Roles of leader and follower cells in collective cell migration

Collective cell migration is a widely observed phenomenon during animal development, tissue repair, and cancer metastasis. Considering its broad involvement in biological processes, it is essential to understand the basics behind the collective movement. Based on the topology of migrating populations, tissue-scale kinetics, called the “leader–follower” model, has been proposed for persistent directional collective movement. Extensive in vivo and in vitro studies reveal the characteristics of leader cells, as well as the special mechanisms leader cells employ for maintaining their positions in collective migration. However, follower cells have attracted increasing attention recently due to their important contributions to collective movement. In this Perspective, the current understanding of the molecular mechanisms behind the “leader–follower” model is reviewed with a special focus on the force transmission and diverse roles of leaders and followers during collective cell movement.     

Mechanobiology of leader–follower dynamics in epithelial cell migration

Collective cell migration is fundamental to biological form and function. It is also relevant to the formation and repair of organs and to various pathological situations, including metastatic propagation of cancer. Technological, experimental, and computational advancements have allowed the researchers to explore various aspects of collective migration, spanning from biochemical signalling to inter-cellular force transduction. Here, we summarize our current understanding of the mechanobiology of collective cell migration, limiting to epithelial tissues. On the basis of recent studies, we describe how cells sense and respond to guidance signals to orchestrate various modes of migration and identify the determining factors dictating leader–follower interactions. We highlight how the inherent mechanics of dense epithelial monolayers at multicellular length scale might instruct individual cells to behave collectively. On the basis of these findings, we propose that mechanical resilience, obtained by a certain extent of cell jamming, allows the epithelium to perform efficient collective migration during wound healing.     

Collective cell migration of primary zebrafish keratocytes

Fish keratocytes are an established model in single cell motility but little is known about their collective migration. Initially, sheets migrate from the scale at ~145 μm/h but over the course of 24 h the rate of leading edge advance decreases to ~23 μm/h. During this period, leader cells retain their ability to migrate rapidly when released from the sheet and follower cell area increases. After the addition of RGD peptide, leader cell lamellae are lost, altering migratory forces within the sheet, resulting in rapid retraction. Leader and follower cell states interconvert within minutes with changes in cell–cell adhesions. Leader cells migrate as single cells when they detach from the leading edge and single cells appear to become leader cells if they rejoin the sheet. Follower cells rapidly establish leader cell morphology during closing of holes formed during sheet expansion and revert to follower cell morphology after hole-closure. Inhibition of Rho associated kinase releases leader cells and halts advancement of the leading edge suggesting an important role for the intercellular actomyosin cable at the leading edge. In addition, the presence of the stationary scale orients direction of sheet migration which is characterized by a more uniform advance of the leading edge than in some cell line systems. These data establish fish keratocyte explant cultures as a collective cell migration system and suggest that cell–cell interactions determine the role of keratocytes within the migrating sheet.     

Fellow travellers: emergent properties of collective cell migration

Cells can migrate individually or collectively. Collective movement is common during normal development and is also a characteristic of some cancers. This review discusses recent insights into features that are unique to collective cell migration, as well as properties that emerge from these features. The first feature is that cells of the collective affect each other through adhesion, force‐dependent and signalling interactions. The second feature is that cells of the collective differ from one another: leaders from followers, tip from stalk and front from back. These are dynamic differences that are important for directional movement. Last, an unexpected property is discussed: epithelial cells can rotate persistently in constrained spaces.     

