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Collective cell migration is crucial to maintain epithelium integrity during developmental and repair processes. It requires a tight regulation of mechanical coordination between neighboring cells. This coordination embraces different features including mechanical self-propulsion of individual cells within cellular colonies and large-scale force transmission through cell–cell junctions. This review discusses how the plasticity of biomechanical interactions at cell–cell contacts could help cellular systems to perform coordinated motions and adapt to the properties of the external environment.

Traction force microscopy (TFM) is an important family of techniques used to measure and study the role of cellular traction forces (CTFs) associated with many biological processes. However, current standard TFM methods rely on imaging techniques that do not provide the experimental capabilities necessary to study CTFs within 3D collective and dynamic systems embedded within optically scattering media. Traction force optical coherence microscopy (TF-OCM) was developed to address these needs, but has only been demonstrated for the study of isolated cells embedded within optically clear media. Here, we present computational 4D-OCM methods that enable the study of dynamic invasion behavior of large tumor spheroids embedded in collagen. Our multi-day, time-lapse imaging data provided detailed visualizations of evolving spheroid morphology, collagen degradation, and collagen deformation, all using label-free scattering contrast. These capabilities, which provided insights into how stromal cells affect cancer progression, significantly expand access to critical data about biophysical interactions of cells with their environment, and lay the foundation for future efforts toward volumetric, time-lapse reconstructions of collective CTFs with TF-OCM.

Collective behaviour emerges from interacting units within communities, such as migrating herds, swimming fish schools, and cells within tissues. At the microscopic level, collective behaviours include collective cell migration in development and cancer invasion, rhythmic gene expression in pattern formation, cell competition in homeostasis and cancer, force generation and mechano-sensing in morphogenesis. Studying the initiation and the maintenance of collective cell behaviours is key to understand the principles of development, regeneration and disease. However, the manifold influences of contributing factors in in vivo environments challenge the dissection of causalities in animal models. As an alternative model that has emerged to overcome this difficulty, in vitro three-dimensional organoid cultures provide a reductionist approach yet retain similarities with the in vivo tissue in cellular composition and tissue organisation. Here, we focus on recent progresses in studying collective behaviours in different organoid systems and discuss their advantages and the possibility of improvement for future applications.

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.

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.

The epithelial-to-mesenchymal transition (EMT) and the unjamming transition (UJT) each comprises a gateway to cellular migration, plasticity and remodeling, but the extent to which these core programs are distinct, overlapping, or identical has remained undefined. Here, we triggered partial EMT (pEMT) or UJT in differentiated primary human bronchial epithelial cells. After triggering UJT, cell-cell junctions, apico-basal polarity, and barrier function remain intact, cells elongate and align into cooperative migratory packs, and mesenchymal markers of EMT remain unapparent. After triggering pEMT these and other metrics of UJT versus pEMT diverge. A computational model attributes effects of pEMT mainly to diminished junctional tension but attributes those of UJT mainly to augmented cellular propulsion. Through the actions of UJT and pEMT working independently, sequentially, or interactively, those tissues that are subject to development, injury, or disease become endowed with rich mechanisms for cellular migration, plasticity, self-repair, and regeneration., The authors acknowledge the support of the Northeastern University Discovery Cluster and TIER 1 Seed Grant for interdisciplinary research, P01HL120839, R01HL148152, U01CA202123, T32HL007118, P30DK065988, the Parker B. Francis Foundation, American Heart Association (13SDG 14320004), the Spanish Ministry of Science, Research, and Innovation (RTI2018-096501-B-I00).

BACKGROUND: The actin bundling protein Fascin is essential for developmental cell migrations and promotes cancer metastasis. In addition to bundling actin, Fascin has several actin-independent roles; how these other functions contribute to cell migration remains unclear. Border cell migration during Drosophila oogenesis provides an excellent model to study Fascin’s various roles during invasive, collective cell migration. RESULTS: On-time border cell migration during Stage 9 requires Fascin (Drosophila Singed). Fascin functions not only within the migrating border cells, but also within the nurse cells, the substrate for this migration. Fascin genetically interacts with the actin elongation factor Enabled to promote on-time Stage 9 migration and overexpression of Enabled suppresses the defects seen with loss of Fascin. Loss of Fascin results in increased, shorter and mislocalized protrusions during migration. Additionally, loss of Fascin inhibits border cell delamination and increases E-Cadherin (Drosophila Shotgun) adhesions on both the border cell clusters and nurse cells. CONCLUSIONS: Overall, Fascin promotes on-time border cell migration during Stage 9 and contributes to multiple aspects of this invasive, collective cell migration, including both protrusion dynamics and delamination. These findings have implications beyond Drosophila, as border cell migration has emerged as a model to study mechanisms mediating cancer metastasis.

Epithelial bending is a fundamental process that shapes organs during development. Previously known mechanisms involve cells locally changing shape from columnar to wedge-shaped. Here we report a different mechanism that occurs without cell wedging. In mammalian salivary glands and teeth, we show that initial invagination occurs through coordinated vertical cell movement: cells towards the periphery of the placode move vertically upwards while their more central neighbours move downwards. Movement is achieved by active cell-on-cell migration: outer cells migrate with apical, centripetally polarised leading edge protrusions but remain attached to the basal lamina, depressing more central neighbours to “telescope” the epithelium downwards into underlying mesenchyme. Inhibiting protrusion formation by Arp2/3 protein blocks invagination. FGF and Hedgehog morphogen signals are required, with FGF providing a directional cue. These findings show that epithelial bending can be achieved by a morphogenetic mechanism of coordinated cell rearrangement quite distinct from previously recognised invagination processes., Mechanisms that regulate epithelial bending mainly link to cell shape changes, for example, the formation of wedge shaped cells. Here, the authors identify a different cell behaviour in the salivary glands and teeth where initial invagination arises by a coordinated vertical cell movement.

miR-126, an endothelial-specific microRNA, is associated to vascular integrity and angiogenesis. It is well established that angiogenesis plays a critical role in burn wound healing. However, there was a lack of understanding of the mechanism by which miR-126 regulates angiogenesis during burn wound healing. HOX transcript antisense intergenic RNA (HOTAIR) is a well-characterized long non-coding RNA (lncRNA) involved in cell proliferation, apoptosis, migration, and invasion of cancer cells. Sciellin (SCEL), a precursor to the cornified envelope of human keratinocytes, has been shown to inhibit migration and invasion capabilities of colorectal cancer cells. In this study, a cohort of 20 burn wound tissues and paired adjacent normal tissues were collected. LncRNA and messenger RNA expression profiles were screened by microarray analysis in five pairs of samples with mostly increased miR-126 levels. miR-126 was highly expressed in burn wound tissues and human umbilical vein endothelial cells (HUVECs) exposed to heat stress, whereas HOTAIR and SCEL were down-regulated after thermal injury. Bioinformatic analysis, dual luciferase reporter assay, and quantitative real-time PCR were conducted to validate that HOTAIR and SCEL competitively bind to miR-126 to function as the competitive endogenous RNA. miR-126 promoted endothelial cell proliferation, migration, and angiogenesis, but suppressed apoptosis, while HOTAIR and SCEL exerted opposite effects in HUVECs. The biological functions were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, Annexin-V-FITC/PI (propidium iodide/fluorescein isothiocyanate) staining, transwell migration, and tube formation assays. Collectively, our study revealed that HOTAIR/miR-126/SCEL axis contributes to burn wound healing through mediating angiogenesis.

