Collective Cell Movement Research Articles

Abstract 1397: The effects of mevalonate pathway inhibitors on the motility and invasion of aggressive breast cancers

Abstract The mevalonate pathway is a metabolic pathway responsible for synthesizing prenyl (both farnesyl and geranylgeranyl) groups and activating small GTPase proteins through transfer of these groups by covalent modification. These GTPase proteins include RhoA, RhoC, Rac and Cdc42, which regulate the actin cytoskeleton during cellular migration and are over-expressed in many cancers. This project explores the effects of three mevalonate pathway inhibitors–zoledronic acid, atorvastatin and geranylgeranyl transferase inhibitor (GGTI)–on the single-cell motility, collective cell migration and invasion of an aggressive breast cancer line (MDA-MB-231) and an inflammatory breast cancer derived (IBC) line (SUM149). We are using MCF10A immortalized breast cells as our “normal-like” control. We hypothesize that these cancers rely on this pathway for cellular motility, invasion and the structure of the actin cytoskeleton more than normal epithelial cells. We observed significant decreases in total quantities of Rac, RhoC and Cdc42 and a striking increase in RhoA upon treatment with each separate inhibitor, observed by western blot. The effect is much more potent with atorvastatin and GGTI, resulting in an almost complete loss of lamellipodia and filopodia. We assayed for invasion through 3D culture transwell chambers, individual cell motility using a bead motility assay and live-cell imaging, and collective cell migration using a wound healing assay. In general, all three drugs were highly effective at inhibiting both types of cell migration and invasion in MDA-MB-231 cells. While all drugs inhibited the collective cell migration of SUM149 cells, only GGTI additionally inhibited invasion and single cell migration. This result suggests that an approach targeting geranylgeranylation may be more effective for inhibition of IBC cells. Typically IBC tumors use collective cell migration mechanisms to metastasize, whereas MDA-MB-231 tumors metastasize through single-cell migration. Our results show that aggressive breast cancers heavily rely on the mevalonate pathway for cellular motility in comparison to normal epithelial cells, and that inhibitors of this pathway have significant potential to inhibit the metastatic properties of these two tumor types, given their mechanism of tissue invasion. We are continuing to study collective cell movement by assaying for cell-cell adhesion molecules upon treatment with our three inhibitors. In addition, we are studying the effects of these inhibitors on metastasis in vivo using xenograft mouse models and Her2Neu transgenic mouse models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1397. doi:10.1158/1538-7445.AM2011-1397

Collective Cell Movements during Vasculogenesis

Group motility of endothelial cells is studied in vivo using a recently developed TIE1-YFP transgenic quail line and in cell cultures. Statistical analysis of motility data show that endothelial cell movements are guided by intercellular contacts, both during vasculogenic sprout formation and within the walls of large early vessels like the dorsal aortae and vitelline arteries. This coordinated, but unoriented cell motility turns into an organized migration towards the heart after the onset of circulation. To interpret our empirical findings, we propose a computational model of endothelial cell motility which includes a positive feedback between cell polarity and cytoskeletal expansion, as well as response to micromechanical cues from the environment.

Nonlinear hyperbolic-elliptic systems in the bounded domain

In the article we study a hyperbolic-elliptic system ofPDE. The system can describe two different physical phenomena: 1st one isthe motion of magnetic vortices in the II-type superconductor and 2nd oneis the collective motion of cells. Motivated by real physics, we considerthis system with boundary conditions, describing the flux of vortices (andcells, respectively) through the boundary of the domain. We prove the globalsolvability of this problem. To show the solvability result we use a'viscous' parabolic-elliptic system. Since the viscous solutions do not havea compactness property, we justify the limit transition on a vanishingviscosity, using a kinetic formulation of our problem. As the final resultof all considerations we have solved a very important question related witha so-called 'boundary layer problem', showing the strong convergence of theviscous solutions to the solution of our hyperbolic-elliptic system.

