matrices interact with a rigid substratum at theventral surface of the cell. The binding of integrin ligands andthe differences in exogenous tension (matrix rigidity) betweenthe dorsal and ventral cell surfaces can selectively trigger focaladhesion formation at the ventral surface, thereby inducing cellpolarity (Giannone and Sheetz, 2006). Fibroblasts in 3Dmatrices are typically not exposed to these large differences inexogenous tension, and hence lack dorsal–ventral polarity.However, observations in 2D cultures suggest that regionalvariations in 3D-matrix exogenous tension (rigidity) mayinfluence the distribution of cell–matrix adhesions and cellbehavior in vivo (Ingber, 2006). Intriguing new insights into theeffects of 3D matrix on cell behavior elucidate the synergisticrelationship of cell and ECM in vivo and the dynamic function ofcell–matrix adhesions in a 3D environment.

Cell morphology

Three-dimensional ECMs can have striking effects on cellmorphology, which differ depending on whether the cells areepithelial cells or fibroblasts. Mammary epithelial cells grown in2D versus 3D matrices have dramatic differences inorganization. In 3D, the mammary epithelial cells aggregate,form cell–cell contacts, polarize, and establish spherical acini(Debnath andBrugge, 2005). In contrast, when cultured on a 2DECM, these cells grow as a simple monolayer (Nelson andBissell, 2006). Fibroblasts in vivo are normally embedded withina collagen-rich ECM. Fibroblasts adherent to a 2D matrixattach, spread out, and flatten with large prominentlamellipodia.Whenplaced back into a 3Dmatrix, the fibroblasts


re-acquire an elongated spindle-shaped phenotype devoid oflarge, flat lamellipodia. The differences between fibroblast andepithelial cell morphologies in 2D and 3D suggest that thephysical configuration of thematrix itself provides spatial signalsthat control cell morphology (Larsen et al., 2006). In fact,mechanically flattening a 3D matrix to a relatively 2D surfacereturns morphology to a 2D phenotype even though the samemolecules and growth factors are present (Cukierman et al.,2001) yet sandwiching cells between two2D surfaces canmimica 3D environment (Beningo et al., 2004).

Cell migration

Migration of cells adherent to 2Dmatrices is based on cycles oflamellipodial extension, attachment, cell body translocation,and retraction of the cell rear. There are at least two majormodes of cell migration through 3D matrices. In themesenchymal 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). Forboth 2D and 3D mesenchymal migration, cell traction forcesare established by matrix-bound integrin receptors andtransmitted by the actin cytoskeleton, and subsequentactomyosin contractility induces centripetal movements ofactin and adhesion structures from the front and rear of cells. Asecond mode of 3D cell migration is termed amoeboid, similarto the process used by amoebae and leukocytes; this migrationmode depends on non-adhesive conformational adaptation ofcell shape to the local surrounding matrix. Cells adapt theirshape to match the path of least resistance within the matrix,and migration is achieved by propulsive squeezing forwardthrough gaps in the matrix (Friedl and Wolf, 2003). Fibroblastsin a 3D cell-derived matrix appear to migrate in a mesenchymalmode. Thus, the regulation of membrane protrusion,retraction, and endogenous tension by cell–matrix adhesionsare likely to be important factors in fibroblast migration in 3Dmatrices. Levels of total Rac activity are lower in fibroblasts in a3D matrix, resulting in fewer lamellae and more directionalmigration (Pankov et al., 2005).

An additional mode of cell–matrix interaction has beendefined for cells on a 2D substratum interacting with a collagenfiber. In this mode, the cell translocates the collagen fibertoward the cell body using cycles of membrane protrusion toestablish fiber contact and membrane retraction with the fiberattached. The repetitive protrusion and retraction cycles elicit a‘‘hand-over-hand’’ membrane dynamic associated withmovement of the collagen fiber (Meshel et al., 2005). It will beinteresting to determine the role of hand-over-handmembranedynamics in 3D matrices.