Angiogenic sprouting relies on collective migration and coordinated rearrangements of endothelial leader and follower cells. VE-cadherin-based adherens junctions have emerged as key cell-cell contacts that transmit forces between cells and trigger signals during collective cell migration in angiogenesis. However, the underlying molecular mechanisms that govern these processes and their functional importance for vascular development still remain unknown. We previously showed that the F-BAR protein PACSIN2 is recruited to tensile asymmetric adherens junctions between leader and follower cells. Here we report that PACSIN2 mediates the formation of endothelial sprouts during angiogenesis by coordinating collective migration. We show that PACSIN2 recruits the trafficking regulators EHD4 and MICAL-L1 to the rear end of asymmetric adherens junctions to form a recycling endosome-like tubular structure. The junctional PACSIN2/EHD4/MICAL-L1 complex controls local VE-cadherin trafficking and thereby coordinates polarized endothelial migration and angiogenesis. Our findings reveal a molecular event at force-dependent asymmetric adherens junctions that occurs during the tug-of-war between endothelial leader and follower cells, and allows for junction-based guidance during collective migration in angiogenesis.

Directed flows of cellsin vivoare essential in morphogenesis. They shape living matter in phenomena involving cell mechanics and regulations of the acto-myosin cytoskeleton. However the onset of coherent motion is still poorly understood. Here we show that coherence is associated with spontaneous alignments of cell polarity by designing cellular rings of controlled dimensions. A tug-of-war between polarities dictates the onset of coherence, as assessed by tracking live cellular shapes and motions in various experimental conditions. In addition, we identify an internally driven constraint set by cellular acto-myosin cables at boundaries as essential to ensure coherence, and active force is generated as evaluated by the high RhoA activity. The cables are required to trigger coherence as shown by our numerical simulations based on a novel Vicsek-type model including free active boundaries. We quantitatively reproducein silicocoherence onsets and we predict criteria leading to coherence. Altogether, spontaneous coherent motion results from basic competitions between cell orientations and active cables at boundaries.

Lysosomal signaling facilitates the migration of immune cells by releasing Ca2+ to activate the actin-based motor myosin II at the cell rear. However, how the actomyosin cytoskeleton physically associates to lysosomes is unknown. We have previously identified myosin II as a direct interactor of Rab7b, a small GTPase that mediates the transport from late endosomes/lysosomes to the trans-Golgi network (TGN). Here, we show that Rab7b regulates the migration of dendritic cells (DCs) in one- and three-dimensional environments. DCs are immune sentinels that transport antigens from peripheral tissues to lymph nodes to activate T lymphocytes and initiate adaptive immune responses. We found that the lack of Rab7b reduces myosin II light chain phosphorylation and the activation of the transcription factor EB (TFEB), which controls lysosomal signaling and is required for fast DC migration. Furthermore, we demonstrate that Rab7b interacts with the lysosomal Ca2+ channel TRPML1 (also known as MCOLN1), enabling the local activation of myosin II at the cell rear. Taken together, our findings identify Rab7b as the missing physical link between lysosomes and the actomyosin cytoskeleton, allowing control of immune cell migration through lysosomal signaling. This article has an associated First Person interview with the first author of the paper., Summary: The small GTPase Rab7b bridges the lysosomal Ca2+ channel TRPML1 to myosin II, thus enabling the local activation of myosin II at the cell rear and promoting fast migration of dendritic cells.

The airway epithelium serves as the interface between the host and external environment. In many chronic lung diseases, the airway is the site of substantial remodeling after injury. While, idiopathic pulmonary fibrosis (IPF) has traditionally been considered a disease of the alveolus and lung matrix, the dominant environmental (cigarette smoking) and genetic (gain of function MUC5B promoter variant) risk factor primarily affect the distal airway epithelium. Moreover, airway-specific pathogenic features of IPF include bronchiolization of the distal airspace with abnormal airway cell-types and honeycomb cystic terminal airway-like structures with concurrent loss of terminal bronchioles in regions of minimal fibrosis. However, the pathogenic role of the airway epithelium in IPF is unknown. Combining biophysical, genetic, and signaling analyses of primary airway epithelial cells, we demonstrate that healthy and IPF airway epithelia are biophysically distinct, identifying pathologic activation of the ERBB-YAP axis as a specific and modifiable driver of prolongation of the unjammed-to-jammed transition in IPF epithelia. Furthermore, we demonstrate that this biophysical state and signaling axis correlates with epithelial-driven activation of the underlying mesenchyme. Our data illustrate the active mechanisms regulating airway epithelial-driven fibrosis and identify targets to modulate disease progression., Environmental and genetic risk factors affect the distal airway epithelium in idiopatic pulmonary fibrosis (IPF) but the role of the epithelium in IPF remains unclear. Here the authors show that pathologic activation of the ERBB-YAP axis induces dynamic and structural dysfunction in the distal airway epithelium eliciting a pro-fibrotic phenotype in mesenchymal cells.

Significance As collective cellular migration is critical for multicellular processes such as healing, approaches to reprogram it are of great interest. However, little is known about what happens when external “commands” clash with natural collective behaviors in a tissue. We investigate this question using a bioelectric stimulus—electrotaxis—to externally program cell migration in large, cultured layers of primary mouse skin monolayers, demonstrating how strong endogenous cell coordination competes with external migration commands, reducing tissue responsiveness to the commands and even causing significant cellular damage. However, we show that specifically weakening natural cell coordination by disrupting E-cadherin–mediated cell–cell adhesion improves our ability to control collective cell migration. These results offer a general approach to externally controlling highly coordinated tissues., As collective cell migration is essential in biological processes spanning development, healing, and cancer progression, methods to externally program cell migration are of great value. However, problems can arise if the external commands compete with strong, preexisting collective behaviors in the tissue or system. We investigate this problem by applying a potent external migratory cue—electrical stimulation and electrotaxis—to primary mouse skin monolayers where we can tune cell–cell adhesion strength to modulate endogenous collectivity. Monolayers with high cell–cell adhesion showed strong natural coordination and resisted electrotactic control, with this conflict actively damaging the leading edge of the tissue. However, reducing preexisting coordination in the tissue by specifically inhibiting E-cadherin–dependent cell–cell adhesion, either by disrupting the formation of cell–cell junctions with E-cadherin–specific antibodies or rapidly dismantling E-cadherin junctions with calcium chelators, significantly improved controllability. Finally, we applied this paradigm of weakening existing coordination to improve control and demonstrate accelerated wound closure in vitro. These results are in keeping with those from diverse, noncellular systems and confirm that endogenous collectivity should be considered as a key quantitative design variable when optimizing external control of collective migration.

Tumor clonal heterogeneity drives treatment resistance. But robust models are lacking that permit eavesdropping on the basic interaction network of tumor clones. We developed an in vitro, functional model of clonal cooperation using U87MG glioblastoma cells, which isolates fundamental clonal interactions. In this model pre-labeled clones are co-cultured to track changes in their individual motility, growth, and drug resistance behavior while mixed. This highly reproducible system allowed us to address a new class of fundamental questions about clonal interactions. We demonstrate that (i) a single clone can switch off the motility of the entire multiclonal U87MG cell line in 3D culture, (ii) maintenance of clonal heterogeneity is an intrinsic and influential cancer cell property, where clones coordinate growth rates to protect slow growing clones, and (iii) two drug sensitive clones can develop resistance de novo when cooperating. Furthermore, clonal communication for these specific types of interaction did not require diffusible factors, but appears to depend on cell-cell contact. This model constitutes a straightforward but highly reliable tool for isolating the complex clonal interactions that make up the fundamental “hive mind” of the tumor. It uniquely exposes clonal interactions for future pharmacological and biochemical studies.

