Leader Cell Formation Research Articles

Angiomotin cleavage promotes leader formation and collective cell migration.

Collective cell migration (CCM) is involved in multiple biological processes, including embryonic morphogenesis, angiogenesis, and cancer invasion. However, the molecular mechanisms underlying CCM, especially leader cell formation, are poorly understood. Here, we show that a signaling pathway regulating angiomotin (AMOT) cleavage plays a role in CCM, using mammalian epithelial cells and mouse models. In a confluent epithelial monolayer, full-length AMOT localizes at cell-cell junctions and limits cell motility. After cleavage, the C-terminal fragment of AMOT (AMOT-CT) translocates to the cell-matrix interface to promote the maturation of focal adhesions (FAs), generate traction force, and induce leader cell formation. Meanwhile, decreased full-length AMOT at cell-cell junctions leads to tissue fluidization and coherent migration of cell collectives. Hence, the cleavage of AMOT serves as a molecular switch to generate polarized contraction, promoting leader cell formation and CCM.

Substrate stress relaxation regulates monolayer fluidity and leader cell formation for collectively migrating epithelia.

Groups of cells must coordinate their movements in order to sculpt organs during development and maintain tissues. The mechanical properties of the underlying substrate on which cells reside are known to influence key aspects of single and collective cell migration. Despite being a nearly universal feature of biological tissues, the role of viscoelasticity (i.e., fluid-like and solid-like behavior) in collective cell migration is unclear. Using tunable engineered biomaterials, we demonstrate that sheets of epithelial cells display enhanced migration on slower-relaxing (more elastic) substrates relative to faster-relaxing (more viscous) substrates. Building our understanding of tissue-substrate interactions and collective cell dynamics provides insights into approaches for tissue engineering and regenerative medicine, and therapeutic interventions to promote health and treat disease.

PIEZO1 regulates leader cell formation and cellular coordination during collective keratinocyte migration.

The collective migration of keratinocytes during wound healing requires both the generation and transmission of mechanical forces for individual cellular locomotion and the coordination of movement across cells. Leader cells along the wound edge transmit mechanical and biochemical cues to ensuing follower cells, ensuring their coordinated direction of migration across multiple cells. Despite the observed importance of mechanical cues in leader cell formation and in controlling coordinated directionality of cell migration, the underlying biophysical mechanisms remain elusive. The mechanically-activated ion channel PIEZO1 was recently identified to play an inhibitory role during the reepithelialization of wounds. Here, through an integrative experimental and mathematical modeling approach, we elucidate PIEZO1's contributions to collective migration. Time-lapse microscopy reveals that PIEZO1 activity inhibits leader cell formation at the wound edge. To probe the relationship between PIEZO1 activity, leader cell formation and inhibition of reepithelialization, we developed an integrative 2D continuum model of wound closure that links observations at the single cell and collective cell migration scales. Through numerical simulations and subsequent experimental validation, we found that coordinated directionality plays a key role during wound closure and is inhibited by upregulated PIEZO1 activity. We propose that PIEZO1-mediated retraction suppresses leader cell formation which inhibits coordinated directionality between cells during collective migration.

Photoactivatable substrates show diverse phenotypes of leader cells in collective migration when moving along different extracellular matrix proteins.

In cancer metastasis, collectively migrating clusters are discriminated into leader and follower cells that move through extracellular matrices (ECMs) with different characteristics. The impact of changes in ECM protein types on leader cells and migrating clusters is unknown. To address this, we investigated the response of leader cells and migrating clusters upon moving from one ECM protein to another using a photoactivatable substrate bearing photocleavable PEG (PCP), whose surface changes from protein-repellent to protein-adhesive in response to light. We chose laminin and collagen I for our study since they are abundant in two distinct regions in living tissues, namely basement membrane and connective tissue. Using the photoactivatable substrates, the precise deposition of the first ECM protein in the irradiated areas was achieved, followed by creating well-defined cellular confinements. Secondary irradiation enabled the deposition of the second ECM protein in the new irradiated regions, resulting in region-selective heterogeneous and homogenous ECM protein-coated surfaces. Different tendencies in leader cell formation from laminin into laminin compared to those migrating from laminin into collagen were observed. The formation of focal adhesion and actin structures for cells within the same cluster in the ECM proteins responded according to the underlying ECM protein type. Finally, integrin β1 was crucial for the appearance of leader cells for clusters migrating from laminin into collagen. However, when it came to laminin into laminin, integrin β1 was not responsible. This highlights the correlation between leader cells in collective migration and the biochemical signals that arise from underlying extracellular matrix proteins.