Three-dimensional collective cell motions in an acinus-like lumen

Collective cell migration is an essential process in embryo development, wound healing, inflammatory response, and cancer invasion. Although cell motions in two-dimensional (2D) monolayers have been studied previously, three-dimensional (3D) collective cell migration, which constantly occurs during embryogenesis such as the establishment of ducts and acini in vivo, remains elusive. In this paper, we develop a cell-based model incorporating cell mechanics and cell motility to address coherent cell motions in a spherical acinus-like lumen with different cell populations. It is found that the interplays between cell persistence, random fluctuation, and geometrical confinement may engender rich and novel migratory modes. In a 3D spherical lumen, two cells may undergo stripe-like or cross-circular coherent rotations, whereas multiple cells can form dynamic twisting or circulating bands, leaving sparse cells at the center or even a hollow cavity in the cell aggregate. The cell density is found to profoundly influence the collective cell migration modes. Our model can reproduce the fundamental features observed in experiments and highlight the role of mechanics in steering 3D collective cell dynamics during mammary acinar morphogenesis.     

Complement fragment C3a controls mutual cell attraction during collective cell migration

Collective cell migration is a mode of movement crucial for morphogenesis and cancer metastasis. However, little is known about how migratory cells coordinate collectively. Here we show that mutual cell-cell attraction (named here coattraction) is required to maintain cohesive clusters of migrating mesenchymal cells. Coattraction can counterbalance the natural tendency of cells to disperse via mechanisms such as contact inhibition and epithelial-to-mesenchymal transition. Neural crest cells are coattracted via the complement fragment C3a and its receptor C3aR, revealing an unexpected role of complement proteins in early vertebrate development. Loss of coattraction disrupts collective and coordinated movements of these cells. We propose that coattraction and contact inhibition act in concert to allow cell collectives to self-organize and respond efficiently to external signals, such as chemoattractants and repellents.     

What makes leader cells arise: Intrinsic properties and support from neighboring cells

Cancer cells collectively invading as a cohesive and polarized group is termed collective invasion, which is a fundamental property of many types of cancers. In this multicellular unit, cancer cells are heterogeneous, consisting of two morphologically and functionally distinct subpopulations, leader cells and follower cells. Leader cells at the invasive front are responsible for exploring the microenvironment, paving the way, and transmitting information to follower cells. Here, in this review, we will describe the important role of leader cells in collective invasion and the emerging underlying mechanisms of leader cell formation including intrinsic properties and the support from neighboring cells. It will help us to elucidate the essence of collective invasion and provide new anticancer therapeutic clues.     

Dynamic myosin activation promotes collective morphology and migration by locally balancing oppositional forces from surrounding tissue

Migrating cells need to overcome physical constraints from the local microenvironment to navigate their way through tissues. Cells that move collectively have the additional challenge of negotiating complex environments in vivo while maintaining cohesion of the group as a whole. The mechanisms by which collectives maintain a migratory morphology while resisting physical constraints from the surrounding tissue are poorly understood. Drosophila border cells represent a genetic model of collective migration within a cell-dense tissue. Border cells move as a cohesive group of 6−10 cells, traversing a network of large germ line–derived nurse cells within the ovary. Here we show that the border cell cluster is compact and round throughout their entire migration, a shape that is maintained despite the mechanical pressure imposed by the surrounding nurse cells. Nonmuscle myosin II (Myo-II) activity at the cluster periphery becomes elevated in response to increased constriction by nurse cells. Furthermore, the distinctive border cell collective morphology requires highly dynamic and localized enrichment of Myo-II. Thus, activated Myo-II promotes cortical tension at the outer edge of the migrating border cell cluster to resist compressive forces from nurse cells. We propose that dynamic actomyosin tension at the periphery of collectives facilitates their movement through restrictive tissues.     

Therapeutic cell engineering: designing programmable synthetic genetic circuits in mammalian cells

Cell therapy approaches that employ engineered mammalian cells for on-demand production of therapeutic agents in the patient’s body are moving beyond proof-of-concept in translational medicine. The therapeutic cells can be customized to sense user-defined signals, process them, and respond in a programmable and predictable way. In this paper, we introduce the available tools and strategies employed to design therapeutic cells. Then, various approaches to control cell behaviors, including open-loop and closed-loop systems, are discussed. We also highlight therapeutic applications of engineered cells for early diagnosis and treatment of various diseases in the clinic and in experimental disease models. Finally, we consider emerging technologies such as digital devices and their potential for incorporation into future cell-based therapies.     