Collective cell migration is involved in development, wound healing and metastasis. In the Drosophila ovary, border cells (BC) form a small cluster that migrates collectively through the egg chamber. To achieve directed motility, the BC cluster coordinates the formation of protrusions in its leader cell and contractility at the rear. Restricting protrusions to leader cells requires the actin and plasma membrane linker Moesin. Herein, we show that the Ste20-like kinase Misshapen phosphorylates Moesin in vitro and in BC. Depletion of Misshapen disrupts protrusion restriction, thereby allowing other cells within the cluster to protrude. In addition, we show that Misshapen is critical to generate contractile forces both at the rear of the cluster and at the base of protrusions. Together, our results indicate that Misshapen is a key regulator of BC migration as it coordinates two independent pathways that restrict protrusion formation to the leader cells and induces contractile forces., Directed motility of cell clusters requires coordination of protrusion formation at the front of leader cells and contractility at the rear. Here the authors show that the kinase Misshapen restricts protrusions to the leader cell and promotes contractile forces at the rear of the cluster.

Tumor metastasis is the predominant cause of lethality in cancer. We found that Neobractatin (NBT), a natural compound isolated from Garcinia bracteata, could efficiently inhibit breast and lung cancer cells metastasis. However, the mechanisms of NBT inhibiting cancer metastasis remain unclear. Based on the RNA-sequencing result and transcriptome analysis, Muscleblind-like 2 (MBNL2) was found to be significantly upregulated in the cells treated with NBT. The Cancer Genome Atlas (TCGA) database analysis indicated that the expression of MBNL2 in breast and lung carcinoma tumor tissues was significantly lower compared to normal tissues. We thus conducted to investigate the antimetastatic role of MBNL2. MBNL2 overexpression mimicked the effect of NBT on breast cancer and lung cancer cell motility and metastasis, in addition significantly enhanced the inhibition effect of NBT. MBNL2 knockdown furthermore partially eliminated the inhibitory effect of NBT on metastasis. Mechanistically, we demonstrated that NBT- and MBNL2-mediated antimetastasis regulation significantly correlated with the pAKT/epithelial-mesenchymal transition (EMT) pathway. Subsequent in vivo study showed the same metastasis inhibition effect in NBT and MBNL2 in MDA-MB-231 xenografts mouse model. This study suggest that NBT possesses significant antitumor activity in breast and lung cancer cells that is partly mediated through the MBNL2 expression and enhancement in metastasis via the pAKT/EMT signaling pathway.

Proliferation and migration of endothelial cells play an important role in many biological activities, and they can be regulated by various microenvironmental factors. In this paper, a novel microfluidic collective cell migration assay is developed to study endothelial cell migration and proliferation under combinations of three oxygen conditions: normoxia, oxygen gradient, and hypoxia and three medium compositions: normal growth medium, the medium with cytochalasin-D for actin polymerization inhibition, and with YC-1 for hypoxia-inducible factor (HIF) inhibition. The microfluidic device designed in the paper allows cell patterns formed with consistent dimensions using laminar flow patterning. In addition, stable oxygen gradients can be generated within the device by a spatially confined chemical reaction method. The device can be operated in conventional cell incubators with minimal chemical reagents and instrumentation for practical applications. The results show directional collective cell migration of the endothelial cells under the oxygen gradients for all the medium compositions. The directional behavior has never been discussed before, and indicates critical roles of oxygen gradients in guiding endothelial cell migration during various biological activities. The developed assay provides a practical yet powerful tool for further in vitro study of endothelial cell behaviors under various physiological microenvironments.

Significance Noonan syndrome belongs to a group of related developmental disorders termed the RASopathies, and both the M-Ras GTPase and its effector Shoc2 are signaling proteins mutated in specific subtypes of this disorder. Previously, the M-Ras/Shoc2 complex has been shown to act as a positive regulator of growth factor-mediated ERK cascade activation, and, here, we demonstrate a function for M-Ras/Shoc2/ERK cascade signaling in the dynamic control of cell–cell adhesion that is required for collective cell migration. Given the essential role of collective cell migration in human embryogenesis and organ development, our elucidation of M-Ras/Shoc2 function in this highly regulated process may provide insight regarding the cellular basis of defects associated with Noonan syndrome and other RASopathies., Collective cell migration is required for normal embryonic development and contributes to various biological processes, including wound healing and cancer cell invasion. The M-Ras GTPase and its effector, the Shoc2 scaffold, are proteins mutated in the developmental RASopathy Noonan syndrome, and, here, we report that activated M-Ras recruits Shoc2 to cell surface junctions where M-Ras/Shoc2 signaling contributes to the dynamic regulation of cell–cell junction turnover required for collective cell migration. MCF10A cells expressing the dominant-inhibitory M-RasS27N variant or those lacking Shoc2 exhibited reduced junction turnover and were unable to migrate effectively as a group. Through further depletion/reconstitution studies, we found that M-Ras/Shoc2 signaling contributes to junction turnover by modulating the E-cadherin/p120-catenin interaction and, in turn, the junctional expression of E-cadherin. The regulatory effect of the M-Ras/Shoc2 complex was mediated at least in part through the phosphoregulation of p120-catenin and required downstream ERK cascade activation. Strikingly, cells rescued with the Noonan-associated, myristoylated-Shoc2 mutant (Myr-Shoc2) displayed a gain-of-function (GOF) phenotype, with the cells exhibiting increased junction turnover and reduced E-cadherin/p120-catenin binding and migrating as a faster but less cohesive group. Consistent with these results, Noonan-associated C-Raf mutants that bypass the need for M-Ras/Shoc2 signaling exhibited a similar GOF phenotype when expressed in Shoc2-depleted MCF10A cells. Finally, expression of the Noonan-associated Myr-Shoc2 or C-Raf mutants, but not their WT counterparts, induced gastrulation defects indicative of aberrant cell migration in zebrafish embryos, further demonstrating the function of the M-Ras/Shoc2/ERK cascade signaling axis in the dynamic control of coordinated cell movement.

International audience; The specific structure of the extracellular matrix (ECM), and in particular the density and orientation of collagen fibres, plays an important role in the evolution of solid cancers. While many experimental studies discussed the role of ECM in individual and collective cell migration, there are still unanswered questions about the impact of nonlocal cell sensing of other cells on the overall shape of tumour aggregation and its migration type. There are also unanswered questions about the migration and spread of tumour that arises at the boundary between different tissues with different collagen fibre orientations. To address these questions, in this study we develop a hybrid multi-scale model that considers the cells as individual entities and ECM as a continuous field. The numerical simulations obtained through this model match experimental observations, confirming that tumour aggregations are not moving if the ECM fibres are distributed randomly, and they only move when the ECM fibres are highly aligned. Moreover, the stationary tumour aggregations can have circular shapes or irregular shapes (with finger-like protrusions), while the moving tumour aggregations have elongate shapes (resembling to clusters, strands or files). We also show that the cell sensing radius impacts tumour shape only when there is a low ratio of fibre to non-fibre ECM components. Finally, we investigate the impact of different ECM fibre orientations corresponding to different tissues, on the overall tumour invasion of these neighbouring tissues.

