Extracellular Matrix Dynamics: a Key Regulator of Cell Migration across Length-Scales and Systems

Abstract

The interactions between cells and their surrounding extracellular matrix (ECM) are dynamic and play critical roles in cell migration during development, health, and diseases. Recent advances have highlighted the complexity and diversity of ECM compositions, or “matrisomes”, of tissues resulting in ECMs of different physical, mechanical, and biochemical properties. Investigating the effects of these properties on cell-ECM interactions in the context of cell migration have led to a better understanding of the principles underlying tissue morphogenesis, wound healing, immune response, or cancer metastasis. These new insights into the interplay between ECM dynamics and cell migration can lead to the identification of unique opportunities for therapeutic interventions.

INTRODUCTION

The extracellular matrix (ECM) is a complex 3-dimensional (3D) meshwork of proteins that provides structural support to cells, tissues, organs, and organisms[1]. In addition, the ECM provides physical, mechanical, and chemical cues that regulate proliferation, migration, differentiation, and function during development, tissue morphogenesis, and homeostasis[2]. Thus, alterations in ECM composition results in the perturbation of its overall architectural properties and associated signaling functions, leading to various pathologies including developmental defects, fibrosis, and cancer[3–5].

The completion of genome sequencing has led to the conceptualization of the “matrisome”, the ensemble of genes, ranging from over 600 in Drosophila to over 1000 in zebrafish and mammals, encoding structural ECM proteins, such as collagens and proteoglycans, and associated regulatory factors, such as ECM remodeling enzymes or ECM-bound growth factors or morphogens (see Glossary and https://matrisome.org)[1,6]. In parallel, proteomic studies have shown that the ECM of any given tissue is composed of over 100 distinct matrisome proteins present in varying abundances and giving rise to unique architectures across tissues[7,8]. ECM protein isoforms resulting from alternative splicing, post-translational modifications, and enzymatic and non-enzymatic remodeling further amplify the complexity and diversity of the ECM imparting it with distinct physical, mechanical, and chemical properties[2,9–11].

Cell migration is essential for development, tissue maintenance, immune function, and is fatal during pathologies such as cancer metastasis[12]. Abundant studies have uncovered factors that regulate cell migration on 2D substrates. In the last decade, more studies have focused on understanding cell migration in 3D scaffolds made from collagen I, reconstituted basement matrix (Matrigel^®) and synthetic/semi-synthetic materials. However, until recently, only few studies have used systems that fully recapitulate the complexity of in-vivo ECMs (e.g., decellularized ECM from organs or tissues)[13]. Thus, little is known about how the ECM meshwork, as a whole, modulates cell migration. Using examples taken from different organisms and pathophysiological states, this review discusses recent studies that have shone light on how ECM deposition and organization, and the resulting mechanical and chemical signals contribute to modulating cell migration (Figure 1).

Figure 1: ECM regulation of cell migration.

The human matrisome consists of over 1000 genes coding for 195 glycoproteins (fibronectin, laminins, fibrillins, tenascins, etc.), 44 collagens, 35 proteoglycans and over 750 matrisome-associated proteins. These genes are translated, secreted and subsequently assembled extracellularly to form through interactions with cell surface receptors and other ECM proteins. The ECM exists in two main forms, a sheet-like basement membrane or an interstitial meshwork. The ECM further undergoes enzymatic and non-enzymatic remodeling, mainly mediated by matrisome-associated proteins. ECM organization results in physical, mechanical, and biochemical properties which are all interdependent. Collectively, these properties regulate different modes of cell migration during tissue morphogenesis and repair, immune response, and cancer metastasis. Figure created with BioRender.com.

