Polarized interfacial tension induces collective migration of cells,as a cluster,in a 3D tissue

In embryogenesis and cancer invasion, cells collectively migrate as a cluster in 3D tissues. Many studies have elucidated mechanisms of either individual or collective cell migration on 2D substrates; however, it remains unclear how cells collectively migrate as a cluster through 3D tissues. To address this issue, we considered the interfacial tension at cell-cell boundaries expressing cortical actomyosin contractions and cell-cell adhesive interactions. The strength of this tension is polarized; i.e., spatially biased within each cell according to a chemoattractant gradient. Using a 3D vertex model, we performed numerical simulations of multicellular dynamics in 3D space. The simulations revealed that the polarized interfacial tension enables cells to migrate collectively as a cluster through a 3D tissue. In this mechanism, interfacial tension induces unidirectional flow of each cell surface from the front to the rear along the cluster surface. Importantly, this mechanism does not necessarily require convection of cells, i.e., cell rearrangement, within the cluster. Moreover, several migratory modes were induced, depending on the strengths of polarity, adhesion, and noise; i.e., cells migrate either as single cells, as a cluster, or aligned like beads on a string, as occurs in embryogenesis and cancer invasion. These results indicate that the simple expansion and contraction of cell-cell boundaries enables cells to move directionally forward and to produce the variety of collective migratory movements observed in living systems.     

Roles of p38 MAPKs in invasion and metastasis

Cells from primary tumours need to go through several steps to become fully metastatic. During this process, cancer cells acquire the ability to invade, migrate across the surrounding tissue, enter into the circulation and colonize distant organs. In the present paper, we review recent progress in understanding how the p38 MAPK (mitogen-activated protein kinase) signalling pathway participates in the different steps of metastasis. Experimental evidence suggests that tumour cells need to modulate p38 MAPK activity levels to successfully metastasize.     

Molecular mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and cofilin

Invadopodia are actin-rich membrane protrusions with a matrix degradation activity formed by invasive cancer cells. We have studied the molecular mechanisms of invadopodium formation in metastatic carcinoma cells. Epidermal growth factor (EGF) receptor kinase inhibitors blocked invadopodium formation in the presence of serum, and EGF stimulation of serum-starved cells induced invadopodium formation. RNA interference and dominant-negative mutant expression analyses revealed that neural WASP (N-WASP), Arp2/3 complex, and their upstream regulators, Nck1, Cdc42, and WIP, are necessary for invadopodium formation. Time-lapse analysis revealed that invadopodia are formed de novo at the cell periphery and their lifetime varies from minutes to several hours. Invadopodia with short lifetimes are motile, whereas long-lived invadopodia tend to be stationary. Interestingly, suppression of cofilin expression by RNA interference inhibited the formation of long-lived invadopodia, resulting in formation of only short-lived invadopodia with less matrix degradation activity. These results indicate that EGF receptor signaling regulates invadopodium formation through the N-WASP-Arp2/3 pathway and cofilin is necessary for the stabilization and maturation of invadopodia.     

Biophysical interactions between components of the tumor microenvironment promote metastasis

During metastasis, tumor cells need to adapt to their dynamic microenvironment and modify their mechanical properties in response to both chemical and mechanical stimulation. Physical interactions occur between cancer cells and the surrounding matrix including cell movements and cell shape alterations through the process of mechanotransduction. The latter describes the translation of external mechanical cues into intracellular biochemical signaling. Reorganization of both the cytoskeleton and the extracellular matrix (ECM) plays a critical role in these spreading steps. Migrating tumor cells show increased motility in order to cross the tumor microenvironment, migrate through ECM and reach the bloodstream to the metastatic site. There are specific factors affecting these processes, as well as the survival of circulating tumor cells (CTC) in the blood flow until they finally invade the secondary tissue to form metastasis. This review aims to study the mechanisms of metastasis from a biomechanical perspective and investigate cell migration, with a focus on the alterations in the cytoskeleton through this journey and the effect of biologic fluids on metastasis. Understanding of the biophysical mechanisms that promote tumor metastasis may contribute successful therapeutic approaches in the fight against cancer.     