Microvascular smooth muscle cells exhibit divergent phenotypic switching responses to platelet-derived growth factor and insulin-like growth factor 1

ObjectiveVascular smooth muscle cell (VSMC) phenotypic switching is critical for normal vessel formation, vascular stability, and healthy brain aging. Phenotypic switching is regulated by mediators including platelet derived growth factor (PDGF)-BB, insulin-like growth factor (IGF-1), as well as transforming growth factor-β (TGF-β) and endothelin-1 (ET-1), but much about the role of these factors in microvascular VSMCs remains unclear. MethodsWe used primary rat microvascular VSMCs to explore PDGF-BB- and IGF-1-induced phenotypic switching. ResultsPDGF-BB induced an early proliferative response, followed by formation of polarized leader cells and rapid, directionally coordinated migration. In contrast, IGF-1 induced cell hypertrophy, and only a small degree of migration by unpolarized cells. TGF-β and ET-1 selectively inhibit PDGF-BB-induced VSMC migration primarily by repressing migratory polarization and formation of leader cells. Contractile genes were downregulated by both growth factors, while other genes were differentially regulated by PDGF-BB and IGF-1. ConclusionsThese studies indicate that PDGF-BB and IGF-1 stimulate different types of microvascular VSMC phenotypic switching characterized by different modes of cell migration. Our studies are consistent with a chronic vasoprotective role for IGF-1 in VSMCs in the microvasculature while PDGF is more involved in VSMC proliferation and migration in response to acute activities such as neovascularization. Better understanding of the nuances of the phenotypic switching induced by these growth factors is important for our understanding of a variety of microvascular diseases.

Long noncoding RNA MALAT1 is dynamically regulated in leader cells during collective cancer invasion

Cancer cells collectively invade using a leader-follower organization, but the regulation of leader cells during this dynamic process is poorly understood. Using a dual double-stranded locked nucleic acid (LNA) nanobiosensor that tracks long noncoding RNA (lncRNA) dynamics in live single cells, we monitored the spatiotemporal distribution of lncRNA during collective cancer invasion. We show that the lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) is dynamically regulated in the invading fronts of cancer cells and patient-derived spheroids. MALAT1 transcripts exhibit distinct abundance, diffusivity, and distribution between leader and follower cells. MALAT1 expression increases when a cancer cell becomes a leader and decreases when the collective migration process stops. Transient knockdown of MALAT1 prevents the formation of leader cells and abolishes the invasion of cancer cells. Taken together, our single-cell analysis suggests that MALAT1 is dynamically regulated in leader cells during collective cancer invasion.

PIEZO1 regulates leader cell formation and cellular coordination during collective cell migration: An integrative multiscale modeling and experimental study.

The collective migration of keratinocytes during wound healing requires both the generation and transmission of mechanical forces for individual cellular locomotion as well as for the coordination of movement across cells. Leader cells initiated along the wound edge transmit mechanical and biochemical cues to ensuing follower cells, ensuring their uniform polarization and coordinated direction of migration, or directionality. Despite the observed importance of mechanical cues in leader cell formation and governing directionality, the underlying biophysical mechanisms remain elusive. The mechanically activated ion channel PIEZO1 was recently identified to play an inhibitory role during the reepithelialization of wounds through retraction of keratinocytes located along the wound edge. Here, through an integrative experimental and mathematical modeling approach, we elucidate PIEZO1's contributions to collective migration. Time-lapse microscopy reveals that PIEZO1 activity inhibits leader cell formation along the wound edge. To probe the relationship between PIEZO1 activity, leader cell formation and inhibition of reepithelialization, we developed an integrative 2D-multiscale model of wound closure that links observations at the single cell and collective cell migration scales. Through numerical simulations and subsequent experimental validation, we found that directionality plays a key role during wound closure and is inhibited by PIEZO1 activity. We propose that PIEZO1-mediated retraction suppresses leader cell formation which inhibits the coordination of directionality between cells during collective migration.

Dynamic Regulation of MALAT1 in Leader Cell Formation and Collective Cancer Invasion

Dynamic Regulation of MALAT1 in Leader Cell Formation and Collective Cancer Invasion

Identification of leader cells by filopodia in collective cell migration using computer vision.