Molecular composition of cell–matrix adhesions

At least four different types of adhesion structures have beendefined in fibroblasts, termed focal complexes, focal adhesions,fibrillar adhesions, and 3D-matrix adhesions. We will focus onmatrix–integrin–actin adhesion structures that are importantcontributors to regulating endogenous and exogenous tension(Katsumi et al., 2004). Focal complexes are small, transientmatrix contact structures that provide early cell attachment atthe leading edge. If stabilized, they will subsequently form focaladhesions,which can in turn transition to fibrillar adhesions. Forcells on 2D matrices, the assembly and disassembly of matrixadhesions are regulated in a dynamic fashion in response to cellsignals and the exogenous tension (matrix rigidity). Thebiological relevance of focal adhesions was initially questioned,since equivalent structures to these prominent 2D adhesionstructures were not observed in most tissues. However, focaladhesions have been found at points of high fluid shear stress inblood vessels (Romer et al., 2006). The fourth adhesionstructure was identified in fibroblasts cultured within a cell-


derived 3D matrix and was also detected in tissues, indicatingthat adhesion structures containing integrins and actin doindeed form in 3D and in vivo, albeit with differing morphologyand composition (Cukierman et al., 2001).

The ability of integrins to localize to adhesion structures isnot restricted to a particular integrin receptor. However,certain integrin receptors are preferentially concentrated atdifferent cell–matrix adhesion structures. For example,fibroblasts adherent to a 2D fibronectin matrix will form focalcomplexes and focal adhesions that are rich inavb3.Whilea5b1

is often excluded from the focal adhesion core, fibrillaradhesions contain a5b1. Three-dimensional-matrix adhesionscontain primarily a5b1, but avb3 can be observed at theadhesion periphery. It seems likely that different integrinreceptors will recruit different cytoplasmic factors anddifferentially control cell signaling and cellular tension(Cukierman et al., 2001).

Subsets of proteins are recruited to different adhesionstructures suggesting that adhesions may have signalingspecificity. For instance, focal adhesions contain vinculin andnumerous tyrosine-phosphorylated proteins including FAK andpaxillin. Fibrillar adhesions contain abundant tensin, low levelsof protein tyrosine phosphorylation, and a5b1 instead of avb3

integrins. Three-dimensional-matrix adhesions are similar tofibrillar adhesions regarding a5b1 localization; however,3D-matrix adhesions contain high levels of vinculin, a-actinin,and phosphorylated paxillin. Proteins that are tyrosinephosphorylated localize to both 3D-matrix adhesions and focaladhesions, but levels of FAK Y397 phosphorylation are low in3D-matrix adhesions, indicating that integrin signaling can differsubstantially in 3D compared to 2D environments (Fig. 3)(Zamir and Geiger, 2001; Yamada et al., 2003).

Levels of particular integrin receptors can be altered invarious diseases, including cancer progression. Normally,integrin b6 is restricted in expression to epithelial cells duringembryonic development and is not typically expressed in adulttissues. Integrinb6 is expressed at the cell surface in associationwith av as a receptor for fibronectin, tenascin, and vitronectin.Expression of avb6 is induced on epithelial cells during woundhealing and in carcinomas of the colon, lung, oral cavity, breast,and cervix (Janes andWatt, 2006). Increased expression of theintegrin b1 subunit correlates with decreased breast cancersurvival. Other integrin receptors such as avb3 and a6b4 areinduced in highly metastatic melanoma cells and pancreaticadenocarcinoma progression, respectively. The shifting profileof integrin receptor expression may influence endogenous(contractility) or exogenous (matrix rigidity) tension orfacilitate tumor cell survival and migration in multiple tissueswith different matrix compositions (Guo and Giancotti, 2004;Danen, 2005; Wilhelmsen et al., 2006).

Changes in the expression of the integrin-associatedproteins FAK, paxillin, a-actinin, and vinculin are also observedduring tumor progression. Tumor cells appear to takeadvantage of the ability of FAK to regulate pathways importantfor cell proliferation, cell migration, gene expression, andsurvival (Mitra et al., 2005; Slack-Davis et al., 2007). Forinstance, FAK expression and activity are enhanced inmetastatic tumors of the oral cavity, colon, rectum, thyroid,prostate, and cervix. In ovarian cancer, increased FAKexpression correlates with decreased patient survival. In breastcarcinoma, FAK activity is important for VEGF expression andtumor angiogenesis (McLean et al., 2005). Similar to FAK,increased expression of paxillin is observed in breastcarcinoma, and a-actinin expression levels are increased inmelanomas and in tumor cell lines with faster migration rates(Vadlamudi et al., 1999). In contrast, vinculin expressionappears to have an inverse relationship with tumor metastasis.Vinculin expression is elevated in weakly metastatic melanomacells. In highly metastatic melanoma cell lines, vinculin