LOCAL ECM DEPOSITION & ORGANIZATION REGULATES CELL MIGRATION

Cells secrete and deposit their ECM and actively organize it through cell surface receptors to facilitate their migration (Figure 2A). Fibronectin is one of the most abundant and well-studied fibrillar ECM proteins, and the mechanisms leading to its assembly mediated by integrin receptors is now well characterized in vitro[14]. Interestingly, recent experiments on the developing chicken embryo and associated computational modeling have demonstrated a novel mode of fibronectin organization mediated by migrating neural crest cells (NCCs). During NCC migration into the cranial region, leader cells remodel the unorganized and punctate fibronectin into a linear and filamentous form. Newly organized fibronectin then acts as a directional cue for efficient migration of follower cells[15] but also contributes to the assembly of other ECM proteins affecting overall ECM architecture. Similarly, during craniofacial skeleton development in mice, the interaction between skeletal progenitor cells and the collagen-rich ECM regulates proliferation, migration, and differentiation[16]. Loss of expression or mutations in discoidin domain receptor 2 (DDR2), a known collagen receptor, causes craniofacial malformations[17]. Yet, the molecular mechanisms through which DDR2 affects craniofacial development is not clear. In a recent study, Ddr2 expression was shown to be required for the homogenous deposition and linear organization of collagen II by suture stem cells and chondrocytes, since disruption in the distribution and orientation of collagen II resulted in abnormal cell migration and bone development[18]. The above-mentioned studies show how the organizations of two of the most abundant ECM proteins, fibronectin and collagens, regulate cell migration independently. It is well established that fibronectin acts as a scaffold for procollagen processing and thereby collagen fiber assembly[19–21]. As a result, the organization of the fibronectin meshwork translates to the organization of the entire ECM. Moving forward, it will be important to understand the interplay between different protein meshworks within the ECM and how this interplay modulates cell migration.

Figure 2: Interdependent properties of the ECM and impact on cell migration.

A) ECM organization, remodeling and signaling. Cells secrete and organize ECM proteins, in part, through interaction with cell surface receptors such as the integrins. ECM organization regulates cell migration along with imparting physical and mechanical properties. Upon binding to cell surface receptors, ECM proteins elicit biochemical signaling leading to cytoskeleton remodeling and the modulation cell migration. In addition, cells secrete enzymes that remodel the ECM (e.g., crosslinking, degradation) which further impacts ECM-mediated physical, mechanical, and chemical signaling.

B) Physical properties of the ECM regulating cell migration. Physical properties such as pore size and fiber bundling are derived from ECM organization and remodeling. Alterations in ECM composition, organization, crosslinking, or degradation changes pore size which in turn effects immune cell infiltration. Similarly, fiber bundling due to increased crosslinking effects cancer cell migration during metastasis.

C) Mechanical properties of the ECM affecting cell migration. Variations in the mechanical properties of the ECM, such as stiffness, viscoelasticity, and topography, are dependent on ECM organization and physical properties. Viscoelasticity of the ECM allows cells to generate transient gradients in fiber alignment and deformation leading to efficient cell migration. Cancer cells adapt to the surrounding ECM stiffness by switching between different modes of cell migration during metastasis. Figure created with BioRender.com.

In addition to regulating cell migration during pre-natal development (see above), the ECM also plays a key role in post-natal tissue morphogenesis. For example, in Drosophila oocyte development, the local deposition of a basement membrane ECM on the basal side by the follicular epithelium is critical for the subsequent assembly of ECM proteins secreted by the fat body on ovariole and stalk cells. This “mosaic” basement membrane organization is key for stalk cell dynamics, organization, and normal morphogenesis of the ovariole[22,23]. In mammals, post-natal mammary gland development involves extensive branching morphogenesis during pregnancy where terminal end buds (TEBs) branch and penetrate farther into the fat pad as ducts elongate. Pre-patterned collagen I networks in the fat pad have been proposed to bias ductal growth along the major axis of the developing gland[24]. Yet, the temporal aspects of collagen I network alignment in the fat pad and how aligned collagen I fibers might direct TEB extension remains unclear. Recent experimental and computational approaches have now revealed that it is the local ECM accumulation confines the branching angle of TEBs and drives the global bias in epithelial orientation along the major axis of the fat pad[25]. These observations present a challenge to the conventional view that global signals regulate the orientation of the mammary epithelium. Instead, local ECM accumulation and resulting mechanical signals are sufficient to dictate the global tissue patterning of the mammary gland.

In tumors, we now know that both epithelial cancer cells and cancer-associated fibroblasts (CAFs) contribute to the secretion and the organization of the tumor ECM[5,8]. Recent evidence shows that breast cancer cells continuously organize and align collagen fibers using the DDR1 receptor[26]. Collagen I fibers in breast cancer can also be stabilized by other ECM proteins, including other collagens like collagen XII [27]. In pancreatic ductal adenocarcinoma, tumor cells actively remodel the tumor ECM by secreting proteases and other enzymes[28,29]. For example, downregulation of MMP1 in cancer cells results in the partial processing of procollagen leading to alteration of the organization of the ECM meshwork[29]. Heterogenous cancer-cell-derived collagen fibers not only promote cancer metastasis but also hinder immune cell infiltration (Figure 2A)[26,30]. A hypothesis for this observation is that T cells prefer to navigate through loosely packed ECM composed of thin fibers rather than dense ECM meshworks composed of thick fibers. In recent study, Sadjadi and coworkers report that T cells can move through the collagen network, by displacing or stretching collagen fibers resulting in a channel. These channels facilitate the movement of other T cells, such that cells entering already existing channels move faster and tend to remain in the existing channel network[31]. The examples discussed in this section highlight the interplay between ECM composition and organization mediated by cell surface receptors in regulating cell migration during tissue morphogenesis and disease progression.