Imaging of cells and cellular organelles has been of great interest among researchers and medical staff because it can provide useful information on cell physiology and pathology. Many researches related to collective cell migration have been established and leader cells seem to be the ones that regulate the migration, however, the identification of leader cells is very time-consuming. This study utilized computer vision with deep learning to segment cell shape and to identify leader cells through filopodia. Healthy Madin-Darby Canine Kidney (MDCK) cells cultured in a Polydimethylsiloxane (PDMS) microchannel device allowed collective cell migration as well as the formation of leader cells. The cells were stained, and cell images were captured to train the computer using UNet++ together with their corresponding masks created using Photoshop for automated cell segmentation. Lastly, cell shape and filopodia were filtered out using Filopodyan and FiloQuant were detected. The segmentation of cell shape and the identification of filopodia were successful and produced accurate results in less than one second per image. The proposed approach of image analysis would be a great help in the field of cell science, engineering, and diagnosis.

Persistence of leader cell behaviour can lead to malignant transformation in oral submucous fibrosis

Betel quid (BQ) chewing-related oral submucous fibrosis (OSMF) is a common, potentially fatal condition in Southeast Asian countries. The literature has proved that BQ is associated with the development of micro-trauma-related injury to the oral mucosa. This is mainly attributed to the coarseness of the BQ and its abrasive action. Since, BQ chewing is a chronic habit in OSMF patients, it leads to the frequent development of micro-trauma and its repeated healing. This mechanism has been implicated in the pathogenesis of OSMF and its malignant transformation. However, the micro-trauma related mechanism for malignant transformation of OSMF is poorly understood. Leader cell formation is a well-known phenomenon in wound healing characterized by migration guidance to other cells, leading to epithelial repair. Due to repeated trauma and micro-wound formation in OSMF patients, we proposed the persistence of leader cells in the oral mucosa. This causes continuous activation of the p53-p21-CDK axis, which is an integral mechanism required for the transformation of normal epithelial cells into leader cells. We also propose that carcinogens derived from BQ chewing cause genetic and epigenetic changes in the leader cells. Overall, the persistence of leader cells in the oral epithelial population could be a major driver for malignant transformation in OSMF and a determinant of intra-tumoral heterogeneity. Strategies to curtail micro-trauma to the oral mucosa and target the leader cells hold good promise for preventing malignant transformation in OSMF.

Nrf2 Modulates the Hybrid Epithelial/Mesenchymal Phenotype and Notch Signaling During Collective Cancer Migration.

Hybrid epithelial/mesenchymal cells (E/M) are key players in aggressive cancer metastasis. It remains a challenge to understand how these cell states, which are mostly non-existent in healthy tissue, become stable phenotypes participating in collective cancer migration. The transcription factor Nrf2, which is associated with tumor progression and resistance to therapy, appears to be central to this process. Here, using a combination of immunocytochemistry, single cell biosensors, and computational modeling, we show that Nrf2 functions as a phenotypic stability factor for hybrid E/M cells by inhibiting a complete epithelial-mesenchymal transition (EMT) during collective cancer migration. We also demonstrate that Nrf2 and EMT signaling are spatially coordinated near the leading edge. In particular, computational analysis of an Nrf2-EMT-Notch network and experimental modulation of Nrf2 by pharmacological treatment or CRISPR/Cas9 gene editing reveal that Nrf2 stabilizes a hybrid E/M phenotype which is maximally observed in the interior region immediately behind the leading edge. We further demonstrate that the Nrf2-EMT-Notch network enhances Dll4 and Jagged1 expression at the leading edge, which correlates with the formation of leader cells and protruding tips. Altogether, our results provide direct evidence that Nrf2 acts as a phenotypic stability factor in restricting complete EMT and plays an important role in coordinating collective cancer migration.

Asymmetric Stratification-Induced Polarity Loss and Coordinated Individual Cell Movements Drive Directional Migration of Vertebrate Epithelium

SUMMARYCollective migration is essential for development, wound repair, and cancer metastasis. For most collective systems, “leader cells” determine both the direction and the power of the migration. It has remained unclear, however, how the highly polarized vertebrate epithelium migrates directionally during branching morphogenesis. We show here that, unlike in other systems, front-rear polarity of the mammary epithelium is set up by preferential cell proliferation in the front in response to the FGF10 gradient. This leads to frontal stratification, loss of apicobasal polarity, and leader cell formation. Leader cells are a dynamic population and move faster and more directionally toward the FGF10 signal than do follower cells, partly because of their intraepithelial protrusions toward the signal. Together, our data show that directional migration of the mammary epithelium is a unique multistep process and that, despite sharing remarkable cellular and molecular similarities, vertebrate and invertebrate epithelial branching are fundamentally distinct processes.