Nuclear Motility in Glioma Cells Reveals a Cell-Line Dependent Role of Various Cytoskeletal Components

Nuclear migration is a general term for the movement of the nucleus towards a specific site in the cell. These movements are involved in a number of fundamental biological processes, such as fertilization, cell division, and embryonic development. Despite of its importance, the mechanism of nuclear migration is still poorly understood in mammalian cells. In order to shed light on the mechanical processes underlying nuclear movements, we adapted a micro-patterning based assay. C6 rat and U87 human glioma cells seeded on fibronectin patterns - thereby forced into a bipolar morphology - displayed oscillatory movements of the nucleus or the whole cell, respectively. We found that both the actomyosin system and microtubules are involved in the nuclear/cellular movements of both cell lines, but their contributions are cell-/migration-type specific. Dynein activity was necessary for nuclear migration of C6 cells but active myosin-II was dispensable. On the other hand, coupled nuclear and cellular movements of U87 cells were driven by actomyosin contraction. We explain these cell-line dependent effects by the intrinsic differences in the overall mechanical tension due to the various cytoskeletal elements inside the cell. Our observations showed that the movements of the nucleus and the centrosome are strongly correlated and display large variation, indicating a tight but flexible coupling between them. The data also indicate that the forces responsible for nuclear movements are not acting directly via the centrosome. Based on our observations, we propose a new model for nuclear oscillations in C6 cells in which dynein and microtubule dynamics are the main drivers of nuclear movements. This mechanism is similar to the meiotic nuclear oscillations of Schizosaccharomyces pombe and may be evolutionary conserved.     

Lymphatic vessels transition to state of summation above a critical contraction frequency

Although behavior of lymphatic vessels is analogous to that of ventricles, which completely relax between contractions, and blood vessels, which maintain a tonic constriction, the mixture of contractile properties can yield behavior unique to lymphatic vessels. In particular, because of their limited refractory period and slow rate of relaxation, lymphatic vessels lack the contractile properties that minimize summation in ventricles. We, therefore, hypothesized that lymphatic vessels transition to a state of summation when lymphatic vessel contraction frequency exceeds a critical value. We used an isovolumic, controlled-flow preparation to compare the time required for full relaxation with the time available to relax during diastole. We measured transmural pressure and diameter on segments of spontaneously contracting bovine mesenteric lymphatic vessels during 10 isovolumic volume steps. We found that beat-to-beat period (frequency(-1)) decreased with increases in diameter and that total contraction time was constant or slightly increased with diameter. We further found that the convergence of beat-to-beat period and contraction cycle duration predicted a critical transition value, beyond which the vessel does not have time to fully relax. This incomplete relaxation and resulting mechanical summation significantly increase active tension in diastole. Because this transition occurs within a physiological range, contraction summation may represent a fundamental feature of lymphatic vessel function.     

Tensional Homeostasis in Single Fibroblasts

Adherent cells generate forces through acto-myosin contraction to move, change shape, and sense the mechanical properties of their environment. They are thought to maintain defined levels of tension with their surroundings despite mechanical perturbations that could change tension, a concept known as tensional homeostasis. Misregulation of tensional homeostasis has been proposed to drive disorganization of tissues and promote progression of diseases such as cancer. However, whether tensional homeostasis operates at the single cell level is unclear. Here, we directly test the ability of single fibroblast cells to regulate tension when subjected to mechanical displacements in the absence of changes to spread area or substrate elasticity. We use a feedback-controlled atomic force microscope to measure and modulate forces and displacements of individual contracting cells as they spread on a fibronectin-patterned atomic-force microscope cantilever and coverslip. We find that the cells reach a steady-state contraction force and height that is insensitive to stiffness changes as they fill the micropatterned areas. Rather than maintaining a constant tension, the fibroblasts altered their contraction force in response to mechanical displacement in a strain-rate-dependent manner, leading to a new and stable steady-state force and height. This response is influenced by overexpression of the actin crosslinker α-actinin, and rheology measurements reveal that changes in cell elasticity are also strain- rate-dependent. Our finding of tensional buffering, rather than homeostasis, allows cells to transition between different tensional states depending on how they are displaced, permitting distinct responses to slow deformations during tissue growth and rapid deformations associated with injury.     