Tight junctions (TJs) are essential components of epithelial tissues connecting neighboring cells to provide protective barriers. While their general function to seal compartments is well understood, their role in collective cell migration is largely unexplored. Here, the importance of the TJ zonula occludens (ZO) proteins ZO1 and ZO2 for epithelial migration is investigated employing video microscopy in conjunction with velocimetry, segmentation, cell tracking, and atomic force microscopy/spectroscopy. The results indicate that ZO proteins are necessary for fast and coherent migration. In particular, ZO1 and 2 loss (dKD) induces actomyosin remodeling away from the central cortex towards the periphery of individual cells, resulting in altered viscoelastic properties. A tug‐of‐war emerges between two subpopulations of cells with distinct morphological and mechanical properties: 1) smaller and highly contractile cells with an outward bulging apical membrane, and 2) larger, flattened cells, which, due to tensile stress, display a higher proliferation rate. In response, the cell density increases, leading to crowding‐induced jamming and more small cells over time. Co‐cultures comprising wildtype and dKD cells migrate inefficiently due to phase separation based on differences in contractility rather than differential adhesion. This study shows that ZO proteins are necessary for efficient collective cell migration by maintaining tissue fluidity and controlling proliferation., In this article, it is shown that tight junction zonula occludens (ZO) proteins maintain epithelial cell sheet fluidity and thereby ensure efficient and coherent migration. Cells lacking these proteins lose viscoelastic tissue integrity due to actomyosin remodeling and increase proliferation, inducing cellular crowding. Particularly, upon ZO protein loss, overly contractile small cells at high cell densities eventually impair migration by causing jamming.

Optical flow algorithms have seen poor adoption in the biological community compared with particle image velocimetry for quantifying cellular dynamics because of the lack of proper validation and an intuitive user interface. To address these challenges, we present OpFlowLab, an integrated platform that integrates our motion estimation workflow. Using routines in our workflow, we demonstrate that optical flow algorithms are more accurate than PIV in simulated images of the movement of nuclei. Qualitative assessment with actual nucleus images further supported this finding. Additionally, we show that refinement of the optical flow velocities is possible with a simple object-matching procedure, opening up the possibility of obtaining reasonable velocity estimates under less ideal imaging conditions. To visualize velocity fields, we employ artificial tracers to allow for the drawing of pathlines. Through the adoption of OpFlowLab, we are confident that optical flow algorithms will allow for the exploration of dynamic biological systems in greater accuracy and detail.

Collective dynamics in multicellular systems such as biological organs and tissues plays a key role in biological development, regeneration, and pathological conditions. Collective tissue dynamics—understood as population behaviour arising from the interplay of the constituting discrete cells—can be studied with on- and off-lattice agent-based models. However, classical on-lattice agent-based models, also known as cellular automata, fail to replicate key aspects of collective migration, which is a central instance of collective behaviour in multicellular systems. To overcome drawbacks of classical on-lattice models, we introduce an on-lattice, agent-based modelling class for collective cell migration, which we call biological lattice-gas cellular automaton (BIO-LGCA). The BIO-LGCA is characterised by synchronous time updates, and the explicit consideration of individual cell velocities. While rules in classical cellular automata are typically chosen ad hoc, rules for cell-cell and cell-environment interactions in the BIO-LGCA can also be derived from experimental cell migration data or biophysical laws for individual cell migration. We introduce elementary BIO-LGCA models of fundamental cell interactions, which may be combined in a modular fashion to model complex multicellular phenomena. We exemplify the mathematical mean-field analysis of specific BIO-LGCA models, which allows to explain collective behaviour. The first example predicts the formation of clusters in adhesively interacting cells. The second example is based on a novel BIO-LGCA combining adhesive interactions and alignment. For this model, our analysis clarifies the nature of the recently discovered invasion plasticity of breast cancer cells in heterogeneous environments., Author summary Pattern formation during embryonic development and pathological tissue dynamics, such as cancer invasion, emerge from individual intercellular interactions. In order to study the impact of single cell dynamics and cell-cell interactions on tissue behaviour, one needs to develop space-time-dependent on- or off-lattice agent-based models (ABMs), which consider the behaviour of individual cells. However, classical on-lattice agent-based models also known as cellular automata fail to replicate key aspects of collective migration, which is a central instance of collective behaviour in multicellular systems. Here, we present the rule- and lattice-based BIO-LGCA modelling class which allows for (i) rigorous derivation of rules from biophysical laws and/or experimental data, (ii) mathematical analysis of collective migration, and (iii) computationally efficient simulations.

The signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive cellular responses occur is unclear. Here, we report development of 3-Dimensional culture analyses to separately quantify growth and invasion. We identify that alternate variants of IQSEC1, an ARF GTPase Exchange Factor, act as switches to promote invasion over growth by controlling phosphoinositide metabolism. All IQSEC1 variants activate ARF5- and ARF6-dependent PIP5-kinase to promote PI(3,4,5)P3-AKT signalling and growth. In contrast, select pro-invasive IQSEC1 variants promote PI(3,4,5)P3 production to form invasion-driving protrusions. Inhibition of IQSEC1 attenuates invasion in vitro and metastasis in vivo. Induction of pro-invasive IQSEC1 variants and elevated IQSEC1 expression occurs in a number of tumour types and is associated with higher-grade metastatic cancer, activation of PI(3,4,5)P3 signalling, and predicts long-term poor outcome across multiple cancers. IQSEC1-regulated phosphoinositide metabolism therefore is a switch to induce invasion over growth in response to the same external signal. Targeting IQSEC1 as the central regulator of this switch may represent a therapeutic vulnerability to stop metastasis., The signalling pathways underpinning cell growth and invasion use overlapping components, yet how mutually exclusive responses occur is unclear. Here, the authors show that alternate isoforms of the ARF GTPase exchange factor IQSEC1 direct phosphoinositide metabolism to control this switch.

This review focuses on novel applications based on multifunctional materials to actuate biological processes. The first section of the work revisits the current knowledge on mechanically dependent biological processes across several scales from subcellular and cellular level to the cellcollective scale (continuum approaches). This analysis presents a wide variety of mechanically dependent biological processes on nervous system behaviour; bone development and healing; collective cell migration. In the second section, this review presents recent advances in smart materials suitable for use as cell substrates or scaffolds, with a special focus on magneto-active polymers (MAPs). Throughout the manuscript, both experimental and computational methodologies applied to the different treated topics are reviewed. Finally, the use of smart polymeric materials in bioengineering applications is discussed. This work has been supported by the Madrid Government (Comunidad de Madrid) under the Multiannual Agreement with UC3M in the line of "Fostering Young Doctors Research" (BIOMASKIN-CM-UC3M), and in the context of the V PRICIT (Regional Programme of Research and Technological Innovation, and support from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 947723). DGG acknowledges support from the Talent Attraction grant (CM 2018-2018-T2/IND-9992) from the Comunidad de Madrid. Publicado

Understanding tissue dynamics and morphogenesis is one of the big challenges of modern biology and has already been studied in detail on the molecular and genetic level during the past decades. The results of these studies led to an integrated view, where the molecular and genetic changes affect force generation and mechanical properties of cells and tissue. However, during the rise of mechanobiology, it turned out that also vice versa, the mechanical properties of the environment largely influence the genetic and molecular composition of many cells. In the context of tissue dynamics and morphogenesis, this single cell–level view needs to be extended to the tissue scale. In this chapter, we will discuss the recent advances in understanding how tissue mechanical changes are the driving forces for different morphogenic collective cell migration during early zebrafish development. We will discuss the first onset of collective migration during zebrafish epiboly, study recent wetting approaches, and review a model that considers viscous effects to explain the doming of the yolk cell. Moreover, we will introduce symmetry-breaking events that are a key element of gastrulation, and finally, we describe how a jamming transition helps in forming the somites of the zebrafish tail.

