Emerging modes of collective cell migration induced by geometrical constraints

The role of geometrical confinement on collective cell migration has been recognized but has not been elucidated yet. Here, we show that the geometrical properties of the environment regulate the formation of collective cell migration patterns through cell-cell interactions. Using microfabrication techniques to allow epithelial cell sheets to migrate into strips whose width was varied from one up to several cell diameters, we identified the modes of collective migration in response to geometrical constraints. We observed that a decrease in the width of the strips is accompanied by an overall increase in the speed of the migrating cell sheet. Moreover, large-scale vortices over tens of cell lengths appeared in the wide strips whereas a contraction-elongation type of motion is observed in the narrow strips. Velocity fields and traction force signatures within the cellular population revealed migration modes with alternative pulling and/or pushing mechanisms that depend on extrinsic constraints. Force transmission through intercellular contacts plays a key role in this process because the disruption of cell-cell junctions abolishes directed collective migration and passive cell-cell adhesions tend to move the cells uniformly together independent of the geometry. Altogether, these findings not only demonstrate the existence of patterns of collective cell migration depending on external constraints but also provide a mechanical explanation for how large-scale interactions through cell-cell junctions can feed back to regulate the organization of migrating tissues.     

From the Cover: Microfabricated tissue gauges to measure and manipulate forces from 3D microtissues

Physical forces generated by cells drive morphologic changes during development and can feedback to regulate cellular phenotypes. Because these phenomena typically occur within a 3-dimensional (3D) matrix in vivo, we used microelectromechanical systems (MEMS) technology to generate arrays of microtissues consisting of cells encapsulated within 3D micropatterned matrices. Microcantilevers were used to simultaneously constrain the remodeling of a collagen gel and to report forces generated during this process. By concurrently measuring forces and observing matrix remodeling at cellular length scales, we report an initial correlation and later decoupling between cellular contractile forces and changes in tissue morphology. Independently varying the mechanical stiffness of the cantilevers and collagen matrix revealed that cellular forces increased with boundary or matrix rigidity whereas levels of cytoskeletal and extracellular matrix (ECM) proteins correlated with levels of mechanical stress. By mapping these relationships between cellular and matrix mechanics, cellular forces, and protein expression onto a bio-chemo-mechanical model of microtissue contractility, we demonstrate how intratissue gradients of mechanical stress can emerge from collective cellular contractility and finally, how such gradients can be used to engineer protein composition and organization within a 3D tissue. Together, these findings highlight a complex and dynamic relationship between cellular forces, ECM remodeling, and cellular phenotype and describe a system to study and apply this relationship within engineered 3D microtissues.     

Lifestyle Interventions to Reduce Diabetes and Cardiovascular Disease Risk Among Children

Type 1 diabetes (T1D) results from progressive immune cell-mediated destruction of pancreatic β cells. As immune cells migrate into the islets, they pass through the extracellular matrix (ECM). This ECM is composed of different macromolecules localized to different compartments within and surrounding islets; however, the involvement of this ECM in the development of human T1D is not well understood. Here, we summarize our recent findings from human and mouse studies illustrating how specific components of the islet ECM that constitute basement membranes and interstitial matrix of the islets, and surprisingly, the intracellular composition of islet β cells themselves, are significantly altered during the pathogenesis of T1D. Our focus is on the ECM molecules laminins, collagens, heparan sulfate/heparan sulfate proteoglycans, and hyaluronan, as well as on the enzymes that degrade these ECM components. We propose that islet and lymphoid tissue ECM composition and organization are critical to promoting immune cell activation, islet invasion, and destruction of islet β cells in T1D.     