Tensional Homeostasis in Single Fibroblasts

Adherent cells generate forces through acto-myosin contraction to move, change shape, and sense the mechanical properties of their environment. They are thought to maintain defined levels of tension with their surroundings despite mechanical perturbations that could change tension, a concept known as tensional homeostasis. Misregulation of tensional homeostasis has been proposed to drive disorganization of tissues and promote progression of diseases such as cancer. However, whether tensional homeostasis operates at the single cell level is unclear. Here, we directly test the ability of single fibroblast cells to regulate tension when subjected to mechanical displacements in the absence of changes to spread area or substrate elasticity. We use a feedback-controlled atomic force microscope to measure and modulate forces and displacements of individual contracting cells as they spread on a fibronectin-patterned atomic-force microscope cantilever and coverslip. We find that the cells reach a steady-state contraction force and height that is insensitive to stiffness changes as they fill the micropatterned areas. Rather than maintaining a constant tension, the fibroblasts altered their contraction force in response to mechanical displacement in a strain-rate-dependent manner, leading to a new and stable steady-state force and height. This response is influenced by overexpression of the actin crosslinker α-actinin, and rheology measurements reveal that changes in cell elasticity are also strain- rate-dependent. Our finding of tensional buffering, rather than homeostasis, allows cells to transition between different tensional states depending on how they are displaced, permitting distinct responses to slow deformations during tissue growth and rapid deformations associated with injury.     

A Coupled Mathematical Model of Cell Migration,Vessel Cooption and Tumour Microenvironment during the Initiation of Micrometastases

We propose a coupled mathematical model for the detailed quantitative analyses of initial microtumour and micrometastases formation by including cancer cell migration, host vessel cooption and changes in microenvironment. Migrating cells are included as a new phenotype to describe the migration behaviour of malignant tumour cells. Migration probability of a migrating cell is assumed to be influenced by local chemical microenvironment. Pre-existing vessel cooption and remodelling are introduced according to the local haemodynamical microenvironment, such as interstitial pressure and vessel wall permeability. After the tumour cells and tumour vessels distribution are updated, the chemical substances are coupled calculated with the haemodynamical environment. The simulation results clearly reproduce the tumour cells migrate and proliferate along the pre-existing vessels at the very early stage of growth, which are consistent with many published experimental observations. In addition, the model demonstrates the interactions of tumour cells with the pre-existing vessels, which are believed to be essential for initial adhesion, proliferation, invasion, and micrometastases establishment. Quantitative analysis of tumour expansion in longitudinal and transverse directions shows that the cooption and migration along host vessels will be inhibited once angiogenesis phase occurs. The influences of the ability of cell migration and the inclusion of vessel cooption on the formation of micrometastases are discussed.     

Cancer cell invasion is enhanced by applied mechanical stimulation

Metastatic cells migrate from the site of the primary tumor, through the stroma, into the blood and lymphatic vessels, finally colonizing various other tissues to form secondary tumors. Numerous studies have been done to identify the stimuli that drive the metastatic cascade. This has led to the identification of multiple biochemical signals that promote metastasis. However, information on the role of mechanical factors in cancer metastasis has been limited to the affect of compliance. Interestingly, the tumor microenvironment is rich in many cell types including highly contractile cells that are responsible for extensive remodeling and production of the dense extracellular matrix surrounding the cancerous tissue. We hypothesize that the mechanical forces produced by remodeling activities of cells in the tumor microenvironment contribute to the invasion efficiency of metastatic cells. We have discovered a significant difference in the extent of invasion in mechanically stimulated verses non-stimulated cell culture environments. Furthermore, this mechanically enhanced invasion is dependent upon substrate protein composition, and influenced by topography. Finally, we have found that the protein cofilin is needed to sense the mechanical stimuli that enhances invasion. We conclude that other types of mechanical signals in the tumor microenvironment, besides the rigidity, can enhance the invasive abilities of cancer cells in vitro. We further propose that in vivo, non-cancerous cells located within the tumor micro-environment may be capable of providing the necessary mechanical stimulus during the remodeling of the extracellular matrix surrounding the tumor.     

The motor units of cat medial gastrocnemius: electrical and mechanical properties as a function of muscle length.