Automated inference of 4‐D cell polarization fields in an agent‐based model of early vertebrate embryogenesis

We present a theoretical model of animal morphogenesis construed as a self‐organized phenomenon emerging from a complex system made of a myriad of individual cell behaviors. It is implemented in an agent‐based simulation centered on the mechanic‐chemical coupling between cellular and genetic dynamics. The goal is to integrate the collective motion of cells and the dynamics of their gene expression underlying the patterning of morphogenetic fields. Within this larger framework, we examine here cell intercalation, a generic process underlying tissue elongation in vertebrate embryogenesis, in particular the convergence and extension movements shaping the embryonic axes during gastrulation. Cell intercalation is thought to arise from polarized cell movements. In our model, we include monopolar and bipolar protrusive activity of polarized cells to investigate the causal bottom‐up link from local cell behavior to global tissue deformation. Conversely, this model also allows reverse inference, e.g., by evolutionary optimization, of the local quantitative features of cell adhesion and polarization from a global 4 D (3 D + time) computational reconstruction of the cell lineage tree, itself based on in toto imaging data of developing zebrafish embryos.

Cell Motility on Varying Substrate Stiffness

Endothelial cell motility has been widely studied, but most research observes single cell motion or collective cell migration on a glass substrate. Although previous findings are informative, there are few studies that observe collective cell migration on soft, physiological substrates. Here, we studied the effects of substrate stiffness on cell migration within a monolayer. To obtain substrates with different mechanical properties, polyacrylamide gels of varying stiffness were made. These gels were coated with the extracellular matrix protein fibronectin, and the surface was plated with a high density of human umbilical vein endothelial cells (HUVEC) or bovine aortic endothelial cells (BAEC). After the cells formed a complete monolayer, they were stained with Hoechst nuclear stain to visualize individual cell movement within the monolayer. We observed that the collective cell movement is slower as the substrate stiffness increases. Also, we noticed an interesting swirling motion within the monolayer, the dynamics of which were substrate-dependent. These results suggest that collective cell migration during tissue morphogenesis depends on the mechanical properties of the cellular environment, which often vary in diseased conditions.

Quantitative In vivo Imaging of the Effects of Inhibiting Integrin Signaling via Src and FAK on Cancer Cell Movement: Effects on E-cadherin Dynamics

Most cancer-related deaths are due to the development of metastatic disease, and several new molecularly targeted agents in clinical development have the potential to prevent disease progression. However, it remains difficult to assess the efficacy of antimetastatic agents in the clinical setting, and an increased understanding of how such agents work at different stages of the metastatic cascade is important in guiding their clinical use. We used optical window chambers combined with photobleaching, photoactivation, and photoswitching to quantitatively measure (a) tumor cell movement and proliferation by tracking small groups of cells in the context of the whole tumor, and (b) E-cadherin molecular dynamics in vivo following perturbation of integrin signaling by inhibiting focal adhesion kinase (FAK) and Src. We show that inhibition of Src and FAK suppresses E-cadherin-dependent collective cell movement in a complex three-dimensional tumor environment, and modulates cell-cell adhesion strength and endocytosis in vitro. This shows a novel role for integrin signaling in the regulation of E-cadherin internalization, which is linked to regulation of collective cancer cell movement. This work highlights the power of fluorescent, direct, in vivo imaging approaches in the preclinical evaluation of chemotherapeutic agents, and shows that inhibition of the Src/FAK signaling axis may provide a strategy to prevent tumor cell spread by deregulating E-cadherin-mediated cell-cell adhesions.

Collective cell motion in endothelial monolayers

Collective cell motility is an important aspect of several developmental and pathophysiological processes. Despite its importance, the mechanisms that allow cells to be both motile and adhere to one another are poorly understood. In this study we establish statistical properties of the random streaming behavior of endothelial monolayer cultures. To understand the reported empirical findings, we expand the widely used cellular Potts model to include active cell motility. For spontaneous directed motility we assume a positive feedback between cell displacements and cell polarity. The resulting model is studied with computer simulations and is shown to exhibit behavior compatible with experimental findings. In particular, in monolayer cultures both the speed and persistence of cell motion decreases, transient cell chains move together as groups and velocity correlations extend over several cell diameters. As active cell motility is ubiquitous both in vitro and in vivo, our model is expected to be a generally applicable representation of cellular behavior.