Multicellular scale front-to-rear polarity in collective migration

Collective cell migration does not only reflect the migration of cells at a similar speed and in the same direction, it also implies the emergence of new properties observed at the level of the cell group. This collective behavior relies on interactions between the cells and the establishment of a hierarchy amongst cells with leaders driving the group of followers. Here, we make the parallel between the front-to-rear polarity axis in single cell and the front-to-rear multicellular-scale polarity of a migrating collective which established through exchange of biochemical and mechanical information from the front to the rear and vice versa. Such multicellular-scale polarity gives the migrating group the possibility to better sense and adapt to energy, biochemical and mechanical constraints and facilitates migration over long distances in complex and changing environments.     

Autocrine galectin-1 promotes collective cell migration of squamous cell carcinoma cells through up-regulation of distinct integrins

We found that high galectin-1 (Gal-1) mRNA levels were associated with invasive squamous cell carcinoma (SCC) cells that expressed Snail, an epithelial-to-mesenchymal transition (EMT) regulator. Both Gal-1 overexpression and soluble Gal-1 treatment accelerated invasion and collective cell migration, along with activation of cdc42 and Rac. Soluble Gal-1 activated c-Jun N-terminal kinase to increase expression levels of integrins α2 and β5, which were essential for Gal-1 dependent collective cell migration and invasiveness. Soluble Gal-1 also increased the incidence of EMT in Snail-expressing SCC cells; these were a minor population with an EMT phenotype under growing conditions. Our findings indicate that soluble Gal-1 promotes invasiveness through enhancing collective cell migration and increasing the incidence of EMT.     

Cell traction in collective cell migration and morphogenesis: The chase and run mechanism

Directional collective cell migration plays an important role in development, physiology, and disease. An increasing number of studies revealed key aspects of how cells coordinate their movement through distances surpassing several cell diameters. While physical modeling and measurements of forces during collective cell movements helped to reveal key mechanisms, most of these studies focus on tightly connected epithelial cultures. Less is known about collective migration of mesenchymal cells. A typical example of such behavior is the migration of the neural crest cells, which migrate large distances as a group. A recent study revealed that this persistent migration is aided by the interaction between the neural crest and the neighboring placode cells, whereby neural crest chase the placodes via chemotaxis, but upon contact both populations undergo contact inhibition of locomotion and a rapid reorganization of cellular traction. The resulting asymmetric traction field of the placodes forces them to run away from the chasers. We argue that this chase and run interaction may not be specific only to the neural crest system, but could serve as the underlying mechanism for several morphogenetic processes involving collective cell migration.     

Quantum dot-based cell motility assay

Motility and migration are measurable characteristics of cells that are classically associated with the invasive potential of cancer cells, but in vitro assays of invasiveness have been less than perfect. We previously developed an assay to monitor cell motility and migration using water-soluble CdSe/ZnS nanocrystals; cells engulf the fluorescent nanocrystals as they crawl across them and leave behind a fluorescent-free trail. We show here that semiconductor nanocrystals can also be used as a sensitive two-dimensional in vitro invasion assay. We used this assay to compare the behavior of seven different adherent human cell lines, including breast epithelial MCF 10A, breast tumor MDA-MB-231, MDA-MB-435S, MCF 7, colon tumor SW480, lung tumor NCI H1299, and bone tumor Saos-2, and observed two distinct behaviors of cancer cells that can be used to further categorize these cells. Some cancer cell lines demonstrate fibroblastic behaviors and leave long fluorescent-free trails as they migrate across the dish, whereas other cancer cells leave clear zones of varying sizes around their periphery. This assay uses fluorescence detection, requires no processing, and can be used in live cell studies. These features contribute to the increased sensitivity of this assay and make it a powerful new tool for discriminating between non-invasive and invasive cancer cell lines.     