Single-cell response to stiffness exhibits muscle-like behavior

Living cells sense the rigidity of their environment and adapt their activity to it. In particular, cells cultured on elastic substrates align their shape and their traction forces along the direction of highest stiffness and preferably migrate towards stiffer regions. Although numerous studies investigated the role of adhesion complexes in rigidity sensing, less is known about the specific contribution of acto-myosin based contractility. Here we used a custom-made single-cell technique to measure the traction force as well as the speed of shortening of isolated myoblasts deflecting microplates of variable stiffness. The rate of force generation increased with increasing stiffness and followed a Hill force–velocity relationship. Hence, cell response to stiffness was similar to muscle adaptation to load, reflecting the force-dependent kinetics of myosin binding to actin. These results reveal an unexpected mechanism of rigidity sensing, whereby the contractile acto-myosin units themselves can act as sensors. This mechanism may translate anisotropy in substrate rigidity into anisotropy in cytoskeletal tension, and could thus coordinate local activity of adhesion complexes and guide cell migration along rigidity gradients.     

From the Cover: Regulation of cell motile behavior by crosstalk between cadherin- and integrin-mediated adhesions

During normal development and in disease, cohesive tissues undergo rearrangements that require integration of signals from cell adhesions to neighboring cells and to the extracellular matrix (ECM). How a range of cell behaviors is coordinated by these different adhesion complexes is unknown. To analyze epithelial cell motile behavior in response to combinations of cell–ECM and cell–cell adhesion cues, we took a reductionist approach at the single-cell scale by using unique, functionalized micropatterned surfaces comprising alternating stripes of ECM (collagenIV) and adjustable amounts of E-cadherin-Fc (EcadFc). On these surfaces, individual cells spatially segregated integrin- and cadherin-based complexes between collagenIV and EcadFc surfaces, respectively. Cell migration required collagenIV and did not occur on surfaces functionalized with only EcadFc. However, E-cadherin adhesion dampened lamellipodia activity on both collagenIV and EcadFc surfaces and biased the direction of cell migration without affecting the migration rate, all in an EcadFc concentration-dependent manner. Traction force microscopy showed that spatial confinement of integrin-based adhesions to collagenIV stripes induced anisotropic cell traction on collagenIV and migration directional bias. Selective depletion of different pools of αE-catenin, an E-cadherin and actin binding protein, identified a membrane-associated pool required for E-cadherin–mediated adhesion and down-regulation of lamellipodia activity and a cytosolic pool that down-regulated the migration rate in an E-cadherin adhesion-independent manner. These results demonstrate that there is crosstalk between E-cadherin– and integrin-based adhesion complexes and that E-cadherin regulates lamellipodia activity and cell migration directionality, but not cell migration rate.     

Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis

Cell migration on 2D surfaces is governed by a balance between counteracting tractile and adhesion forces. Although biochemical factors such as adhesion receptor and ligand concentration and binding, signaling through cell adhesion complexes, and cytoskeletal structure assembly/disassembly have been studied in detail in a 2D context, the critical biochemical and biophysical parameters that affect cell migration in 3D matrices have not been quantitatively investigated. We demonstrate that, in addition to adhesion and tractile forces, matrix stiffness is a key factor that influences cell movement in 3D. Cell migration assays in which Matrigel density, fibronectin concentration, and beta1 integrin binding are systematically varied show that at a specific Matrigel density the migration speed of DU-145 human prostate carcinoma cells is a balance between tractile and adhesion forces. However, when biochemical parameters such as matrix ligand and cell integrin receptor levels are held constant, maximal cell movement shifts to matrices exhibiting lesser stiffness. This behavior contradicts current 2D models but is predicted by a recent force-based computational model of cell movement in a 3D matrix. As expected, this 3D motility through an extracellular environment of pore size much smaller than cellular dimensions does depend on proteolytic activity as broad-spectrum matrix metalloproteinase (MMP) inhibitors limit the migration of DU-145 cells and also HT-1080 fibrosarcoma cells. Our experimental findings here represent, to our knowledge, discovery of a previously undescribed set of balances of cell and matrix properties that govern the ability of tumor cells to migration in 3D environments.     