P104. Apoptosis drives collective cell movement that is required for the morphogenesis of Drosophila male terminalia

During animal development, dynamic cellular behaviors, including cell movement, divisions and death, are precisely orchestrated. The execution mechanisms of complex behaviors has been mainly deciphered through the identification of conserved signaling pathways that control each cellular movement, however, how cellular behaviors regulate morphogenesis according to the developmental timetable is elusive. To understand the mechanisms of complex morphogenesis, we have investigated the development of Drosophila male terminalia. The Drosophila male terminalia is an asymmetric looping organ; the internal genitalia (spermiduct) loops dextrally around the hindgut. Mutants for apoptotic signal have the orientation defect of their male terminalia, indicating that apoptosis may contribute to looping morphogenesis. We studied the role of cell death in the organogenesis of male terminalia using time-lapse imaging. In normal flies, genitalia rotation accelerated as development proceeded, to complete the full 360° rotation, however, the acceleration was impaired by suppression of apoptosis. The acceleration was produced by two distinct rotations of the A8 segment that surrounded the male genitalia (A9 segment): inner ring primarily rotates with genitalia, and outer ring additionally rotates later to accelerate the primary rotation. Inhibition of apoptosis suppressed this additional rotation. Thus, we found that apoptotic cell death coordinates two independent rotations, which drives the acceleration of genitalia rotation, enabling the complete morphogenesis of male genitalia within a limited developmental time window.

P05. Mechanical force generated by leading edge mesoderm modulates collective cell polarization in axial mesoderm during Xenopus gastrulation

Gastrulation is one of the most important processes during the morphogenesis of early embryo, involving dynamic cell shape change and migration. In chordates, notochord, a rod-shape structure that contributes to body extension and neural tube formation, is formed by collective cell movements of the axial mesoderm (AM) through gastrulation. During notochord formation, AM cells become narrow along mediolateral (M–L) axis and such polarized cells intercalate each other to extend the tissue along anterior–posterior (A–P) axis. This process, called convergent extension (CE), requires the establishment of cell polarity at the same time and in the same direction in AM. Although planar cell polarity (PCP) pathway is essential for cell polarization and regulation of CE, little is known about how the polarity of AM cells is established collectively at the beginning of CE. During Xenopus gastrulation, leading edge mesoderm (LEM), positioned in front of the AM, attach to the blastocoel roof and migrate towards the animal pole. The LEM contributes to the directional migration of involuting marginal zone cells. Therefore, we hypothesized that anterior migration of the LEM generate the pulling force along A–P direction, and the generated force stretch AM cells to establish M–L cell polarity. To test this hypothesis, we first performed mesoderm migration assay in vitro and found that the LEM migrated faster than the AM. We measured the force generated by the LEM in vitro using thin glass pipette. The estimated force varied but ranged from tens to hundreds of nano-newtons. These results suggest that the LEM generate the pulling force to the AM in a significant order of force magnitude. Next, to test whether the pulling force induces collective cell polarization in the AM, we developed stretch assay system using a silicone chamber. To determine the magnitude of stretch force applied to the activin-induced AM on the scale same as we observed, we measured the Young’s modulus of the AM using glass pipette and calculated the force by formula of Hooke’s law. Using these methods, we estimated that 5–15% stretch is probably equal to LEM-generated force. We are currently examining the effect of such stretch on AM with comparable magnitudes of force generated by LEM, and determine if the stretching affects polarity formation process. This study might contribute to better understanding of the distribution of force fields and the function of mechanical factors in animal development.