The effects of changing muscle length on the mechanical properties of 89 motor units from adult cat medial gastrocnemius have been studied in eight experiments. Few differences were found between the effects of length on tetanic tension, twitch tension, twitch-tetanus ratio, twitch contraction time, twitch half relaxation time, rate of force development and electrical activity for fast contracting (twitch contraction time less than or equal to 45 msec) and slowly contracting (greater than 45 msec) units. Those differences that did appear did not persist when these two groups were matched by tetanic tension. It is concluded that the biophysical mechanisms responsible for the changes in mechanical and electrical properties with length must be similar for fast and slow twitch units and not related to potential differences in their muscle fiber type. The effects of changing muscle length on the mechanical properties of the eight whole muscles suggest that changes in force output with length are of minor importance during normal movements as the muscle is found to be electrically active over a relatively narrow range of lengths close to the optimum length for tetanus of the whole muscle. The very shortest muscle lengths at which there is only minimal force development are not used in natural movements, while the declining limb of the length tension curve is at muscle lengths beyond the maximum in situ length.     

Molecular mechanisms of cell segregation and boundary formation in development and tumorigenesis

The establishment and maintenance of precisely organized tissues requires the formation of sharp borders between distinct cell populations. The maintenance of segregated cell populations is also required for tissue homeostasis in the adult, and deficiencies in segregation underlie the metastatic spreading of tumor cells. Three classes of mechanisms that underlie cell segregation and border formation have been uncovered. The first involves differences in cadherin-mediated cell-cell adhesion that establishes interfacial tension at the border between distinct cell populations. A second mechanism involves the induction of actomyosin-mediated contraction by intercellular signaling, such that cortical tension is generated at the border. Third, activation of Eph receptors and ephrins can lead to both decreased adhesion by triggering cleavage of E-cadherin, and to repulsion of cells by regulation of the actin cytoskeleton, thus preventing intermingling between cell populations. These mechanisms play crucial roles at distinct boundaries during development, and alterations in cadherin or Eph/ephrin expression have been implicated in tumor metastasis.     

Melanoma Cells Break Down LPA to Establish Local Gradients That Drive Chemotactic Dispersal

The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient.     

The role of calponin in the gene profile of metastatic cells: inhibition of metastatic cell motility by multiple calponin repeats

Metastasis of diseased cells is the basic event leading to death in individuals with cancer. Establishment of metastasis requires that tumour cells migrate from the site of the primary tumour into the circulation system, escape from the vasculature and form secondary tumours at novel sites. These processes depend to a large degree on cytoskeletal remodeling. We show here that multiple copies of the short actin-binding module CLIK(23) from human or Caenorhabditis elegans calponin proteins effectively inhibit cell motility on two dimensional matrices and suppress soft agar colony formation of metastatic melanoma and adenocarcinoma cells of murine and human origin. Ectopic expression of CLIK(23) modules for 30 days results in the formation of multinucleated cells. The repeat displays true modular behaviour, resulting in increased cytoskeletal effects in direct correlation with the increase in number of modules. Our results demonstrate that the role of calponin in the signature profile of metastasising cells is that of a mechanical stabiliser of the actin cytoskeleton, which interferes with actin turnover by binding at a unique interface along the actin filament.     

Computer modelling of gastrulation and neurulation in amphibian embryos based on mechanical tension fields

The values of cell wall tensions were calculated with an assumption of mechanical equilibrium of every cell apex on schematic diagrams of histological sections of the common frog embryonic tissues. The maps of the main (the strongest) tensions were drawn for the early gastrula and early neurula. The further course of gastrulation and neurulation was simulated with an assumption that the cell apices are displaced due to active contractions of the most tensed cell walls (variant A of the model). In addition, a suggestion was studied that the capacity for contraction falls in the most extended cell walls (variant B). Up to seven steps of model morphogenesis were simulated and the tension field was recalculated at every step. The course of gastrulation and neurulation was reproduced during simulation with sufficient details, including regional peculiarities of neurulation in the trunk and head regions. Both variants gave roughly similar results for gastrulation, whereas variant B ensured a faster course of model morphogenesis for neurulation. A conclusion was drawn that the mechanical tension fields established by the onset of gastrulation and neurulation represent a sufficient informational base for their further course.     