Contractility of the cell rear drives invasion of breast tumor cells in 3D Matrigel

Cancer cells use different modes of migration, including integrin-dependent mesenchymal migration of elongated cells along elements of the 3D matrix as opposed to low-adhesion-, contraction-based amoeboid motility of rounded cells. We report that MDA-MB-231 human breast adenocarcinoma cells invade 3D Matrigel with a characteristic rounded morphology and with F-actin and myosin-IIa accumulating at the cell rear in a uropod-like structure. MDA-MB-231 cells display neither lamellipodia nor bleb extensions at the leading edge and do not require Arp2/3 complex activity for 3D invasion in Matrigel. Accumulation of phospho-MLC and blebbing activity were restricted to the uropod as reporters of actomyosin contractility, and velocimetric analysis of fluorescent beads embedded within the 3D matrix showed that pulling forces exerted to the matrix are restricted to the side and rear of cells. Inhibition of actomyosin contractility or β1 integrin function interferes with uropod formation, matrix deformation, and invasion through Matrigel. These findings support a model whereby actomyosin-based uropod contractility generates traction forces on the β1 integrin adhesion system to drive cell propulsion within the 3D matrix, with no contribution of lamellipodia extension or blebbing to movement.     

Collective mesendoderm migration relies on an intrinsic directionality signal transmitted through cell contacts

Collective cell migration is key to morphogenesis, wound healing, or cancer cell migration. However, its cellular bases are just starting to be unraveled. During vertebrate gastrulation, axial mesendoderm migrates in a group, the prechordal plate, from the embryonic organizer to the animal pole. How this collective migration is achieved remains unclear. Previous work has suggested that cells migrate as individuals, with collective movement resulting from the addition of similar individual cell behavior. Through extensive analyses of cell trajectories, morphologies, and polarization in zebrafish embryos, we reveal that all prechordal plate cells show the same behavior and rely on the same signaling pathway to migrate, as expected if they do so individually. However, by using cell transplants, we demonstrate that prechordal plate migration is a true collective process, as isolated cells do not migrate toward the animal pole. They are still polarized and motile but lose directionality. Directionality is restored upon contact with the endogenous prechordal plate. This contact dependent orientation relies on E-cadherin, Wnt-PCP signaling, and Rac1. Importantly, groups of cells also need contact with the endogenous plate to orient correctly, showing an instructive role of the plate in establishing directionality. Overall, our results lead to an original model of collective migration in which directional information is contained within the moving group rather than provided by extrinsic cues, and constantly maintained in cells by contacts with their neighbors. This self-organizing model could account for collective invasion of new territories, as observed in cancer strands, without requirement for any attractant in the colonized tissue.     

Insulin-like growth factor binding protein-2 binding to extracellular matrix plays a critical role in neuroblastoma cell proliferation, migration, and invasion

IGF binding proteins (IGFBPs) modulate IGF cellular bioavailability and may directly regulate tumor growth and invasion. We have previously shown that IGFBP-2 binds and localizes IGF-I to the pericellular matrix and have provided some evidence suggesting that the heparin binding domain (HBD) or the arginine-glycine-aspartic acid (RGD) integrin binding motif may be involved in these interactions. However, the precise mechanisms involved remain to be elucidated. We therefore mutated the HBD or RGD sequence of IGFBP-2 and investigated consequent effects on extracellular matrix (ECM) binding, IGF-induced proliferation, and migration of neuroblastoma cells. IGFBP-2 and its arginine-glycine-glutamic acid (RGE) mutant similarly bound ECM components, whereas binding of mutant HBD-IGFBP-2 to each of the ECM substrates was markedly reduced by 70-80% (P < 0.05). IGF-I (100 ng/ml) increased incorporation of 3H-thymidine in neuroblastoma SK-N-SHEP cells by approximately 30%, an effect blunted by exogenously added native or either mutant IGFBP-2. Overexpression of IGFBP-2 and its RGE mutant potently promoted SHEP cell proliferation (5-fold), whereas SHEP cell proliferation was negligible when HBD-IGFBP-2 was overexpressed. Addition or overexpression of IGFBP-2 and its RGE mutant potently (P < 0.05) enhanced SHEP cell migration/invasion through the ECM. However, overexpression of the HBD-IGFBP-2 mutant potently inhibited (50-60%) SHEP cell invasion through ECM. Thus, IGFBP-2, which binds to the ECM, enhances proliferation and metastatic behavior of neuroblastoma cells, functions that directly or indirectly use the HBD but not the integrin binding sequence. Our novel findings thus point to a key role for the HBD of IGFBP-2 in the control and regulation of neuroblastoma growth and invasion.     