Planar Cell Polarity Acts Through Septins to Control Collective Cell Movement and Ciliogenesis

The planar cell polarity (PCP) signaling pathway governs collective cell movements during vertebrate embryogenesis, and certain PCP proteins are also implicated in the assembly of cilia. The septins are cytoskeletal proteins controlling behaviors such as cell division and migration. Here, we identified control of septin localization by the PCP protein Fritz as a crucial control point for both collective cell movement and ciliogenesis in Xenopus embryos. We also linked mutations in human Fritz to Bardet-Biedl and Meckel-Gruber syndromes, a notable link given that other genes mutated in these syndromes also influence collective cell movement and ciliogenesis. These findings shed light on the mechanisms by which fundamental cellular machinery, such as the cytoskeleton, is regulated during embryonic development and human disease.

Individual Cell Movement, Asymmetric Colony Expansion, Rho-Associated Kinase, and E-Cadherin Impact the Clonogenicity of Human Embryonic Stem Cells

Clonality is, at present, the only means by which the self-renewal potential of a given stem cell can be determined. To assess the clonality of human embryonic stem cells (hESC), a protocol involving seeding wells at low cell densities is commonly used to surmount poor cloning efficiencies. However, factors influencing the accuracy of such an assay have not been fully elucidated. Using clonogenic assays together with time-lapse microscopy, numerical analyses, and regulated gene expression strategies, we found that individual and collective cell movements are inherent properties of hESCs and that they markedly impact the accuracy of clonogenic assays. Analyses of cell motility using mean-square displacement and paired migration correlation indicated that cell movements become more straight-line or ballistic and less random-walk as separation distance decreases. Such motility-induced reaggregation (rather than a true clone) occurs ∼70% of the time if the distance between two hESCs is <6.4 μm, and is not observed if the distance is >150 μm. Furthermore, newly formed small hESC colonies have a predisposition toward the formation of larger colonies through asymmetric colony expansion and movement, which would not accurately reflect self-renewal and proliferative activity of a true hESC clone. Notably, inhibition of Rho-associated kinase markedly upregulated hESC migration and reaggregation, producing considerable numbers of false-positive colonies. Conversely, E-cadherin upregulation significantly augmented hESC clonogenicity via improved survival of single hESCs without influencing cell motility. This work reveals that individual cell movement, asymmetric colony expansion, Rho-associated kinase, and E-cadherin all work together to influence hESC clonogenicity, and provides additional guidance for improvement of clonogenic assays in the analysis of hESC self-renewal.

Light-mediated activation reveals a key role for Rac in collective guidance of cell movement in vivo.

The small GTPase Rac induces actin polymerization, membrane ruffling and focal contact formation in cultured single cells but can either repress or stimulate motility in epithelial cells depending on the conditions. The role of Rac in collective epithelial cell movements in vivo, which are important for both morphogenesis and metastasis, is therefore difficult to predict. Recently, photoactivatable analogues of Rac (PA-Rac) have been developed, allowing rapid and reversible activation or inactivation of Rac using light. In cultured single cells, light-activated Rac leads to focal membrane ruffling, protrusion and migration. Here we show that focal activation of Rac is also sufficient to polarize an entire group of cells in vivo, specifically the border cells of the Drosophila ovary. Moreover, activation or inactivation of Rac in one cell of the cluster caused a dramatic response in the other cells, suggesting that the cells sense direction as a group according to relative levels of Rac activity. Communication between cells of the cluster required Jun amino-terminal kinase (JNK) but not guidance receptor signalling. These studies further show that photoactivatable proteins are effective tools in vivo.

The Role of Cell-Cell Adhesion in the Formation of Multicellular Sprouts.

Collective cell motility and its guidance via cell-cell contacts is instrumental in several morphogenetic and pathological processes such as vasculogenesis or tumor growth. Multicellular sprout elongation, one of the simplest cases of collective motility, depends on a continuous supply of cells streaming along the sprout towards its tip. The phenomenon is often explained as leader cells pulling the rest of the sprout forward via cell-cell adhesion. Building on an empirically demonstrated analogy between surface tension and cell-cell adhesion, we demonstrate that such a mechanism is unable to recruit cells to the sprout. Moreover, the expansion of such hypothetical sprouts is limited by a form of the Plateau-Taylor instability. In contrast, actively moving cells - guided by cell-cell contacts - can readily populate and expand linear sprouts. We argue that preferential attraction to the surfaces of elongated cells can provide a generic mechanism, shared by several cell types, for multicellular sprout formation.