International audience; The ability of organisms to spontaneously generate order relies on the intricate interplay of mechanical and biochemical signals. If the general consensus is that chemical signaling governs the behavior of cells, an increasing amount of evidence points towards the impact of mechanical factors into differentiation, proliferation, motility and cancer progression. In this context, several studies recently highlighted the existence of long-range mechanical excitations (i.e. waves) at the supra-cellular level. In this review, we discuss all recent observations of mechanical oscillations on in vitro monolay-ers on a planar geometry, and their potential role for the patterning of cellular behavior at large scales. We focus on the importance of externally imposed confinement on the appearance of different phenotypes, namely traveling and standing waves, and how these mechanical stimuli might have a different potential for future research. We tackle the debate of whether oscillations are innate to the cell or only dependent on the boundary conditions, and propose a framework to reconcile these two points of view. We conclude with an outlook on the future of this field, and the directions to be explored in order to give mechanical oscillations a biological role.

The migration of activated T cells across the blood–brain barrier (BBB) is a critical step in central nervous system (CNS) immune surveillance and inflammation. Whereas T cell diapedesis across the intact BBB seems to occur preferentially through the BBB cellular junctions, impaired BBB integrity during neuroinflammation is accompanied by increased transcellular T cell diapedesis. The underlying mechanisms directing T cells to paracellular versus transcellular sites of diapedesis across the BBB remain to be explored. By combining in vitro live-cell imaging of T cell migration across primary mouse brain microvascular endothelial cells (pMBMECs) under physiological flow with serial block-face scanning electron microscopy (SBF-SEM), we have identified BBB tricellular junctions as novel sites for T cell diapedesis across the BBB. Downregulated expression of tricellular junctional proteins or protein-based targeting of their interactions in pMBMEC monolayers correlated with enhanced transcellular T cell diapedesis, and abluminal presence of chemokines increased T cell diapedesis through tricellular junctions. Our observations assign an entirely novel role to BBB tricellular junctions in regulating T cell entry into the CNS. This article has an associated First Person interview with the first author of the paper., Highlighted Article: Ultrastructural analysis of T cell migration across the blood–brain barrier (BBB) under physiological flow identifies BBB tricellular junctions as sites of T cell diapedesis.

Collective cell migration (CCM), a highly coordinated and fine-tuned migratory mode, is involved in a plethora of biological processes, such as embryogenesis, tissue repair and cancer invasion. Although a good comprehension of how cells collectively migrate by following molecular rules has been generated, the impact of cellular rearrangements on collective migration remains less understood. Thus, considering CCM from a multi-scale quantitative approach could result in a powerful tool to address the contribution of cellular rearrangements in CCM and help to understand this important but still controversial topic. In this work, a review of existing literature in CCM modeling at different scales is given along with assortment of published experimental findings, to invite experimentalists to test given theoretical considerations in multicellular systems. In addition, three different time and space scales (free or weakly connected cells, cluster of cells and collision fronts of different cells clusters) are considered and the multi-scale nature of those processes was discussed with special emphasis of jamming and unjamming states.

Mathematical and computational models can assist in gaining an understanding of cell behavior at many levels of organization. Here, we review models in the literature that focus on eukaryotic cell motility at 3 size scales: intracellular signaling that regulates cell shape and movement, single cell motility, and collective cell behavior from a few cells to tissues. We survey recent literature to summarize distinct computational methods (phase-field, polygonal, Cellular Potts, and spherical cells). We discuss models that bridge between levels of organization, and describe levels of detail, both biochemical and geometric, included in the models. We also highlight links between models and experiments. We find that models that span the 3 levels are still in the minority., Comment: 39 pages, 5 figures

The Zebrafish Posterior Lateral Line primordium migrates in a channel between the skin and somites. Its migration depends on the coordinated movement of its mesenchymal-like leading cells and trailing cells, which form epithelial rosettes, or protoneuromasts. We describe a superficial population of flat primordium cells that wrap around deeper epithelialized cells and extend polarized lamellipodia to migrate apposed to the overlying skin. Polarization of lamellipodia extended by both superficial and deeper protoneuromast-forming cells depends on Fgf signaling. Removal of the overlying skin has similar effects on superficial and deep cells: lamellipodia are lost, blebs appear instead, and collective migration fails. When skinned embryos are embedded in Matrigel, basal and superficial lamellipodia are recovered; however, only the directionality of basal protrusions is recovered, and migration is not rescued. These observations support a key role played by superficial primordium cells and the skin in directed migration of the Posterior Lateral Line primordium.

The movement of collective cells is affected through changes in physical interactions of cells in response to external mechanical stimuli, including fluid flow. Most tissues are affected by fluid flow at the interstitial level, but few studies have investigated the physical effects in collective cells affected by a low flow rate. In this study, collective cell migration of Madin–Darby canine kidney (MDCK) epithelial cells was investigated under static or interstitial flow (0, 0.1, and 1 μL/min) using a traction microfluidic device. The optimization of calculation of cellular traction forces was first achieved by changing interrogation window size from the fluorescent bead images. Migration analysis of cell collectives patterned with a 700 μm circular shape reveals that cells under the slow flow (0.1 and 1 μL/min) showed the inhibitory migration by decreasing cell island size and cellular speed compared to that of static condition. Analysis of cellular forces shows that level of traction forces was lower in the slow flow condition (~20 Pa) compared to that of static condition (~50 Pa). Interestingly, the standard deviation of traction force of cells was dramatically decreased as the flow rate increased from 0 to 1 μL/min, which indicates that flow affects the distribution of cellular traction forces among cell collectives. Cellular tension was increased by 50% in the cells under the fluid flow rate of 1 μL/min. Treatment of calcium blocker increased the migratory speed of cells under the flow condition, whereas there is little change of cellular forces. In conclusion, it has been shown that the interstitial flow inhibited the collective movement of epithelial cells by decreasing and re-distributing cellular forces. These findings provide insights into the study of the effect of interstitial flow on cellular behavior, such as development, regeneration, and morphogenesis.

Despite the fact that different genetic programmes drive metastasis of solid tumours, the ultimate outcome is the same: tumour cells are empowered to pass a series of physical hurdles to escape the primary tumour and disseminate to other organs. Epithelial-to-mesenchymal transition (EMT) has been proposed to drive the detachment of individual cells from primary tumour masses and facilitate the subsequent establishment of metastases in distant organs. However, this concept has been challenged by observations from pathologists and from studies in animal models, in which partial and transient acquisition of mesenchymal traits is seen but tumour cells travel collectively rather than as individuals. In this review, we discuss how crosstalk between a hybrid E/M state and variations in the mechanical aspects of the tumour microenvironment can provide tumour cells with the plasticity required for strategies to navigate surrounding tissues en route to dissemination. Targeting such plasticity provides therapeutic opportunities to combat metastasis.