Evidence of a large-scale mechanosensing mechanism for cellular adaptation to substrate stiffness

Cell migration plays a major role in many fundamental biological processes, such as morphogenesis, tumor metastasis, and wound healing. As they anchor and pull on their surroundings, adhering cells actively probe the stiffness of their environment. Current understanding is that traction forces exerted by cells arise mainly at mechanotransduction sites, called focal adhesions, whose size seems to be correlated to the force exerted by cells on their underlying substrate, at least during their initial stages. In fact, our data show by direct measurements that the buildup of traction forces is faster for larger substrate stiffness, and that the stress measured at adhesion sites depends on substrate rigidity. Our results, backed by a phenomenological model based on active gel theory, suggest that rigidity-sensing is mediated by a large-scale mechanism originating in the cytoskeleton instead of a local one. We show that large-scale mechanosensing leads to an adaptative response of cell migration to stiffness gradients. In response to a step boundary in rigidity, we observe not only that cells migrate preferentially toward stiffer substrates, but also that this response is optimal in a narrow range of rigidities. Taken together, these findings lead to unique insights into the regulation of cell response to external mechanical cues and provide evidence for a cytoskeleton-based rigidity-sensing mechanism.     

Cell-ECM traction force modulates endogenous tension at cell-cell contacts

Cells in tissues are mechanically coupled both to the ECM and neighboring cells, but the coordination and interdependency of forces sustained at cell-ECM and cell-cell adhesions are unknown. In this paper, we demonstrate that the endogenous force sustained at the cell-cell contact between a pair of epithelial cells is approximately 100?nN, directed perpendicular to the cell-cell interface and concentrated at the contact edges. This force is stably maintained over time despite significant fluctuations in cell-cell contact length and cell morphology. A direct relationship between the total cellular traction force on the ECM and the endogenous cell-cell force exists, indicating that the cell-cell tension is a constant fraction of the cell-ECM traction. Thus, modulation of ECM properties that impact cell-ECM traction alters cell-cell tension. Finally, we show in a minimal model of a tissue that all cells experience similar forces from the surrounding microenvironment, despite differences in the extent of cell-ECM and cell-cell adhesion. This interdependence of cell-cell and cell-ECM forces has significant implications for the maintenance of the mechanical integrity of tissues, mechanotransduction, and tumor mechanobiology.     

The Extracellular Matrix and Cell Migration

As we know all too well, pancreatic cancer has a very poor prognosis largely due to its early tendency to invade, locally and distantly. Recently, scientists in the field have increasingly focused on the desmoplastic reaction, which is characteristic of most pancreatic cancers. This reaction is associated with proliferation of fibroblastic cells, sometimes outnumbering local tumor cells, and consists of abundant extracellular matrix (ECM) proteins. Importantly, the processes of invasion and metastasis take place within this tumor microenvironment. Stroma and tumor cells exchange signals to modify the local ECM, which subsequently stimulates cell migration and promotes proliferation and survival. Even though recognition of the significance of these microenvironment interactions exists, knowledge on the mechanisms ofthe interplay among pancreatic cells, myofibroblasts, and the ECM is lacking. Therefore, this ‘Pancreatology and the Web’ focuses on websites that provide information on the ECM and cell migration.     

Mechanical regulation of cellular phenotype: implications for vascular tissue regeneration

Cells sense a myriad of cues from their surrounding microenvironment to regulate their function. In recent years, it has become clear that physical and mechanical cues are as critical as biochemical factors in regulating cellular function. The geometry of the extracellular matrix (ECM), degree of cell spreading, and ECM rigidity all influence the physical connection between cells and their microenvironment and play a major role in regulating proliferation, differentiation, and migration. Leveraging these findings to promote specific cell behaviours will be paramount to realize the full potential of cellular therapies. In this review, I examine our current understanding of how mechanical cues-specifically, geometric control of cell shape and matrix rigidity-are transduced by stem cells to control their stemness, proliferation, and differentiation. The implications of these findings for vascular smooth muscle cell differentiation and cardiovascular tissue engineering will be highlighted.     