Fig. 3. Comparison of focal adhesions, fibrillar adhesions, and 3D-matrix adhesions. These matrix adhesion structures recruit distinctcytoplasmicproteinsanddiffer insignaling, forexample, inthelevelsofproteintyrosinephosphorylation(pY)ofadaptorandsignalingproteins.Forexample,FAKpY397(phosphorylationattyrosine397)levelsarehighinfocaladhesionsbutsubstantially lowerinfibrillarand3D-matrixadhesions.Paxillin pY 31 and FAK pY 861 levels are high in both focal adhesions and 3D-matrix adhesions but are lower in fibrillar adhesions. Based on thesedifferences, distinct protein complexes are likely to form at each type of matrix adhesion to trigger specific signaling pathways.


expression is reduced, and vinculin localizes to small punctateadhesion structures (Lifschitz-Mercer et al., 1997). It will beimportant to determinewhether these changes in expression ofintegrin-associated proteins contribute directly to tumorprogression or are secondary responses to altered tumor ortumor stromal microenvironments.

Cellular interactions with the matrix are coordinated withlocal actin polymerization and F-actin organization. Fibroblastsadherent to a 2D matrix contain prominent bundles of actinfilaments or stress fibers that insert into focal adhesions. TheArp2/3 complex that nucleates actin polymerization is localizedin a broad band at the leading edgewithin prominent lamellipodia(Vicente-Manzanares et al., 2005). In a 3D matrix, thin stressfibers are located at the periphery of membrane extensions.Focal adhesions are rare and appear as dot-like structures nearthe protrusive edge. The Arp2/3 complex is in foci at the tips ofextensions and lamellipodia are smaller and narrow (Beningoet al., 2004). Actin and nucleators of actin polymerizationsuch as the Arp 2/3 complex assume different spatialconfigurations in cells engaged with 2D and 3D matrices.Thus, dynamics in exogenous tension or rigidity within 3Dmatrices may influence the cellular distribution of actin andthe Arp2/3 complex.

The Influence of Cell–Matrix Interaction on Extracellularand Intracellular CompartmentsMatrix control of cell phenotype

Matrix ligand density and exogenous tension levels.Conversion between motile and stationary phenotypes iscritical for controlling developmental processes and tissueremodeling, for example, the recruitment and organization of


epithelial cells and fibroblasts duringwound healing. The densityof matrix ligands regulates cell migration. There is a biphasicresponse of cell motility to increasing the matrix ligand densityin both 2D and 3Dmatrices. Cells migrate poorly on substratesof relatively low or high matrix ligand density, and theypreferentiallymigrate at an intermediate density (Li et al., 2005).The matrix rigidity or exogenous tension of collagen gelsregulates fibroblast cell migration and cell signaling (Rhee et al.,2007). Fibroblasts migrate efficiently on less rigid matrices withlower exogenous tension. The relative migration rate isimpeded on rigid matrices with high exogenous tension. Thesensitivity of cell migration to the matrix ligand density andmatrix rigidity indicates that cellular mechanisms linkexogenous tension (matrix rigidity) and ligand density to cellmigration. Interestingly, if the migratory path of a cell includesdifferentials in eithermatrix ligand density ormatrix rigidity, thecells will move toward the ligand-dense or more rigid regionsand away from the low ligand density or less rigid matrix inphenomena termed haptotaxis and durotaxis, respectively (Loet al., 2000).

Mammary tumors and the adjacent tumor stroma have highexogenous tension compared to normal mammary gland.Metastatic breast tumors, unlike non-metastatic tumors,frequently express high levels of lysyl oxidase, a collagen-crosslinking enzyme, and form rigid stroma in vivo.Overexpression of an isoform of lysyl oxidase in a non-metastatic breast tumor cell line is sufficient to induce bothtumor fibrosis and tumor invasion (Kirschmann et al., 2002). Inbreast tumor biopsies, invasive potential correlates with thepresence of fibrotic foci associated with the tumor stroma(Hasebe et al., 2002).


The influence of tumor stroma tension on cell phenotypewas recapitulated in 3D cultures using the basementmembraneextract Matrigel crosslinked to polyacrylamide gels of variabletension. Exogenous tension (rigidity) was adjusted to the levelsobserved in normal and tumor-containing tissue by changing thepolyacrylamide gel composition. Elevation of exogenoustension enhances Rho activity, induces cytoskeletal tension,increases focal adhesions, decreases cell–cell contact, perturbstissue polarity, and increases growth. Exogenous tension(rigidity) at levels comparable to the tumor stroma inducesintegrin clustering and activation of intracellular signaling, suchas ERK phosphorylation and ROCK-dependent contractility.Inhibiting Rho and ERK signaling was sufficient to decreaseendogenous tension in cells adhering to a rigid matrix, and thisresulted in decreased formation of focal adhesions andincreased cell–cell contacts (Fig. 4) (Paszek et al., 2005). Thus,the high exogenous tension of the tumor stroma can influencethe endogenous tension and cell–matrix adhesions that form intumor cells (Bershadsky et al., 2006). The levels of exogenoustension or rigidity also regulate preosteoblast proliferation,differentiation, and focal adhesion dynamics (Kong et al., 2005).