Changes in tissue architecture and multicellular organisation contribute to many diseases, including cancer and cardiovascular diseases. Scratch wound assay is a commonly used tool that assesses cells’ migratory ability based on the area of a wound they cover over a certain time. However, analysis of changes in the organisational patterns formed by migrating cells following genetic or pharmacological perturbations are not well explored in these assays, in part because analysing the resulting imaging data is challenging. Here we present DeepScratch, a neural network that accurately detects the cells in scratch assays based on a heterogeneous set of markers. We demonstrate the utility of DeepScratch by analysing images of more than 232,000 lymphatic endothelial cells. In addition, we propose various topological measures of cell connectivity and local cell density (LCD) to characterise tissue remodelling during wound healing. We show that LCD-based metrics allow classification of CDH5 and CDC42 genetic perturbations that are known to affect cell migration through different biological mechanisms. Such differences cannot be captured when considering only the wound area. Taken together, single-cell detection using DeepScratch allows more detailed investigation of the roles of various genetic components in tissue topology and the biological mechanisms underlying their effects on collective cell migration.

Epithelial-mesenchymal transition (EMT) is a cellular biological process involved in migration of primary cancer cells to secondary sites facilitating metastasis. Besides, EMT also confers properties such as stemness, drug resistance and immune evasion which can aid a successful colonization at the distant site. EMT is not a binary process; recent evidence suggests that cells in partial EMT or hybrid E/M phenotype(s) can have enhanced stemness and drug resistance as compared to those undergoing a complete EMT. Moreover, partial EMT enables collective migration of cells as clusters of circulating tumor cells or emboli, further endorsing that cells in hybrid E/M phenotypes may be the ‘fittest’ for metastasis. Here, we review mechanisms and implications of hybrid E/M phenotypes, including their reported association with hypoxia. Hypoxia-driven activation of HIF-1α can drive EMT. In addition, cyclic hypoxia, as compared to acute or chronic hypoxia, shows the highest levels of active HIF-1α and can augment cancer aggressiveness to a greater extent, including enriching for a partial EMT phenotype. We also discuss how metastasis is influenced by hypoxia, partial EMT and collective cell migration, and call for a better understanding of interconnections among these mechanisms. We discuss the known regulators of hypoxia, hybrid EMT and collective cell migration and highlight the gaps which needs to be filled for connecting these three axes which will increase our understanding of dynamics of metastasis and help control it more effectively., Graphical abstract Unlabelled Image

International audience; One contribution of 14 to a theme issue 'Multi-scale analysis and modelling of collective migration in biological systems'. Collective migration has become a paradigm for emergent behaviour in systems of moving and interacting individual units resulting in coherent motion. In biology, these units are cells or organisms. Collective cell migration is important in embryonic development, where it underlies tissue and organ formation, as well as pathological processes, such as cancer invasion and metastasis. In animal groups, collective movements may enhance individuals' decisions and facilitate navigation through complex environments and access to food resources. Mathematical models can extract unifying principles behind the diverse manifestations of collective migration. In biology, with a few exceptions, collective migration typically occurs at a 'mesoscopic scale' where the number of units ranges from only a few dozen to a few thousands, in contrast to the large systems treated by statistical mechanics. Recent developments in multi-scale analysis have allowed linkage of mesoscopic to micro-and macroscopic scales, and for different biological systems. The articles in this theme issue on 'Multi-scale analysis and modelling of collective migration' compile a range of mathematical modelling ideas and multi-scale methods for the analysis of collective migration. These approaches (i) uncover new unifying organization principles of collective behaviour, (ii) shed light on the transition from single to collective migration, and (iii) allow us to define similarities and differences of collective behaviour in groups of cells and organisms. As a common theme, self-organized collective migration is the result of ecological and evolutionary constraints both at the cell and organismic levels. Thereby, the rules governing physiological collective behaviours also underlie pathological processes, albeit with different upstream inputs and consequences for the group. This article is part of the theme issue 'Multi-scale analysis and modelling of collective migration in biological systems'.

Shear stress induces directed endothelial cell (EC) migration in blood vessels leading to vessel diameter increase and induction of vascular maturation. Other factors, such as EC elongation and interaction between ECs and non-vascular areas are also important. Computational models have previously been used to study collective cell migration. These models can be used to predict EC migration and its effect on vascular remodelling during embryogenesis. We combined live time-lapse imaging of the remodelling vasculature of the quail embryo yolk sac with flow quantification using a combination of micro-Particle Image Velocimetry and computational fluid dynamics. We then used the flow and remodelling data to inform a model of EC migration during remodelling. To obtain the relation between shear stress and velocity in vitro for EC cells, we developed a flow chamber to assess how confluent sheets of ECs migrate in response to shear stress. Using these data as an input, we developed a multiphase, self-propelled particles (SPP) model where individual agents are driven to migrate based on the level of shear stress while maintaining appropriate spatial relationship to nearby agents. These agents elongate, interact with each other, and with avascular agents at each time-step of the model. We compared predicted vascular shape to real vascular shape after 4 hours from our time-lapse movies and performed sensitivity analysis on the various model parameters. Our model shows that shear stress has the largest effect on the remodelling process. Importantly, however, elongation played an especially important part in remodelling. This model provides a powerful tool to study the input of different biological processes on remodelling., Author summary Shear stress is known to play a leading role in endothelial cell (EC) migration and hence, vascular remodelling. Vascular remodelling is, however, more complicated than only EC migration. To achieve a better understanding of this process, we developed a computational model in which, shear stress mediated EC migration has the leading role and other factors, such as avascular regions and EC elongation, are also accounted for. We have tested this model for different vessel shapes during remodelling and could study the role that each of these factors play in remodelling. This model gives us the possibility of addition of other factors such as biochemical signals and angiogenesis which will help us in the study of vascular remodelling in both development and disease.

The wound healing assay provides essential information about collective cell migration and cell-to-cell interactions. It is a simple, effective, and widely used tool for observing the effect of numerous chemical treatments on wound healing speed. To perform and analyze a wound healing assay, various imaging techniques have been utilized. However, image acquisition and analysis are often limited in two-dimensional space or require the use of exogenous labeling agents. Here, we present a method for imaging large-scale wound healing assays in a label-free and volumetric manner using optical diffraction tomography (ODT). We performed quantitative high-resolution three-dimensional (3D) analysis of cell migration over a long period without difficulties such as photobleaching or phototoxicity. ODT enables the reconstruction of the refractive index (RI) tomogram of unlabeled cells, which provides both structural and biochemical information about the individual cell at subcellular resolution. Stitching multiple RI tomograms enables long-term (24 h) and large field-of-view imaging (> 800 × 400 μm2) with a lateral resolution of 110 nm. We demonstrated the thickness changes of leading cells and studied the effects of cytochalasin D. The 3D RI tomogram also revealed increased RI values in leading cells compared to lagging cells, suggesting the formation of a highly concentrated subcellular structure.STATEMENT OF SIGNIFICANCEThe wound healing assay is a simple but effective tool for studying collective cell migration (CCM) that is widely used in biophysical studies and high-throughput screening. However, conventional imaging and analysis methods only address two-dimensional properties in a wound healing assay, such as gap closure rate. This is unfortunate because biological cells are complex 3D structures, and their dynamics provide significant information about cell physiology. Here, we presented three-dimensional (3D) label-free imaging for wound healing assays and investigated the 3D dynamics of CCM. High-resolution subcellular structures as well as their collective dynamics were imaged and analyzed quantitatively. Our label-free quantitative 3D analysis method provides a unique opportunity to study the behavior of migrating cells during the wound healing process.