Overexpression of ECM1 contributes to migration and invasion in cholangiocarcinoma cell

Although the expression of extracellular matrix protein-1 (ECM1) has been documented in several tumor models, the function of ECM1 has remained unclear. In this study, expression of ECM1 was detected by real time PCR and immunohistochemistry. The role and mechanism of ECM1 overexpression in cholangiocarcinoma (CCA) cells were assessed by wound-healing, matrigel invasion assay and Western blotting. Expression of ECM1 was significantly elevated in CCA tissues than that in adjacent noncancerous, cholangitis and normal bile duct tissues. Its overexpression was associated with poor differentiation, lymph node metastasis, poor prognosis, and the level of CA199, MMP-9, estrogen receptor. Knockdown of ECM1 suppressed migration and invasion of CCA cells. Using PI3K or IKK inhibitor reduced the level of phospho-Akt or phospho-IκBα as well as ECM1. Taken together, overexpression of ECM1 may contribute to CCA initiation and progression through promoting migration and invasion of CCA cells, its overexpression was associated with Akt/NF-κB signaling axis.     

Measuring dynamic cell–material interactions and remodeling during 3D human mesenchymal stem cell migration in hydrogels

Biomaterials that mimic aspects of the extracellular matrix by presenting a 3D microenvironment that cells can locally degrade and remodel are finding increased applications as wound-healing matrices, tissue engineering scaffolds, and even substrates for stem cell expansion. In vivo, cells do not simply reside in a static microenvironment, but instead, they dynamically reengineer their surroundings. For example, cells secrete proteases that degrade extracellular components, attach to the matrix through adhesive sites, and can exert traction forces on the local matrix, causing its spatial reorganization. Although biomaterials scaffolds provide initially well-defined microenvironments for 3D culture of cells, less is known about the changes that occur over time, especially local matrix remodeling that can play an integral role in directing cell behavior. Here, we use microrheology as a quantitative tool to characterize dynamic cellular remodeling of peptide-functionalized poly(ethylene glycol) (PEG) hydrogels that degrade in response to cell-secreted matrix metalloproteinases (MMPs). This technique allows measurement of spatial changes in material properties during migration of encapsulated cells and has a sensitivity that identifies regions where cells simply adhere to the matrix, as well as the extent of local cell remodeling of the material through MMP-mediated degradation. Collectively, these microrheological measurements provide insight into microscopic, cellular manipulation of the pericellular region that gives rise to macroscopic tracks created in scaffolds by migrating cells. This quantitative and predictable information should benefit the design of improved biomaterial scaffolds for medically relevant applications.Synthetic hydrogel scaffolds have been designed to serve as mimics of the native extracellular matrix (ECM) with the goal of promoting desired cell functions (e.g., proliferation, migration, differentiation), especially for applications in wound healing (1), tissue regeneration (2), and stem cell culture (3, 4). For example, poly(ethylene glycol) (PEG) hydrogels can serve as blank slates in which peptide cues can be systematically introduced in the scaffold to allow integrin binding (5, 6), proteolytic degradation (7, 8), and even local sequestering of growth factors (9). Furthermore, it is well known that cells respond to mechanical stimuli (e.g., stiffness) in their local microenvironment, the so-called pericellular region, and tuning of a scaffold’s mechanical properties can influence how a cell degrades and remodels its surroundings (10–12). The complex cell–matrix interactions that occur in the native ECM are often mimicked in peptide-functionalized hydrogels through the incorporation of adhesive binding peptides (e.g., RGDS, IKVAV) and enzymatically degradable peptide cross-linkers (e.g., GPQGIWGQ, GPLGLWAR), both of which are necessary for cell attachment, spreading (13), and motility (12, 14). However, changes in the local material properties as a result of this cell-mediated remodeling have largely remained a “black box,” limiting interpretation of data and confounding the design of more advanced biomaterials.Macroscopically, cells degrade micrometer-sized channels into scaffolds as they move, an event that begins with microscopic remodeling of their pericellular region and eventually permanently reengineering the scaffold architecture and material properties on a larger scale. If one seeks to design synthetic ECM environments to direct cellular processes, such as migration, it is important to better understand how these inputs are dynamically altered on the local length scale. Such information can help advance biomaterial design, especially for applications focused on the delivery or recruitment of cells, where directing cell–material interactions and migration can be critically important. At present, cell matrices are generally engineered to have certain initial material properties, but the resulting cell motility and cell–material interactions are often only empirically correlated with