PHYSICAL PROPERTIES OF THE ECM INFLUENCE CELL MIGRATION

The assembly of ECM proteins into an organized meshwork dictates the physical properties of the ECM including pore size, fiber bundle thickness and cross-linked geometry (see Glossary). It is becoming increasingly clear that these physical cues regulate cell migration (Figure 2B). New insights have emerged from the use of advanced tools to label ECM components in vivo coupled with the use of sophisticated imaging modalities. In a recent study, Soans et al., have labeled laminin α-1, a major component of the basement membrane, to study the relationship between ECM pore size and cell migration during zebrafish optic cup development[32]. Cells in the rim region migrate in a step-like manner along the basement membrane ECM by extending long cryptic lamellipodia at the leading front. Increasing the pore size by downregulating laminin α-1 or nidogen expression drastically altered the topography of the underlying basement membrane leading to poor cryptic lamellipodia formation and rim cell migration along the ECM. This suggests the lack of a linear relationship between pore size and the efficiency of cryptic lamellipodia formation. This also suggests that there is an optimal porosity of the underlying ECM that allows rim cells to move efficiently using cryptic lamellipodia[32].

ECMs in vivo exist in heterogenous topologies: sheet-like basement membranes and fibrillar interstitial ECM (see Glossary, Figure 1). One way to examine how different ECM topologies regulate cell migration is to use tunable synthetic hydrogels to incorporate fibrous architecture and reconstituted matrices. In a recent study, Hiraki et al., demonstrated that cancer cells solely migrated as single cells in non-fibrous synthetic hydrogel and their migration dynamics was inversely correlated with hydrogel stiffness. Interestingly, incorporation of fibers in non-fibrous hydrogels promoted collective cell migration and the migration dynamics increased with increase in crosslinking and stiffness of fibers[33]. In contrast, using experimental and computational approaches, Pally and coworkers demonstrated that presence of non-fibrous ECM (e.g., reconstituted basement membrane) around breast tumor organoids elicited collective cell migration while fibrous ECM (e.g., collagen I) induced single cell migration[34]. Both studies demonstrate the importance of heterogeneity in stromal ECM topology (non-fibrous and fibrous) in defining tumor-cell migration modality[33,34].

It is important to appreciate the interdependency of different physical properties of the ECM. For example, increased collagen bundling results in increased pore size and stiffens (mechanical property, see below) the ECM which enhances overall invasion of 3D breast cancer organoid[30]. Hence, variations in a given physical property not only affects another physical property but also results in alternations of certain mechanical properties of the ECM. Decoupling the effects of each biophysical properties has been difficult but a recent study by Koorman and co-workers used threose-based advanced glycation end (AGE) to crosslink collagen fibers and study its effects on cell migration without affecting ECM pore size. In this study, the authors show that collective cell invasion is inhibited by increased stiffness irrespective of ECM pore size, whereas single cell invasion is promoted with both increased stiffness and collagen pore size[30]. This suggests that cancer cells can sense changes in stiffness and can respond to these changes by switching between different modes of invasion. In summary, physical properties such as pore size, topology, and cross-linking arising from different ECM compositions and organizations exert significant influence on cell migration in both physiological and pathological contexts.

MECHANICAL CUES FROM THE ECM REGULATE CELL MIGRATION

Mechanical properties of the ECM, such as stiffness, viscoelasticity, and confinement (see Glossary), derive from its compositional and physical characteristics. Growing evidence suggests that mechanical properties influence cell migration via mechano-transduction[10,35]. Yet, we still do not fully understand how active mechanical remodeling of the ECM affects cell migration (Figure 2C).

Viscoelasticity is a characteristic feature of the ECM and, over the past decade, studies i have shown that it affects cellular differentiation, fibrosis, and tumor progression[10]. But how short-term changes in viscoelasticity can affect long-term cellular processes such as cell migration and tissue morphogenesis are still elusive. Recent studies have shown that collectively migrating cells generate transient gradients in collagen density and alignment due to viscoelastic relaxation of the collagen networks[36]. Experimental evidence and theoretical models have revealed that crosslinking of collagen networks or smaller sized cell clusters result in decreased network deformation, shorter viscoelastic relaxation time and smaller gradients at the migratory front resulting in lower migration persistence[37]. This suggests that local reorganization of the collagen network at the leading front results in local asymmetries in force distributions and collagen stiffness which further promote finger like protrusions in leader cells to persist migration during tissue morphogenesis and invasion[36,37].