It is interesting to speculate that due to durotaxis, thegradient in stroma exogenous tension at the tumorboundary—high at the tumor and low in surrounding tissue—may induce a migratory response of cells in the surroundingtissue, leading to preferential recruitment of cells to the tumorand its stroma to continually increase tumor size. The idea ofcells responding to the presence of a rigid body in amatrix is notnovel. Chicken heart fibroblasts are known to align and inducemembrane protrusions toward an embedded rigid body fromadjacent compliant areas (Boocock, 1989).

Composition of the ECM. Rarely will cells encounteronly a single isolated ECM protein in vivo. The basement

Fig. 4. Increasing the exogenous tension (matrix rigidity) hasdramatic effects on intracellular signaling, matrix adhesions, andendogenous tension (contractility). An increase in exogenous tensionalters RhoA activity, the actin cytoskeleton, focal adhesions, cell–cellcontacts, tissue polarity, and importantly the growth rate. The figureshows a comparison of cellular phenotypes on matrices of differentrigidity (" indicates relative increase and # relative decrease). Thus,dense ‘‘desmoplastic’’ tumor stroma will influence the endogenoustension, matrix adhesion structures, and cell signaling pathways. Ifsome mechanism could be developed to reduce both the exogenoustension (rigidity) and endogenous tension (contractility) of rigidtumors, it might be possible to inhibit tumor growth or progression.[Color figure can be viewed in the online issue, which is available atwww.interscience.wiley.com.]


membrane and connective tissue are specialized matricescontaining networks of multiple proteins including severalcollagen isoforms, laminin isoforms, and fibronectin. Migrationfrom a collagen plug and collagen gel contractility by fibroblastsadherent to collagen or mixtures of collagen and fibronectinindicate that the matrix mixture elicits a cell phenotype that isdistinct from cells adherent to only collagen (Greiling andClark,1997; Liu et al., 2006). Consequently, this and many otherstudies indicate that the composition of the ECM, rather thansimply the presence of an extracellular scaffold, is critical forregulating cell phenotype.

Cell control of the matrix

Matrix porosity. The matrix is a meshwork of fibrillar andnon-fibrillar proteins containing pores. At a constant matrixconcentration, crosslinking of the matrix will reduce pore sizeand enhance the barrier function of thematrix (McKegney et al.,2001). Lysyl oxidase is a cell-derived collagen-crosslinkingenzyme that associates with fibronectin to form a complex thatretains crosslinking activity (Fogelgren et al., 2005). Thus, cell-derived lysyl oxidase may utilize fibronectin as a scaffold tolocally increase collagen crosslinking and decrease matrixporosity.

Matrix cleavage. Cells use matrix proteases to remodelthe matrix. ECM remodeling is required for normalphysiological processes such as embryonic development,morphogenesis, and wound repair. Tissues normally harborlow levels of matrix protease activity that are controlled byinhibitors 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 followingtissue damage, there is a shift favoring matrix protease activity,and matrix remodeling ensues. The triple-helical structure offibrillar collagen confers resistance to many proteasesexcept collagenase. Collagenase promotes uncoiling of thetriple helix and exposes additional sites that becomesusceptible to proteolysis. Thus, the activity of collagenases candisrupt the fibrous meshwork of the ECM and increase matrixporosity.

Endothelial cells provide an excellent example of inherentdifferences between 2D and 3D matrix adhesions andassociated downstreampathways that regulatematrix proteaseactivity and cell migration. Endothelial cell migration occursindependent of matrix protease activity in a 2D matrix. Incontrast, in a 3Dmatrix environment, endothelial cell migrationis dependent on protease activity. Interestingly, the activation ofmatrix metalloproteinase-2 also occurs selectively forendothelial cells cultured in a 3D matrix. Thus, 2D and 3Dmatrix adhesions induce different cell signaling pathways thatcontrol 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 displayenhanced matrix protease activities. Tumor-associated matrixproteases can promote cell invasion, in part, by increasingmatrix porosity or generating pro-migratory matrix peptides(Giannelli et al., 1997; Hotary et al., 2006). However, therequirement formatrix remodeling during tumor cell migrationin vivo is controversial, since cells can reportedly shift betweenprotease-dependent and independent migration modes (Friedland Wolf, 2003; Wolf et al., 2003). Nonetheless, carcinomacells acquire the ability to invade through the basementmembrane and connective tissues, potentially utilizing bothmatrix protease-dependent and independent modes of cellmigration.