these design parameters (7, 15). To overcome this obstacle and provide an in situ measurement of scaffold degradation, microrheological measurements have been used to fingerprint and understand changes in material properties in the pericellular region during cell motility.Although real-time measurements of material properties near a cell are difficult, investigations have focused on developing techniques to access this information. In two dimensions, forces that cells exert when seeded on hydrogel surfaces have been measured using deflection of beds of microneedles (15) and deformation of gel surfaces (16). For example, Tan et al. (15) developed a measurement technique that exploits independent deflection of microneedles of varying lengths (and therefore stiffnesses) to measure the distribution of subcellular traction forces of both smooth muscle cells and fibroblasts. The main conclusion was that cellular spreading and morphology control the magnitude of the traction forces (15). The traction force of confluent cell sheets interacting with a gel surface was also analyzed, toward understanding how cellular processes are coordinated over large length scales. Using endothelial, epithelial, and breast cancer cell sheets, results showed that collective migration was due to a transmittance of normal stress across cell–cell junctions with migration orientated in the direction of the minimal intercellular shear stress (16).Cell-mediated degradation of the local microenvironment plays a critical role in permitting cellular migration and invasion in vivo. These processes are important during development, wound regeneration, and pathophysiological states facilitated by proteolytic events via cellular protease secretion. Previous work has begun to elucidate the length scales and spatial effects of secreted proteases in relation to migrating tumor cells during collagen matrix remodeling (17–19). For example, Packard et al. (20) used matrix metalloproteinase (MMP)-sensitive biosensors to visualize protease activity in the pericellular region of migrating tumor cells in collagen, finding increased activity at the polarized leading edge. These seminal works have elucidated the spatial presence and local activity of proteases in relation to individual migrating cells. However, how migrating cells temporally degrade and remodel the local microenvironment on larger length scales remains relatively unknown.Although 2D studies add to our understanding of cell–matrix interactions, 2D environments can unnaturally polarize cells, and some aspects of cell motility can be quite different in 2D versus 3D environments (21, 22). For these reasons, recent developments have focused on strategies to measure cell–material interactions in three dimensions (e.g., cell-laden hydrogels). Traction force microscopy measures spatial interfacial forces by quantifying the elastic deformation of a substrate (21). If the modulus of the material is known, this technique quantifies the forces cells exert in three dimensions calculated from embedded bead displacement. This approach has identified patterns of forces generated around distinct morphological regions during cellular invasion into a scaffold (21). Additionally, Bloom et al. (23) investigated the degradation of a collagen scaffold during the migration of a fibrosarcoma cell line (HT1080s) using embedded particle displacements. The authors showed that the hydrogel was reversibly deformed at the cell’s leading edge, but irreversibly remodeled at the trailing edge. Collectively, these pioneering investigations have provided insight into aspects of the complex interplay between cells and scaffold materials; however, complementary techniques that allow characterization of dynamic and local changes in mechanical properties, degradation, and scaffold erosion would be beneficial in further advancing our understanding of mechanotransduction, mechanisms of cell motility, and even biomaterials design.In this contribution, multiple particle tracking microrheology (MPT) is used to measure how human mesenchymal stem cells (hMSCs) remodel peptide cross-linked PEG hydrogels as they migrate. hMSC migration is characterized by significant remodeling of the local environment through attachment, enzymatic degradation, and cellular traction. Furthermore, hMSCs are observed to degrade the synthetic network through two pathways, MMP secretion that cleaves the peptide cross-linker and myosin II-regulated adhesion and reversible remodeling of the network. We find that MPT has the sensitivity to capture the temporal transition of the hydrogel from an elastic gel to a viscous liquid, during hMSC-mediated degradation. MPT simultaneously provides information about the spatial region, proximal to the cell, over which this matrix remodeling occurs. The technique and measurements enhance our understanding of cell–material interactions in three dimensions and enable visualization of dynamic cell-mediated matrix degradation, the so-called fourth dimension. On longer timescales, these microscopic changes give rise to the creation of macroscopic channels in the hydrogel that are important for hMSC motility. We believe that this approach and characterization can provide an important link for better understanding outside-in signaling experienced by cells when embedded in 3D environments.     