Movement Directionality in Collective Migration of Germ Layer Progenitors

Collective cell migration, the simultaneous movement of multiple cells that are connected by cell-cell adhesion, is ubiquitous in development, tissue repair, and tumor metastasis [1, 2]. It has been hypothesized that the directionality of cell movement during collective migration emerges as a collective property [3, 4]. Here we determine how movement directionality is established in collective mesendoderm migration during zebrafish gastrulation. By interfering with two key features of collective migration, (1) having neighboring cells and (2) adhering to them, we show that individual mesendoderm cells are capable of normal directed migration when moving as single cells but require cell-cell adhesion to participate in coordinated and directed migration when moving as part of a group. We conclude that movement directionality is not a de novo collective property of mesendoderm cells but rather a property of single mesendoderm cells that requires cell-cell adhesion during collective migration.

What makes cells move: requirements and obstacles for spontaneous cell motility

Movement of individual cells and of cellular cohorts, chains or sheets requires physical forces that are established through interactions of cells with their environment. In vivo, migration occurs extensively during embryonic development and in adults during wound healing and tumorigenesis. In order to identify the molecular events involved in cell movement, in vitro systems have been developed. These have contributed to the definition of a number of molecular pathways put into play in the course of migratory behaviours, such as mesenchymal and amoeboid movement. More recently, our knowledge of migratory modes has been enriched by analyses of cells exploring and moving through three-dimensional (3D) matrices. While the cells' morphologies differ in 2D and 3D environments, the basic mechanisms that put a cellular body into motion are remarkably similar. Thus, in both 2D and 3D, the polarity of the migrating cell is initially defined by a specific subcellular localization of signalling molecules and components of molecular machines required for motion. While the polarization can be initiated either in response to extracellular signalling or be a chance occurrence, it is reinforced and sustained by positive feedback loops of signalling molecules. Second, adhesion to a substratum is necessary to generate forces that will propel the cell engaged in either mesenchymal or ameboid migration. For collective cell movement, intercellular coordination constitutes an additional requirement: a cell cohort remains stationary if individual cells pull in opposite directions. Finally, the availability of space to move into is a general requirement to set cells into motion. Lack of free space is probably the main obstacle for migration of most healthy cells in an adult multicellular organism. Thus, the requirements for cell movement are both intrinsic to the cell, involving coordinated signalling and interactions with molecular machines, and extrinsic, imposed by the physicochemical nature of the environment. In particular, the geometry and stiffness of the support act on a range of signalling pathways that induce specific cell migratory responses. These issues are discussed in the present review in the context of published work and our own data on collective migration of hepatocyte cohorts.

Osteopathy and (hatha) yoga

Differences and points of contact between osteopathy and yoga as regards their history and practical application are outlined. Both seek to promote healing. Yoga seeks the attainment of consciousness; osteopathy aims for providing support to health. One fundamental difference is the personal involvement of the individual in yoga. Teacher and student alike are challenged to re-examine the attitudes of mind they have adopted toward their lives. Osteopathy generally involves a relatively passive patient while the osteopath is active in providing treatment. Practical examples are used to highlight points of contact between yoga and osteopathy. The text includes a discussion of the importance of physicality and a description of ways of using it in healing processes. Furthermore, processes of attaining consciousness are outlined. Possible reductionist misconceptions in yoga and osteopathy are also pointed out. Fundamental attitudes and focus that complement each other are presented, taking the concept of stillness as a particular example.