We previously reported that corneal fibroblasts within 3D fibrin matrices secrete, bind, and organize fibronectin into tracks that facilitate cell spreading and migration. Other cells use these fibronectin tracks as conduits, which leads to the development of an interconnected cell/fibronectin network. In this study, we investigate how cell-induced reorganization of fibrin correlates with fibronectin track formation in response to two growth factors present during wound healing: PDGF BB, which stimulates cell spreading and migration, and TGF&beta, 1, which stimulates cellular contraction and myofibroblast transformation. Both PDGF BB and TGF&beta, 1 stimulated global fibrin matrix contraction (p &lt, 0.005), however, the cell and matrix patterning were different. We found that, during PDGF BB-induced cell spreading, fibronectin was organized simultaneously with the generation of tractional forces at the leading edge of pseudopodia. Over time this led to the formation of an interconnected network consisting of cells, fibronectin and compacted fibrin tracks. Following culture in TGF&beta, 1, cells were less motile, produced significant local fibrin reorganization, and formed fewer cellular connections as compared to PDGF BB (p &lt, 0.005). Although bands of compacted fibrin tracks developed in between neighboring cells, fibronectin labeling was not generally present along these tracks, and the correlation between fibrin and fibronectin labeling was significantly less than that observed in PDGF BB (p &lt, 0.001). Taken together, our results show that cell-induced extracellular matrix (ECM) reorganization can occur independently from fibronectin patterning. Nonetheless, both events seem to be coordinated, as corneal fibroblasts in PDGF BB secrete and organize fibronectin as they preferentially spread along compacted fibrin tracks between cells, producing an interconnected network in which cells, fibronectin and compacted fibrin tracks are highly correlated. This mechanism of patterning could contribute to the formation of organized cellular networks that have been observed following corneal injury and refractive surgery.

Collective cell migration is an essential phenomenon in many naturally occurring pathophysiological processes, as well as in tissue engineering applications. Cells in tissues and organs are known to sense chemical and mechanical signals from the microenvironment and collectively respond to these signals. For the last few decades, the effects of chemical signals such as growth factors and therapeutic agents on collective cell behaviors in the context of tissue engineering have been extensively studied, whereas those of the mechanical cues have only recently been investigated. The mechanical signals can be presented to the constituent cells in different forms, including topography, substrate stiffness, and geometrical constraint. With the recent advancement in microfabrication technology, researchers have gained the ability to manipulate the geometrical constraints by creating 3D structures to mimic the tissue microenvironment. In this study, we simulate the pore curvature as presented to the cells within 3D-engineered tissue-scaffolds by developing a device that features tortuous microchannels with geometric variations. We show that both cells at the front and rear respond to the varying radii of curvature and channel amplitude by altering the collective migratory behavior, including cell velocity, morphology, and turning angle. These findings provide insights into adaptive migration modes of collective cells to better understand the underlying mechanism of cell migration for optimization of the engineered tissue-scaffold design.

Cells mitigate ER stress through the unfolded protein response (UPR). Here, we report formation of ER whorls as an effector mechanism of the ER stress response. We found that strong ER stress induces formation of ER whorls, which contain ER-resident proteins such as the Sec61 complex and PKR-like ER kinase (PERK). ER whorl formation is dependent on PERK kinase activity and is mediated by COPII machinery, which facilitates ER membrane budding to form tubular-vesicular ER whorl precursors. ER whorl precursors then go through Sec22b-mediated fusion to form ER whorls. We further show that ER whorls contribute to ER stress-induced translational inhibition by possibly modulating PERK activity and by sequestering translocons in a ribosome-free environment. We propose that formation of ER whorls reflects a new type of ER stress response that controls inhibition of protein translation.

Accumulating evidence indicates that intratumoral heterogeneity contributes to the development of resistance to anticancer therapeutics. Fibroblasts, which are components of the paraneoplastic stroma, play a crucial role in the wound-healing process. Activated fibroblasts accumulate in the wound and are involved in many aspects of the tissue remodeling cascade that initiates the repair process and prevents further tissue damage. The pathophysiological roles of cancer-associated fibroblasts (CAFs) in the heterogeneous tumor microenvironment have attracted increasing interest. CAFs play crucial roles in tumor progression and the response to chemotherapy. Several cytokines and chemokines are involved in the conversion of normal fibroblasts into CAFs, and some of these form a feedback loop between cancer cells and CAFs. In addition, the physical force between tumor cells and CAFs promotes cooperative invasion or co-migration of both types of cells. Pro-inflammatory cytokines, such as leukemia inhibitory factor (LIF) and interleukin-6 (IL-6), are secreted by both cancer cells and CAFs, and mediate the epigenetic modification of CAFs. This enhances the pro-tumorigenic function of CAFs mediated by promoting actomyosin contractility and extracellular matrix remodeling to form the tracks used for collective cancer cell migration. The concept of intra-tumoral CAF heterogeneity refers to the presence of inflammatory CAFs with low levels of α-smooth muscle actin (α-SMA) and high levels of IL-6 expression, which are in striking contrast to transforming growth factor-β (TGF-β)-dependent myofibroblastic CAFs with high α-SMA expression levels. CAF populations that suppress tumor growth and progression through stroma-specific Hedgehog (Hh) activation have been detected in different murine tumor models including those of the bladder, colon, and pancreas. A new therapeutic strategy targeting CAFs is the “stromal switch,” in which tumor-promoting CAFs are changed into tumor-retarding CAFs with attenuated stromal stiffness. Several molecular mechanisms that can be exploited to design personalized anticancer therapies targeting CAFs remain to be elucidated. Strategies aimed at targeting the tumor stroma as well as tumor cells themselves have attracted academic attention for their application in precision medicine. This novel review discusses the role of the activation of EGFR, Wnt/β-catenin, Hippo, TGF-β, and JAK/STAT cascades in CAFs in relation to the chemoresistance and invasive/metastatic behavior of cancer cells. For instance, although activated EGFR signaling contributes to collective cell migration in cooperation with CAFs, an activated Hippo pathway is responsible for stromal stiffness resulting in the collapse of neoplastic blood vessels. Therefore, identifying the signaling pathways that are activated under specific conditions is crucial for precision medicine.

Summary During collective cell migration, front cells tend to extend a predominant leading protrusion, which is rarely present in cells at the side or rear positions. Using Drosophila border cells (BCs) as a model system of collective migration, we revealed the presence of a supracellular actomyosin network at the peripheral surface of BC clusters. We demonstrated that the Myosin II-mediated mechanical tension as exerted by this peripheral supracellular network not only mediated cell-cell communication between leading BC and non-leading BCs but also restrained formation of prominent protrusions at non-leading BCs. Further analysis revealed that a cytoplasmic dendritic actin network that depends on the function of Arp2/3 complex interacted with the actomyosin network. Together, our data suggest that the outward pushing or protrusive force as generated from Arp2/3-dependent actin polymerization and the inward restraining force as produced from the supracellular actomyosin network together determine the collective and polarized morphology of migratory BCs., Graphical Abstract, Highlights • A supracellular actomyosin network encompasses the surface of migratory BC cluster • The network mediates cell-cell communication and restrains protrusion formation • Dynamics of myosin patches correlate with protrusion behaviors • A cytoplasmic dendritic actin population interacts with the actomyosin network, Biological Sciences; Cell Biology; Organizational Aspects of Cell Biology; Functional Aspects of Cell Biology

Collective cell migration is crucial in many biological processes such as wound healing, tissue morphogenesis, and tumor progression. The leading front of a collective migrating epithelial cell layer often destabilizes into multicellular finger-like protrusions, each of which is guided by a leader cell at the fingertip. Here, we develop a subcellular-element-based model of this fingering instability, which incorporates leader cells and other related properties of a monolayer of epithelial cells. Our model recovers multiple aspects of the dynamics, especially the traction force patterns and velocity fields, observed in experiments on Madin-Darby canine kidney cells. Our model predicts the necessity of the leader cell and its minimal functions for the formation and maintenance of a stable finger pattern. Meanwhile, our model allows for an analysis of the role of supracellular actin cable on the leading front, predicting that while this observed structure helps maintain the shape of the finger, it is not required in order to form a finger. In addition, we also study the driving instability in the context of continuum active fluid model, which justifies some of our assumptions in the computational approach. In particular, we show that in our model no finger protrusions would emerge in a phenotypically homogenous active fluid and hence the role of the leader cell and its followers are often critical.