Leaders benefit followers in the collective movement of a social sawfly

The challenges of maintaining cohesion while making collective decisions in social or aggregating insects can result in the emergence of a leader or leaders. Larval aggregations of the steel-blue sawfly Perga affinis forage nocturnally, and some larvae lead the aggregation on foraging trips more often than expected by chance. We investigated the relationship between these leader and follower roles by comparing the weight and growth of individual larvae with different roles. Our observations reveal no significant difference between the growth of leaders and followers, suggesting that the role of leadership may not provide direct foraging benefits. However, by experimentally manipulating the social structure of larval aggregations, we found that individuals within aggregations that comprise a mixture of leaders and followers enjoy higher growth rates than those in aggregations comprising a single behavioural type. These data demonstrate, for the first time, individual benefits to maintaining a balance of leader and follower roles within larval aggregations, and highlight the importance of considering the perspectives of both leaders and followers when investigating the evolutionary significance of this behavioural variation within animal groups.     

Leader cell positioning drives wound-directed collective migration in TGFβ-stimulated epithelial sheets

During wound healing and cancer metastasis, cells are frequently observed to migrate in collective groups. This mode of migration relies on the cooperative guidance of leader and follower cells throughout the collective group. The upstream determinants and molecular mechanisms behind such cellular guidance remain poorly understood. We use live-cell imaging to track the behavior of epithelial sheets of keratinocytes in response to transforming growth factor β (TGFβ), which stimulates collective migration primarily through extracellular regulated kinase 1/2 (Erk1/2) activation. TGFβ-treated sheets display a spatial pattern of Erk1/2 activation in which the highest levels of Erk1/2 activity are concentrated toward the leading edge of a sheet. We show that Erk1/2 activity is modulated by cellular density and that this functional relationship drives the formation of patterns of Erk1/2 activity throughout sheets. In addition, we determine that a spatially constrained pattern of Erk1/2 activity results in collective migration that is primarily wound directed. Conversely, global elevation of Erk1/2 throughout sheets leads to stochastically directed collective migration throughout sheets. Our study highlights how the spatial patterning of leader cells (cells with elevated Erk1/2 activity) can influence the guidance of a collective group of cells during wound healing.     

Grunt to go—Vocal coordination of group movements in redfronted lemurs

To remain cohesive as a group, individuals must coordinate their movements between resources. In many species, vocalisations are used in this context. While some species have specific movement calls, others use calls which are also employed in different contexts. The use of such multicontextual calls has rarely been studied quantitatively, especially during both the pre‐departure and departure period associated with collective decisions. We thus investigated the use of close calls (“grunts”) for the coordination of collective movements in four groups of wild redfronted lemurs (Eulemur rufifrons) in Kirindy Forest, Western Madagascar. Group movements are started by an initiator, who moves away from the group and is joined by followers setting out in the same direction. We observed collective movements and recorded vocalisations from 18 focal individuals (54 movements recorded for followers, 21 for initiators). The grunt rate of both initiators and followers was higher in the pre‐departure period than in a control context (i.e., during foraging). Initiators of collective movements grunted more often than followers in the pre‐departure period as well as at individual departure. The latter difference was due to the initiators’ grunt rates increasing earlier than the followers’ and remaining at an elevated level for longer. These observations suggest that grunts serve to coordinate the departure by indicating the individual's readiness to move. The pre‐departure period, in which both initiators and followers showed an elevated grunt rate, may provide the basis for a shared decision on departure time. The difference in initiator and follower call rates suggests that grunts may have a recruitment function, but playback experiments are required to test this potential function. Overall, our study describes how multicontextual close calls can function as movement calls, with changes in call rate providing a potential feedback mechanism for the timing of group movements. This study thus contributes to a more detailed understanding of the mechanisms of group coordination and collective decision‐making.     

Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells

Background

The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen.

Results

Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs) disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion.