Three-Dimensional Microtumors for Probing Heterogeneity of Invasive Bladder Cancer.

Bladder cancer is an increasingly common malignancy, and muscle invasive bladder cancer is associated with particularly high rates of morbidity and mortality. The morphologic and molecular diversity of bladder cancer poses significant challenges in elucidating the invasion mechanisms responsible for disease progression. Furthermore, conventional invasion assays do not provide a physiological context for studying bladder cancer invasion within 3D microenvironments and have limited ability to capture the contribution of cellular phenotypic heterogeneity to disease progression. Here, we describe the development of a 3D microtumor invasion model suitable for the analysis of cellular phenotypic heterogeneity in cell lines and primary tumor cells from bladder cancer patients. This model incorporates a self-assembly approach for recapitulating features of bladder cancer invasion in 3D microenvironments and probing the invasive cell subpopulations. The gene expression profiles of invading microtumors were analyzed by incorporating a gold nanorod-locked nucleic acid biosensor. The incorporation of the single cell biosensor and transient gene knockdown into the system revealed the formation of invasive leader cells with upregulated Delta-like ligand 4 (DLL4) expression as well as the role of NOTCH1-DLL4 signaling in collective bladder cancer invasion. The involvement of DLL4 expressing cells in bladder cancer invasion was also observed in patient samples obtained from transurethral resection. Collectively, our study demonstrates a 3D microtumor invasion model for investigating intracellular heterogeneity of bladder cancer invasion and analyzing patient derived samples toward personalized medicine applications.

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.

Inducible formation of leader cells driven by CD44 switching gives rise to collective invasion and metastases in luminal breast carcinomas

Collective invasion into adjacent tissue is a hallmark of luminal breast cancer, and ~20% of these cases eventually undergo metastasis. How less aggressive luminal-like breast cancer transitions to invasive cancer remains unclear. Our study revealed that CD44hi cancer cells are the leading subpopulation in collectively invading luminal cancer cells and efficiently promote the collective invasion of CD44lo/follower cells. The CD44hi/leader subpopulation showed a specific gene signature of various hybrid epithelial/mesenchymal genes and key functional coregulators of collective invasion, which was distinct from that of CD44lo/follower cells. However, the CD44hi/leader cells, which showed a partial epithelial-mesenchymal transition (EMT) phenotype, readily switched to the CD44lo phenotype along with collective migration and vice versa; this phenomenon was spontaneous and sensitive to the tumor microenvironment. The CD44lo-to-CD44hi conversion was accompanied by a shift in CD44s to CD44v but not a conversion of non-cancer stem cells to cancer stem cells (CSCs). Therefore, the CD44hi leader cells, as currently identified, are not a stable subpopulation in breast tumors. This plasticity and ability to generate CD44hi carcinoma cells with enhanced migratory and invasive behavior might be responsible for the transition from in situ to invasive behavior of luminal-type breast cancer.

ROCK2 inhibition triggers the collective invasion of colorectal adenocarcinomas.

The metastatic progression of cancer is a multi-step process initiated by the local invasion of the peritumoral stroma. To identify the mechanisms underlying colorectal carcinoma (CRC) invasion, we collected live human primary cancer specimens at the time of surgery and monitored them exvivo. This revealed that conventional adenocarcinomas undergo collective invasion while retaining their epithelial glandular architecture with an inward apical pole delineating a luminal cavity. To identify the underlying mechanisms, we used microscopy-based assays on 3D organotypic cultures of Caco-2 cysts as a model system. We performed two siRNA screens targeting Rho-GTPases effectors and guanine nucleotide exchange factors. These screens revealed that ROCK2 inhibition triggers the initial leader/follower polarization of the CRC cell cohorts and induces collective invasion. We further identified FARP2 as the Rac1 GEF necessary for CRC collective invasion. However, FARP2 activation is not sufficient to trigger leader cell formation and the concomitant inhibition of Myosin-II is required to induce invasion downstream of ROCK2 inhibition. Our results contrast with ROCK pro-invasive function in other cancers, stressing that the molecular mechanism of metastatic spread likely depends on tumour types and invasion mode.

Decreasing Wound Edge Stress Enhances Leader Cell Formation during Collective Smooth Muscle Cell Migration.