Invadopodia aremembrane protrusions on the cell surface oftumor cells that mediate matrix cleavage. Many of thecytoplasmic proteins that are recruited to matrix adhesions inprimary cells are also recruited to invadopodia in tumor cells.


Invasive cancer cells can extend multiple invadopodia thatinduce matrix cleavage, leading to increases in matrix porosityand liberation of pro-migratory matrix peptides. Because oftheir matrix remodeling properties, invadopodia are suggestedto 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 clutchmechanism that provides a bi-directional conduit forcontrollingmechanical tension across the cell membrane (Evansand Calderwood, 2007). Endogenous intracellular tensionfluctuates in response to disruption or stabilization of thelinkages between matrix-engaged integrins, the F-actincytoskeleton, and myosin. Actomyosin contractility increasesendogenous cell tension through sliding of actin filaments andsubsequent force applied to matrix adhesions. Increasing thecytoplasmic tension of matrix-engaged integrins can transfertension to the tethered matrix and increase exogenous tension(matrix rigidity) (Smilenov et al., 1999; Brown et al., 2006; Huet al., 2007). As an example, during fibroblast migration, cellularforce applied through matrix adhesions in protrusions at theleading edge will displace the matrix centripetally, and this localmatrix stretch increases the exogenous tension.

Fibronectin is comprised of a series of modular domains thatfold into tertiary structures that can undergo conformationalrearrangements in response to tension. Elevating actomyosincontractility or endogenous tension induces cell-associatedfibronectin to undergo a conformational change that revealspreviously concealed fibronectin-binding sites and triggersfibronectin matrix assembly (Pankov and Yamada, 2002; Maoand Schwarzbauer, 2005). Adjusting either the endogenous(contractility) or exogenous tension (matrix rigidity) canpotentially regulate fibronectin matrix assembly and matrixstructure.

Potential Future Therapeutic Applications

The major advances in knowledge of the mechanisms andsequelae of cell–matrix interactions that we have summarizedin thismini review should lead to new therapeutic approaches inthe future. Principles such as the roles of matrix composition,three-dimensionality, and rigidity, as well as the existence ofdistinct types of cell–matrix adhesions and bi-directionalsignaling responses provide a rational foundation for thedevelopment of novel approaches to tissue repair andintervention in disease processes. For example, there arealready many applications of matrix molecules and syntheticbiomaterials to tissue engineering that speed wound repair andpotentially replace failed organs (Lutolf and Hubbell, 2005;Maskarinec and Tirrell, 2005; Clark et al., 2007; Kong andMooney, 2007; Metcalfe and Ferguson, 2007). Applying newknowledge of the principles of the 3D organization and rigidityof the microenvironment to controlling the cell signalingresponse should accelerate research in engineering tissues.Similarly, increasing the understanding of the roles of localtension and feedbackmechanismsmay lead to the developmentof approaches to facilitate wound repair and prevent scarringand fibrosis (Xiao et al., 2004; Metcalfe and Ferguson, 2007).

Since integrin-mediated adhesion and signaling are crucial forthrombosis and inflammation, there are major efforts alreadyunderway to develop novel therapies by manipulating integrinactivation and functions (Rose et al., 2000; Coller, 2001;Meadows and Bhatt, 2007). Complex diseases with aninflammatory and mechanical component, such asatherosclerosis, may eventually include management of cell–matrix interactions, since they play crucial roles in cellrecruitment, adhesion, and tissue remodeling (Hamm, 2003). Aless-explored area of future application of cell–matrix biologyinvolves cancer, and we propose below a few possible areas of


future research. Although highly speculative, they provideexamples of novel possible approaches based on new findings inmatrix biology.