Phenotypic heterogeneity exists within collectively invading packs of tumor cells, suggesting that cellular subtypes cooperate to drive invasion and metastasis. Here, we take a chemical biology approach to probe cell:cell cooperation within the collective invasion pack. These data reveal metabolic heterogeneity within invasive chains, in which leader cells preferentially utilize mitochondrial respiration and trailing follower cells rely on elevated glucose uptake. We define a pyruvate dehydrogenase (PDH) dependency in leader cells that can be therapeutically exploited with the mitochondria-targeting compound alexidine dihydrochloride. In contrast, follower cells highly express glucose transporter 1 (GLUT1), which sustains an elevated level of glucose uptake required to maintain proliferation. Co-targeting of both leader and follower cells with PDH and GLUT1 inhibitors, respectively, inhibits cell growth and collective invasion. Taken together, our work reveals metabolic heterogeneity within the lung cancer collective invasion pack and provides rationale for co-targeting PDH and GLUT1 to inhibit collective invasion., The presence of phenotypic heterogeneity in collectively invading cells suggests cooperation amongst distinct subtypes of cells to promote invasion and metastasis. Here, the authors use chemical biology tools and report metabolic heterogeneity within the lung cancer collective invasion pack.

The micro- and nanosize surface topography of dental implants has been shown to affect the growth of surrounding cells. In this study, standardized and controlled periodic nanopatterns were fabricated with nanosized surface roughness on titanium substrates, and their influence on bone marrow stromal cells investigated. Cell proliferation assays revealed that the bare substrate with a 1.7 nm surface roughness has lower hydrophilicity but higher proliferation ability than that with a 0.6 nm surface roughness. Further, with the latter substrate, directional cell growth was observed for line and groove patterns with a width of 100 nm and a height of 50 or 100 nm, but not for those with a height of 10 or 25 nm. With the smooth substrate, time-lapse microscopic analyses showed that more than 80% of the bone marrow cells on the line and groove pattern with a height of 100 nm grew and divided along the lines. As the nanosized grain structure controls the cell proliferation rate and the nanosized line and groove structure (50–100 nm) controls cell migration, division, and growth orientation, these standardized nanosized titanium structures can be used to elucidate the mechanisms by which surface topography regulates tissue responses to biomaterials.

The extracellular matrix is known to modulate cell adhesion and migration during tissue regeneration. However, the molecular mechanisms that fine-tune cells to extra-cellular matrix dynamics during regeneration of the peripheral nervous system remain poorly understood. Using the RSC96 Schwann cell line, we show that Sox2 directly controls fibronectin fibrillogenesis in Schwann cells in culture, to provide a highly oriented fibronectin matrix, which supports their organization and directional migration. We demonstrate that Sox2 regulates Schwann cell behaviour through the upregulation of multiple extracellular matrix and migration genes as well as the formation of focal adhesions during cell movement. We find that mouse primary sensory neurons and human induced pluripotent stem cell-derived motoneurons require the Sox2-dependent fibronectin matrix in order to migrate along the oriented Schwann cells. Direct loss of fibronectin in Schwann cells impairs their directional migration affecting the alignment of the axons in vitro. Furthermore, we show that Sox2 and fibronectin are co-expressed in proregenerative Schwann cells in vivo in a time-dependent manner during sciatic nerve regeneration. Taken together, our results provide new insights into the mechanisms by which Schwann cells regulate their own extracellular microenvironment in a Sox2-dependent manner to ensure the proper migration of neurons.

During collective migration of epithelial cells, the migration direction is aligned over a tissue-scale expanse. Although the collective cell migration is known to be directed by mechanical forces transmitted via cell-cell junctions, it remains elusive how the intercellular force transmission is coordinated with intracellular biochemical signaling to achieve collective movements. Here, we show that intercellular coupling of extracellular signal-regulated kinase (ERK)-mediated mechanochemical feedback yields long-distance transmission of guidance cues. Mechanical stretch activates ERK through epidermal growth factor receptor (EGFR) activation, and ERK activation triggers cell contraction. The contraction of the activated cell pulls neighboring cells, evoking another round of ERK activation and contraction in the neighbors. Furthermore, anisotropic contraction based on front-rear polarization guarantees unidirectional propagation of ERK activation, and in turn, the ERK activation waves direct multicellular alignment of the polarity, leading to long-range ordered migration. Our findings reveal that mechanical forces mediate intercellular signaling underlying sustained transmission of guidance cues for collective cell migration., 分子活性の波が細胞集団に伝わる制御機構を解明 --細胞同士の綱引きが情報を遠くに伝える--. 京都大学プレスリリース. 2020-06-04., Cells communicate by doing the 'wave'. 京都大学プレスリリース. 2020-07-22.

Collective cell migration occurs in a diversity of physiological processes such as wound healing, cancer metastasis, and embryonic morphogenesis. In the collective context, cohesive cells may move as a translational solid, swirl as a fluid, or even rotate like a disk, with scales ranging from several to dozens of cells. In this work, an active vertex model is presented to explore the regulatory roles of social interactions of neighboring cells and environmental confinements in collective cell migration in a confluent monolayer. It is found that the competition between two kinds of intercellular social interactions—local alignment and contact inhibition of locomotion—drives the cells to self-organize into various dynamic coherent structures with a spatial correlation scale. The interplay between this intrinsic length scale and the external confinement dictates the migration modes of collective cells confined in a finite space. We also show that the local alignment–contact inhibition of locomotion coordination can induce giant density fluctuations in a confluent cell monolayer without gaps, which triggers the spontaneous breaking of orientational symmetry and leads to phase separation.

During development and in cancer, cells often move together in small to large collectives. To move as a unit, cells within collectives need to stay coupled together and coordinate their motility. How cell collectives remain interconnected and migratory, especially when moving through in vivo environments, is not well understood. The genetically tractable border cell group undergoes a highly polarized and cohesive cluster-type migration in the Drosophila ovary. Here we report that the small GTPase Rap1, through activation by PDZ-GEF, regulates border cell collective migration. We find that Rap1 maintains cell contacts within the cluster, at least in part by promoting the organized distribution of E-cadherin at specific cell–cell junctions. Rap1 also restricts migratory protrusions to the front of the border cell cluster and promotes the extension of protrusions with normal dynamics. Further, Rap1 is required in the outer migratory border cells but not in the central nonmigratory polar cells. Such cell specificity correlates well with the spatial distribution of the inhibitory Rapgap1 protein, which is higher in polar cells than in border cells. We propose that precisely regulated Rap1 activity reinforces connections between cells and polarizes the cluster, thus facilitating the coordinated collective migration of border cells.