Collective cell migration is vital to tissue remodeling in wound repair, development, and cancer invasion. Nevertheless, studies on collective cell migration have largely focused on epithelial growth and repair mechanisms and have only recently expanded to explore coordinated metastatic cancer and smooth muscle cell behaviors. The regulatory mechanisms of smooth muscle cell collective migration, such as leader-follower organization and mechanosensitivity, remain poorly understood. In this study, we demonstrate the involvement of leader cells during collective smooth muscle cell migration using dynamic cell tracking and single cell gene expression analysis. Engineered wound models, including ingrowth, outgrowth, and straight edge geometries, along with traction force microscopy and finite element stress mapping reveal that smooth muscle leader cells are enhanced at the wound edge when the intercellular tension near the cell wound boundary is reduced. Pharmacological perturbation further supports the notion that mechanical force negatively regulates the formation of leader cells. The mechanical regulation of collective smooth muscle cell migration via the formation of leader cells may lead to novel treatment strategies for pathogenic smooth muscle cell conditions in the future.

Cathepsin B defines leader cells during the collective invasion of salivary adenoid cystic carcinoma.

Cathepsin B (CTSB) has been reported to be involved in cancer metastasis by altering extracellular matrix (ECM) remodeling and facilitating invasion. However, the contribution of CTSB to collective cell invasion in salivary adenoid cystic carcinoma (SACC) and the underlying mechanisms remain unclear. The present study demonstrated that collective cell invasion is commonly observed in SACC without a complete epithelial-mesenchymal transition signature. CTSB was found to be overexpressed in the invasive front of SACC compared to the tumor center, and was associated with a poor prognosis of patients with SACC. Subsequently, a 3D spheroid invasion assay was established in order to recapitulate the collective cell invasion of SACC and the results revealed that CTSB was only expressed in leader cells. The knockdown of CTSB by siRNA inhibited the migration and invasion of SACC-83 cells and impaired the formation of leader cells. CTSB knockdown also disrupted cytoskeletal organization, altered cell morphology and inhibited ECM remodeling by downregulating matrix metalloproteinase-9, focal adhesion kinase and Rho/ROCK function. Therefore, the present study provides evidence that CTSB may define leader cells in SACC and is required for collective cell invasion as a potential key regulator of ECM remodeling.

Collective Cell Migration in 3D Epithelial Wound Healing.

Collective cell migration plays a pivotal role in development, wound healing, and metastasis, but little is known about the mechanisms and coordination of cell migration in 3D microenvironments. Here, we demonstrate a 3D wound healing assay by photothermal ablation for investigating collective cell migration in epithelial tissue structures. The nanoparticle-mediated photothermal technique creates local hyperthermia for selective cell ablation and induces collective cell migration of 3D tissue structures. By incorporating dynamic single cell gene expression analysis, live cell actin staining, and particle image velocimetry, we show that the wound healing response consists of 3D vortex motion moving toward the wound followed by the formation of multicellular actin bundles and leader cells with active actin-based protrusions. Inhibition of ROCK signaling disrupts the multicellular actin bundle and enhances the formation of leader cells at the leading edge. Furthermore, single cell gene expression analysis, pharmacological perturbation, and RNA interference reveal that Notch1-Dll4 signaling negatively regulates the formation of multicellular actin bundles and leader cells. Taken together, our study demonstrates a platform for investigating 3D collective cell migration and underscores the essential roles of ROCK and Notch1-Dll4 signaling in regulating 3D epithelial wound healing.

Photoactivatable substrates for systematic study of the impact of an extracellular matrix ligand on appearance of leader cells in collective cell migration

Epithelial cells migrate as multicellular units. The directionality and speed of these units are determined by actively moving leader cells. It is important to understand how external cues affect the appearance of these leader cells in physiological and pathological processes. However, the impact of extracellular matrices (ECMs) is still controversial, because physically-adsorbed ECM proteins are amenable to protein remodeling, and uncontrolled cluster geometry can vary migration phenotypes. Here, we demonstrate a photoactivatable substrate, which we used to study the impact of a cyclic Arg-Gly-Asp (cRGD) ligand on leader cell formation in MDCK cells. This robust platform allowed us to investigate the effect of cRGD density on leader cell formation, in any given cluster geometry, with minimized ECM remodeling. Our results show a biphasic response of leader cell appearance upon reducing the surface cRGD density. The increase, in leader cell appearance, within the higher density range, is not only associated with the weakening of circumferential actomyosin belts, but also reduction of cellular mechanical tension and intercellular junctional E-cadherin. These results indicate that cRGD-mediated cell-ECM interactions positively regulate mechanical and biochemical coupling within cell clusters; both are critical for the coordination of cell collectives and eventual reduction in the appearance of leader cells.