Potential therapeutic approaches based on altering cell–matrix adhesion in cancer

Selective expression of integrin receptors on tumor cellspresents an opportunity for targeting drugs, peptides, orradioisotopes using anti-integrin antibody conjugates. Inparticular, integrin b6 expression is induced on severalcarcinomas, whereas b6 subunit expression is normally onlydetectable in the adult duringwound healing (Jones et al., 1997).Tumor cells retain expression of the b6 subunit at metastaticsites, such as in lymph nodes (Hazelbag et al., 2007). Thus, anti-avb6 antibody conjugates may provide a tool for deliveringcytotoxic agents to the primary tumor and metastatic sites forpotential therapy at both early and late tumor stages (Weinrebet al., 2004; Sheppard, 2005; Hehlgans et al., 2007). Clearly, thisapproach 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 toinvasive tumors or the associated stroma (Kirschmann et al.,2002; Erler and Giaccia, 2006), but the challenge will be how toobtain drug targeting. Invasive tumor cells expressing lysyloxidase might be targeted with a fusion protein containing as afusion partner the collagen substrate domain for lysyl oxidase.The substrate domain could crosslink the fusion protein to thetumor matrix (Lucero and Kagan, 2006).

Once a fusion protein or antibody is targeted to the tumor bytaking advantage of selective tumor cell expression of anintegrin or lysyl oxidase, a number of therapeutic approachesare possible (Fig. 5); some can take further advantage ofknowledge ofmatrix biology. The fusion protein could contain atoxin that is specifically released in active form by engineeringsites specific for matrix metalloprotease(s) known to beexpressed by the particular tumor or its stroma. Alternatively,the fusion protein could carry a selective matrix proteaseinhibitor or non-cleavable substrate to inhibit local matrixremodeling and invasion. The strategy in this case would be torestrain a potentially metastatic tumor within a molecular cagedesigned to prevent cell invasion and tumor cell matrixremodeling. Engineered fusion proteins could be injected intosites of tumor excision after surgery to target any remainingtumor cells.

Breast and other tumors can associate with dense, collagen-rich desmoplastic stroma. The high exogenous tension orrigidity associatedwith breast tumor stroma is known to inducesignaling and cytoskeletal changes, disrupt tissue polarity, andstimulate cell growth (Paszek et al., 2005). Therefore, strategiesto reduce the exogenous and endogenous tension of breast andother tumors characterized by high exogenous tension mayprovide a novel therapeutic opportunity to control tumorprogression or recurrence (Fig. 4). Developing approaches todecrease exogenous and/or endogenous tension of tumors invivo will require creative new technologies.

Conclusions

Over the past few decades, there has been exciting progress inunderstanding the molecular mechanisms that regulate theformation and function of cell–matrix adhesions. Many cellularprocesses are now known to be regulated by signals from cell–matrix adhesion structures that are transmitted bi-directionallyacross the cell membrane and dynamically link the intracellularand extracellular microenvironments. Future studies will clarifyfurther the roles of cell–matrix interactions in determiningcellular fate in vivo. This knowledge should provide a foundationfor developing molecular tools to specifically modify the cell–

Fig. 5. Speculative applications of matrix biology: lysyl oxidase (LOX)-targeted inhibitors and a molecular cage for tumors. Certain metastatictumors frequently express elevated lysyl oxidase, which leads to a rigid stroma with high exogenous tension. This selective expression of thecollagen-crosslinking enzyme lysyl oxidase in metastatic tumors could potentially provide a unique opportunity to target and crosslink proteins tothetumorstroma.Forexample,modularproteins fortargetingcouldcontainthe lysyloxidasecollagensubstratedomain(LOXsubstratedomain)fused to effector domains such as inhibitors of matrix remodeling or toxins to be crosslinked to tumor stroma. The modular protein could beengineered to contain a protease-resistant matrix protein to be crosslinked to the tumor stroma, which together with targeted matrix proteaseinhibitors could trap tumor cells within a molecular cage designed toresist invasion and matrix remodeling. In addition, a targeted toxin could alsobe crosslinked to tumor stroma that is tailored to the matrix protease activity signatureof the tumor for local activation and release. [Color figurecan be viewed in the online issue, which is available at www.interscience.wiley.com.]


matrix interface in order to control particular cellular functionssuch as gene expression, cell migration, and differentiation. Theability to control cellular phenotype in vivo could be translatedto therapeutic technologies to prevent progression of cancerand other diseases.

Acknowledgments

We appreciate the insightful comments of Marinilce dos Santosand Andrew Doyle in the preparation of this article. Thisresearchwas supported by the Intramural Research Programofthe NIH, the National Institute of Dental and CraniofacialResearch, and the National Center on Minority Health and


HealthDisparities.Wewould like to dedicate this review to thememory of Dr. Suzanne Bernier.

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