Conclusions

The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0039-2) contains supplementary material, which is available to authorized users.     

Characterization of cell signaling,morphology, and differentiation potential of human mesenchymal stem cells based on cell adhesion mechanism

It is essential to characterize the cellular properties of mesenchymal stem cell populations to maintain quality specifications and control in regenerative medicine. Biofunctional materials have been designed as artificial matrices for the stimulation of cell adhesion and specific cellular functions. We have developed recombinant maltose-binding protein (MBP)-fused proteins as artificial adhesion matrices to control human mesenchymal stem cell (hMSC) fate by using an integrin-independent heparin sulfate proteoglycans-mediated cell adhesion. In this study, we characterize cell adhesion-dependent cellular behaviors of human adipose-derived stem cells (hASCs) and human bone marrow stem cells (hBMSCs). We used an MBP-fused basic fibroblast growth factor (MF)-coated surface and fibronectin (FN)-coated surface to restrict and support, respectively, integrin-mediated adhesion. The cells adhered to MF exhibited restricted actin cytoskeleton organization and focal adhesion kinase phosphorylation. The hASCs and hBMSCs exhibited different cytoplasmic projection morphologies on MF. Both hASCs and hBMSCs differentiated more dominantly into osteogenic cells on FN than on MF. In contrast, hASCs differentiated more dominantly into adipogenic cells on MF than on FN, whereas hBMSCs differentiated predominantly into adipogenic cells on FN. The results indicate that hASCs exhibit a competitive differentiation potential (osteogenesis vs. adipogenesis) that depends on the cell adhesion matrix, whereas hBMSCs exhibit both adipogenesis and osteogenesis in integrin-mediated adhesion and thus hBMSCs have noncompetitive differentiation potential. We suggest that comparing differentiation behaviors of hMSCs with the diversity of cell adhesion is an important way to characterize hMSCs for regenerative medicine.     

Chemokine‐guided cell migration and motility in zebrafish development

Chemokines are vertebrate‐specific, structurally related proteins that function primarily in controlling cell movements by activating specific 7‐transmembrane receptors. Chemokines play critical roles in a large number of biological processes and are also involved in a range of pathological conditions. For these reasons, chemokines are at the focus of studies in developmental biology and of clinically oriented research aimed at controlling cancer, inflammation, and immunological diseases. The small size of the zebrafish embryos, their rapid external development, and optical properties as well as the large number of eggs and the fast expansion in genetic tools available make this model an extremely useful one for studying the function of chemokines and chemokine receptors in an in vivo setting. Here, we review the findings relevant to the role that chemokines play in the context of directed single‐cell migration, primarily in neutrophils and germ cells, and compare it to the collective cell migration of the zebrafish lateral line. We present the current knowledge concerning the formation of the chemokine gradient, its interpretation within the cell, and the molecular mechanisms underlying the cellular response to chemokine signals during directed migration.     

From Cell Differentiation to Cell Collectives: Bacillus subtilis Uses Division of Labor to Migrate

The organization of cells, emerging from cell–cell interactions, can give rise to collective properties. These properties are adaptive when together cells can face environmental challenges that they separately cannot. One particular challenge that is important for microorganisms is migration. In this study, we show how flagellum-independent migration is driven by the division of labor of two cell types that appear during Bacillus subtilis sliding motility. Cell collectives organize themselves into bundles (called “van Gogh bundles”) of tightly aligned cell chains that form filamentous loops at the colony edge. We show, by time-course microscopy, that these loops migrate by pushing themselves away from the colony. The formation of van Gogh bundles depends critically on the synergistic interaction of surfactin-producing and matrix-producing cells. We propose that surfactin-producing cells reduce the friction between cells and their substrate, thereby facilitating matrix-producing cells to form bundles. The folding properties of these bundles determine the rate of colony expansion. Our study illustrates how the simple organization of cells within a community can yield a strong ecological advantage. This is a key factor underlying the diverse origins of multicellularity.