Production of plasminogen activator by migrating cephalic neural crest cells.

Neural crest cells migrate extensively during embryonic development and differentiate into a wide variety of cell types. Our working hypothesis is that during migration, embryonic cells secrete proteases which modify local microenvironments, thereby facilitating directed cellular movements. In this communication, we report studies on the migration of cephalic neural crest cells in the avian embryo. We demonstrate that these cells produce high levels of the serine protease, plasminogen activator (PA), at the time of their initial migration from the neural tube and during their migration to and colonization of the developing head and neck.     

Morphomechanics: goals, basic experiments and models

Morphomechanics is a branch of developmental biology, studying the generation, space-time patterns and morphogenetic role of mechanical stresses (MS) which reside in embryonic tissues. All the morphogenetically active embryonic tissues studied in this respect have been shown to bear substantial mechanical stresses of tension or pressure. MS are indispensable for organized cell movements, expression of a number of developmentally important genes and the very viability of cells. Even a temporary relaxation of MS leads to an increase in the morphological variability and asymmetry of embryonic rudiments. Moreover, MS may be among the decisive links of morphogenetic feedback required for driving forth embryonic development and providing its regular space-time patterns. We hypothesize that one such feedback is based upon the tendency of cells and tissues to hyperrestore (restore with an overshoot) their MS values after any deviations, either artificial or produced by neighboring morphogenetically active tissues. This idea is supported by a number of observations and experiments performed on the tissue and individual cell levels. We describe also the models demonstrating that a number of biologically realistic stationary shapes and propagating waves can be generated by varying the parameters of the hyperrestoration feedback loop. Morphomechanics is an important and rapidly developing branch of developmental and cell biology, being complementary to other approaches.     

Collective Cell Migration in Embryogenesis Follows the Laws of Wetting

Collective cell migration is a fundamental process during embryogenesis and its initial occurrence, called epiboly, is an excellent in vivo model to study the physical processes involved in collective cell movements that are key to understanding organ formation, cancer invasion, and wound healing. In zebrafish, epiboly starts with a cluster of cells at one pole of the spherical embryo. These cells are actively spreading in a continuous movement toward its other pole until they fully cover the yolk. Inspired by the physics of wetting, we determine the contact angle between the cells and the yolk during epiboly. By choosing a wetting approach, the relevant scale for this investigation is the tissue level, which is in contrast to other recent work. Similar to the case of a liquid drop on a surface, one observes three interfaces that carry mechanical tension. Assuming that interfacial force balance holds during the quasi-static spreading process, we employ the physics of wetting to predict the temporal change of the contact angle. Although the experimental values vary dramatically, the model allows us to rescale all measured contact-angle dynamics onto a single master curve explaining the collective cell movement. Thus, we describe the fundamental and complex developmental mechanism at the onset of embryogenesis by only three main parameters: the offset tension strength, α, that gives the strength of interfacial tension compared to other force-generating mechanisms; the tension ratio, δ, between the different interfaces; and the rate of tension variation, λ, which determines the timescale of the whole process.     

Talin2 mediates secretion and trafficking of matrix metallopeptidase 9 during invadopodium formation

Talin2 plays an important role in transduction of mechanical signals between extracellular matrix and actin cytoskeleton. Recent studies showed that talin2 is localized to invadopodia and regulates their maturation, subsequently cancer cell invasion and metastasis. However, the molecular mechanism whereby talin2 mediates invadopodium maturation is unknown. Here we show that ablation of talin2 in MDA-MB-231 cells inhibited the secretion of matrix metallopeptidase 9 (MMP9), a proteinase involved in extracellular matrix degradation in invadopodium maturation and metastasis. Furthermore, re-expression of talin2^WT in talin2-KO cells rescued MMP9 secretion, but talin2^S339C, a mutant with reduced β-integrin binding, did not, indicating that the talin2-β-integrin interaction is involved in the MMP9 secretion. Moreover, ablation of talin2 caused an accumulation of enlarged MMP9 vesicles. These vesicles co-localized with enlarged early, late endosomes and autophagosomes, suggesting talin2 controls MMP9 trafficking process. Therefore, these data suggest that talin2 regulates extracellular matrix degradation and invadopodium maturation by mediating MMP9 secretion.