Stochastic Collective Movement of Cells and Fingering Morphology: No Maverick Cells

The classical approach to model collective biological cell movement is through coupled nonlinear reaction-diffusion equations for biological cells and diffusive chemicals that interact with the biological cells. This approach takes into account the diffusion of cells, proliferation, death of cells, and chemotaxis. Whereas the classical approach has many advantages, it fails to consider many factors that affect multicell movement. In this work, a multiscale approach, the Glazier-Graner-Hogeweg model, is used. This model is implemented for biological cells coupled with the finite element method for a diffusive chemical. The Glazier-Graner-Hogeweg model takes the biological cell state as discrete and allows it to include cohesive forces between biological cells, deformation of cells, following the path of a single cell, and stochastic behavior of the cells. Where the continuity of the tissue at the epidermis is violated, biological cells regenerate skin to heal the wound. We assume that the cells secrete a diffusive chemical when they feel a wounded region and that the cells are attracted by the chemical they release (chemotaxis). Under certain parameters, the front encounters a fingering morphology, and two fronts progressing against each other are attracted and correlated. Cell flow exhibits interesting patterns, and a drift effect on the chemical may influence the cells' motion. The effects of a polarized substrate are also discussed.

Multi-scale models of cell and tissue dynamics

Cell and tissue movement are essential processes at various stages in the life cycle of most organisms. The early development of multi-cellular organisms involves individual and collective cell movement; leukocytes must migrate towards sites of infection as part of the immune response; and in cancer, directed movement is involved in invasion and metastasis. The forces needed to drive movement arise from actin polymerization, molecular motors and other processes, but understanding the cell- or tissue-level organization of these processes that is needed to produce the forces necessary for directed movement at the appropriate point in the cell or tissue is a major challenge. In this paper, we present three models that deal with the mechanics of cells and tissues: a model of an arbitrarily deformable single cell, a discrete model of the onset of tumour growth in which each cell is treated individually, and a hybrid continuum-discrete model of the later stages of tumour growth. While the models are different in scope, their underlying mechanical and mathematical principles are similar and can be applied to a variety of biological systems.

Migration of Dictyostelium slugs: Anterior‐like cells may provide the motive force for the prespore zone

The collective motion of cells in a biological tissue originates from their individual responses to chemical and mechanical signals. The Dictyostelium slug moves as a collective of up to 100,000 cells with prestalk cells in the anterior 10-30% and prespore cells, intermingled with anterior-like cells (AL cells), in the posterior. We used traction force microscopy to measure the forces exerted by migrating slugs. Wild-type slugs exert frictional forces on their substratum in the direction of motion in their anterior, balanced by motive forces dispersed down their length. StlB- mutants lack the signal molecule DIF-1 and hence a subpopulation of AL cells. They produce little if any motive force in their rear and immediately break up. This argues that AL cells, but not prespore cells, are the motive cells in the posterior zone. Slugs also exert large outward radial forces, which we have analyzed during "looping" movement. Each time the anterior touches down after a loop, the outward forces rapidly develop, approximately normal to the almost stationary contact lines. We postulate that these forces result from the immediate binding of the sheath to the substratum and the subsequent application of outward "pressure," which might be developed in several different ways.

Traction forces during collective cell motion

Collective motion of cell cultures is a process of great interest, as it occurs during morphogenesis, wound healing, and tumor metastasis. During these processes cell cultures move due to the traction forces induced by the individual cells on the surrounding matrix. A recent study [Trepat, et al. (2009). Nat. Phys. 5, 426-430] measured for the first time the traction forces driving collective cell migration and found that they arise throughout the cell culture. The leading 5-10 rows of cell do play a major role in directing the motion of the rest of the culture by having a distinct outwards traction. Fluctuations in the traction forces are an order of magnitude larger than the resultant directional traction at the culture edge and, furthermore, have an exponential distribution. Such exponential distributions are observed for the sizes of adhesion domains within cells, the traction forces produced by single cells, and even in nonbiological nonequilibrium systems, such as sheared granular materials. We discuss these observations and their implications for our understanding of cellular flows within a continuous culture.