The mechanical properties of the ECM not only influence collective cell migration but also modulate single-cell migration. T cells in lymphatic organs migrate either by interacting with ECM through integrins present at the cell surface or by mechanically sensing the ECM topography and transmitting forces[38]. Geometrically irregular environment mechanically loads the cell membrane leading to formation of Wiskott-Aldrich syndrome protein (WASp) mediated actin patches. The retrograde flow of actin patches generates local pushing forces necessary for forward locomotion in obstructive ECM[39,40]. This mechanism is dependent on ECM topography-triggered mechano-transduction and does not require transmembrane receptors to assemble the actin cytoskeleton.

Cancer cells, too, sense mechanical properties of their surrounding ECM. We now know that cancer cells switch between different modes of invasion depending on their state along the epithelium-to-mesenchyme spectrum. Epithelial-like cells with high expression of E-cadherin are thought to invade collectively while mesenchymal-like cells have low or no E-cadherin expression and invade as single cells[41]. But recent studies attribute microenvironmental conditions such as topology and stiffness to regulate such plasticity[33,34,42]. Increasing fiber stiffness beyond a certain range potentially increases the solid stress acting at the invading front leading to confinement[43]. Thus, ECM confinement progressively jams highly motile mesenchymal-like cells to invade collectively as a flock[15,30,43,44].

THE ECM ELICITS SIGNALING CASCADES REGULATING CELL MIGRATION

In addition to the structural, physical, and mechanical properties of the ECM influencing cell migration, the ECM also signals biochemically to cells through receptors (Figure 2A). During Drosophila egg development, collective follicle cell rotations and mechanical stress exerted by collagen IV fibrils in the basement membrane regulate elongation of spherical egg chambers along the antero-posterior axis. Recent evidence suggests that collagen IV secretion and deposition as oriented fibrils on the basal side of follicle epithelium act as a persistent cue, guiding the orientation of F-actin fibers (stress fibers) through the Dystroglycan (Dg)-Dystrophyin (Dy) complex[22,23,45]. Such orientation of stress fibers is essential for egg chamber elongation[45].

ECM signaling is critical for tissue homeostasis and is altered during tissue damage and repair. A large body of work has focused on how the cellular factors such as cell proliferation, and migration contribute to wound healing. On the contrary, very few studies have focused on understanding how ECM signaling regulates tissue repair. Chakraborty et al., have demonstrated that agrin, a proteoglycan whose role is very well characterized in neuromuscular junction during development, is one of the several ECM proteins that are immediately secreted into the wounded area by keratinocytes. Moreover, locally secreted agrin sensitizes keratinocytes to collectively migrate by reorganizing their cytoskeleton and secreting MMP12, which further remodels the ECM for sustained cell migration and successful wound healing in mice (Figure 2A)[46]. While agrin-mediated mechano-transduction via YAP/TAZ in tumors and angiogenesis is very well studied[47], the molecular mechanisms downstream of the novel agrin/MMP12 axis in keratinocyte migration and wound healing are yet to be discovered[46].

Akin to keratinocyte migration during wound healing, epithelial cancer cells rely on cell-cell and cell-ECM interactions for collective invasion during metastasis. We have come across multiple examples in this review highlighting how local physical and mechanical changes in the ECM modulate collective cancer cell invasion. The first evidence linking spatial alteration in ECM organization and biochemical signaling are now emerging. For example, in breast cancer, it was recently shown that cancer cells secrete ECM proteins such as laminin 332, collagen XVII, and collagen IV at the invading front. These localized changes in the ECM composition locally activate the ECM receptors integrins α1 and α2 on cells, resulting in the activation of Src and focal adhesion kinase, which are known drivers of cancer cell invasion[48,49].

CONCLUDING REMARKS AND FUTURE PERSPECTIVES:

Recent research has demonstrated that the highly dynamic and complex interplay between cells and their surrounding ECM plays critical roles in guiding cell migration during tissue morphogenesis, tissue repair, immune response, and cancer metastasis.