Matrix metalloproteinase and alphavbeta3 integrin-dependent vascular smooth muscle cell invasion through a type I collagen lattice

Smooth muscle cell (SMC) migration from the tunica media to the intima is a key event in the development of atherosclerotic lesions and in restenosis after angioplasty. SMCs require not only migratory but also degradative abilities that enable them to migrate through extracellular matrix proteins, which surround and embed these cells. We used a collagen type I lattice as a coating on top of a porous filter as a matrix barrier in a chamber to test the invasive behavior of SMCs in response to a chemoattractant (invasion assay) and compared that behavior with simple SMC migration through collagen type I-coated filters (migration assay). Inhibitors of matrix metalloproteinase, KB-R8301, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), TIMP-2, and peptide 74, attenuated platelet-derived growth factor-BB (PDGF-BB)-directed SMC invasion across the collagen lattice, whereas no effect was seen with these inhibitors on simple SMC migration through collagen-coated filters. RGD peptide inhibited SMC invasion but did not affect SMC migration. Anti-alphavbeta3 integrin antibody attenuated PDGF-BB-directed SMC invasion, whereas other antibodies against RGD-recognizing integrins, namely alphavbeta5 and alpha5, had no effect. None of these antibodies had any effect on simple SMC migration. RGD peptide and anti-alphavbeta3 antibody inhibited the attachment and spreading of SMCs on denatured collagen but not on native collagen. These findings indicate that there is a difference in the mechanisms between simple SMC migration across a collagen-coated filter and SMC invasion through a fibrillar collagen barrier. A proteolytic process is required for SMC invasion, and the degradation of matrix proteins alters the relationship between matrix protein molecules and SMC surface integrins.     

Segregation of lipid raft markers including CD133 in polarized human hematopoietic stem and progenitor cells

During ontogenesis and the entire adult life hematopoietic stem and progenitor cells have the capability to migrate. In comparison to the process of peripheral leukocyte migration in inflammatory responses, the molecular and cellular mechanisms governing the migration of these cells remain poorly understood. A common feature of migrating cells is that they need to become polarized before they migrate. Here we have investigated the issue of cell polarity of hematopoietic stem/progenitor cells in detail. We found that human CD34(+) hematopoietic cells (1) acquire a polarized cell shape upon cultivation, with the formation of a leading edge at the front pole and a uropod at the rear pole; (2) exhibit an amoeboid movement, which is similar to the one described for migrating peripheral leukocytes; and (3) redistribute several lipid raft markers including cholesterol-binding protein prominin-1 (CD133) in specialized plasma membrane domains. Furthermore, polarization of CD34(+) cells is stimulated by early acting cytokines and requires the activity of phosphoinositol-3-kinase as previously reported for peripheral leukocyte polarization. Together, our data reveal a strong correlation between polarization and migration of peripheral leukocytes and hematopoietic stem/progenitor cells and suggest that they are governed by similar mechanisms.     