Classical approaches have involved perturbing a protein to study its effect on cell migration. However, we now know that disrupting one ECM protein has ripple effect on the ECM as a whole: for example, altering collagen IV, laminins, or nidogen expression perturbs the entire basement membrane ECM[32,45]. In addition, various combinations of ECM components and their organization give rise to interdependent physical, mechanical, and chemical features. Hence, future studies must appreciate the complexity of ECM and design orthogonal experiments to dissect the inter-dependent properties.

With the advent of technologies like proteomics, it has also emerged that ECM compositions are much more complex and diverse than originally anticipated and go far beyond the abundant fibronectin and collagens. Thus, future studies should focus on deciphering how other matrisome proteins modulates the physical, mechanical, and signaling properties of the ECM and cell-ECM interactions in the context of cell migration.

Importantly, the diversity of the matrisome also offers many potential opportunities to modulate cell migration, either to enhance migration for example to accelerate wound healing or inhibit migration to prevent metastasis. These opportunities include 1) targeting signaling pathways that result in expression of ECM genes and regulate local ECM deposition[50] 2) targeting ECM remodeling enzymes to perturb ECM organization [51], 3) targeting cell surface receptors to modulate ECM organization [51], or 4) targeting ECM-dependent signaling pathways[52]. Although many inhibitors have been developed to target the ECM (e.g., targeting TGF-β1, integrins, lysyl oxidase, matrix metalloproteinases, focal adhesion kinase[50,53,54]) none have been shown to directly affect cell migration in vivo through changes in the composition, structure, or signaling functions of the ECM. Moreover, none are approved for use in patients yet. Hence, it is critical to gain a better understanding of how the ECM regulates cell migration to design better modulating strategies to achieve a therapeutic response.

HIGHLIGHTS.

• The matrisome is the set of genes encoding extracellular matrix components

• The composition of the extracellular matrix is tissue-specific

• Tissue-specific matrisomes results in unique protein assemblies and ECM architectures

• ECM composition confers interdependent physical, mechanical, and chemical properties

• Changes in ECM composition, assemblies, and properties regulate cell migration

GLOSSARY

Extracellular matrix (ECM)

3-dimensional meshwork of proteins that surrounds and supports cells and tissues

• Basement membrane ECM: Thin (20–50 nm) sheet-like ECM layer that lines the basal side of epithelial and endothelial tissues; essential for tissue homeostasis and function. The major components include laminins, nidogens, collagen IV, and the proteoglycan, perlecan
• Interstitial ECM: The fibrous scaffold that makes up the bulk of connective tissues. The major components include fibrillar collagens (collagens I, II, III, V) and fibronectin that are in large part secreted by fibroblasts, myofibroblasts, and smooth muscle cells, although other cell types can contribute to the secretion of these components

Matrisome

Parts-list of ECM components predicted via sequence analysis. By extension, the term “matrisome” also now refers to the ensemble of ECM and ECM-associated proteins of a given tissue or organ. For a detailed description of all matrisome components, see [1,6]

• Core matrisome: Subset of ECM components comprising glycoproteins, collagens, and proteoglycans that form the structural ECM scaffold. Sequence analysis has shown that in the human genome, 195 genes encode ECM glycoproteins, 44 genes encode collagens, and 35 genes encode proteoglycans
• Matrisome-associated proteins: Subset of matrisome proteins that are either structurally related to ECM proteins or that are known or predicted to bind to or modify core matrisome proteins. Sequence analysis has shown that in the human genome, 171 genes encode ECM-affiliated proteins, 238 genes encode ECM regulators including matrix metalloporteinases (MMPs) and cross-linking enzymes, and 344 genes encode secreted factors

Example of physical properties of the ECM

• Fiber bundle: Collagen self-assemble into micro fibrils (<5 nm thickness) in a head-to-tail orientation. Groups of collagen microfibrils are crosslinked covalently together to form a thicker fiber (~100 nm thickness). Further assembly of such thicker fibers into a microstructure are called fiber bundles (~10 μm thickness). Several core matrisome components also form fibers such as fibronectin, fibrillins, and elastin
• Pore size: The empty spaces that arise due to crosslinking and bundling of ECM fibers. Generally, measured as the distance between adjacent ECM fibers
• Topology: Overall 3-dimensional arrangement and organization of ECM proteins
• Topography: Structural characteristics of ECM organization in tissues or organs such as alignment, texture, porosity etc

Example of mechanical properties of the ECM

• Viscoelasticity: Property of ECM where it behaves like a viscous fluid and elastic material that attempts to return to its original forms when a deforming force is applied
• Stiffness: The extent to which ECM resists deformation when an external force is applied
• ECM confinement: Emergent property of the ECM where progressive increase in stiffness confines singly migrating cells to collectively migrate