Proteolytic-antiproteolytic balance and its regulation in carcinogenesis

Cancer development is essentially a tissue remodeling process in which normal tissue is substituted with cancer tissue. A crucial role in this process is attributed to proteolytic degradation of the extracellular matrix (ECM). Degradation of ECM is initiated by proteases, secreted by different cell types, participating in tumor cell invasion and increased expression or activity of every known class of proteases (metallo-, serine-, aspartyl-, and cysteine) has been linked to malignancy and invasion of tumor cells. Proteolytic enzymes can act directly by degrading ECM or indirectly by activating other proteases, which then degrade the ECM. They act in a determined order, resulting from the order of their activation. When proteases exert their action on other proteases, the end result is a cascade leading to proteolysis. Presumable order of events in this complicated cascade is that aspartyl protease (cathepsin D) activates cysteine proteases (e.g. cathepsin B) that can activate pro-uPA. Then active uPA can convert plasminogen into plasmin. Cathepsin B as well as plasmin are capable of degrading several components of tumor stroma and may activate zymogens of matrix metalloproteinases, the main family of ECM degrading proteases. The activities of these proteases are regulated by a complex array of activators, inhibitors and cellular receptors. In physiological conditions the balance exists between proteases and their inhibitors. Proteolytic-antiproteolytic balance may be of major significance in the cancer development. One of the reasons for such a situation is enhanced generation of free radicals observed in many pathological states. Free radicals react with main cellular components like proteins and lipids and in this way modify proteolytic-antiproteolytic balance and enable penetration damaging cellular membrane. All these lead to enhancement of proteolysis and destruction of ECM proteins and in consequence to invasion and metastasis.     

Host epithelial geometry regulates breast cancer cell invasiveness

Breast tumor development is regulated in part by cues from the local microenvironment, including interactions with neighboring nontumor cells as well as the ECM. Studies using homogeneous populations of breast cancer cell lines cultured in 3D ECM have shown that increased ECM stiffness stimulates tumor cell invasion. However, at early stages of breast cancer development, malignant cells are surrounded by normal epithelial cells, which have been shown to exert a tumor-suppressive effect on cocultured cancer cells. Here we explored how the biophysical characteristics of the host microenvironment affect the proliferative and invasive tumor phenotype of the earliest stages of tumor development, by using a 3D microfabrication-based approach to engineer ducts composed of normal mammary epithelial cells that contained a single tumor cell. We found that the phenotype of the tumor cell was dictated by its position in the duct: proliferation and invasion were enhanced at the ends and blocked when the tumor cell was located elsewhere within the tissue. Regions of invasion correlated with high endogenous mechanical stress, as shown by finite element modeling and bead displacement experiments, and modulating the contractility of the host epithelium controlled the subsequent invasion of tumor cells. Combining microcomputed tomographic analysis with finite element modeling suggested that predicted regions of high mechanical stress correspond to regions of tumor formation in vivo. This work suggests that the mechanical tone of nontumorigenic host epithelium directs the phenotype of tumor cells and provides additional insight into the instructive role of the mechanical tumor microenvironment.     

The role of extracellular matrix stiffness in megakaryocyte and platelet development and function

The extracellular matrix (ECM) is a key acellular structure in constant remodeling to provide tissue cohesion and rigidity. Deregulation of the balance between matrix deposition, degradation, and crosslinking results in fibrosis. Bone marrow fibrosis (BMF) is associated with several malignant and nonmalignant pathologies severely affecting blood cell production. BMF results from abnormal deposition of collagen fibers and enhanced lysyl oxidase‐mediated ECM crosslinking within the marrow, thereby increasing marrow stiffness. Bone marrow stiffness has been recently recognized as an important regulator of blood cell development, notably by modifying the fate and differentiation process of hematopoietic or mesenchymal stem cells. This review surveys the different components of the ECM and their influence on stem cell development, with a focus on the impact of the ECM composition and stiffness on the megakaryocytic lineage in health and disease. Megakaryocyte maturation and the biogenesis of their progeny, the platelets, are thought to respond to environmental mechanical forces through a number of mechanosensors, including integrins and mechanosensitive ion channels, reviewed here.