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Background In light of the growing appreciation for the role of collective cell motility in metastasis, a deeper understanding of the underlying signaling pathways will be critical to translating these observations to the treatment of advanced cancers. Here, we examine the contribution of Wnt/planar cell polarity (Wnt/PCP), one of the non-canonical Wnt signaling pathways and defined by the involvement of the tetraspanin-like proteins Vangl1 and Vangl2, to breast tumor cell motility, collective cell invasiveness and mammary tumor metastasis. Methods Vangl1 and Vangl2 knockdown and overexpression and Wnt5a stimulation were employed to manipulate Wnt/PCP signaling in a battery of breast cancer cell lines representing all breast cancer subtypes, and in tumor organoids from MMTV-PyMT mice. Cell migration was assessed by scratch and organoid invasion assays, Vangl protein subcellular localization was assessed by confocal fluorescence microscopy, and RhoA activation was assessed in real time by fluorescence imaging with an advanced FRET biosensor. The impact of Wnt/PCP suppression on mammary tumor growth and metastasis was assessed by determining the effect of conditional Vangl2 knockout on the MMTV-NDL mouse mammary tumor model. Results We observed that Vangl2 knockdown suppresses the motility of all breast cancer cell lines examined, and overexpression drives the invasiveness of collectively migrating MMTV-PyMT organoids. Vangl2-dependent RhoA activity is localized in real time to a subpopulation of motile leader cells displaying a hyper-protrusive leading edge, Vangl protein is localized to leader cell protrusions within leader cells, and actin cytoskeletal regulator RhoA is preferentially activated in the leader cells of a migrating collective. Mammary gland-specific knockout of Vangl2 results in a striking decrease in lung metastases in MMTV-NDL mice, but does not impact primary tumor growth characteristics. Conclusions We conclude that Vangl-dependent Wnt/PCP signaling promotes breast cancer collective cell migration independent of breast tumor subtype and facilitates distant metastasis in a genetically engineered mouse model of breast cancer. Our observations are consistent with a model whereby Vangl proteins localized at the leading edge of leader cells in a migrating collective act through RhoA to mediate the cytoskeletal rearrangements required for pro-migratory protrusion formation.

The movement of groups of cells by collective cell migration requires division of labor between group members. Therefore, distinct cell identities, unique cell behaviors, and specific cellular roles are acquired by cells undergoing collective movement. A key driving force behind the acquisition of discrete cell states is the precise control of where, when, and how genes are expressed, both at the subcellular and supracellular level. Unraveling the mechanisms underpinning the spatiotemporal control of gene expression in collective cell migration requires not only suitable experimental models but also high-resolution imaging of messenger RNA and protein localization during this process. In recent times, the highly stereotyped growth of new blood vessels by sprouting angiogenesis has become a paradigm for understanding collective cell migration, and consequently this has led to the development of numerous user-friendly in vitro models of angiogenesis. In parallel, single-molecule fluorescent in situ hybridization (smFISH) has come to the fore as a powerful technique that allows quantification of both RNA number and RNA spatial distribution in cells and tissues. Moreover, smFISH can be combined with immunofluorescence to understand the precise interrelationship between RNA and protein distribution. Here, we describe methods for use of smFISH and immunofluorescence microscopy in in vitro angiogenesis models to enable the investigation of RNA and protein expression and localization during endothelial collective cell migration.

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.

Research questionAdenomyosis is a common uterine disorder of uncertain causes. Can transcriptomic analyses of the endometrium and myometrium reveal potential mechanisms underlying adenomyosis pathogenesis?DesignTranscriptomic profiles of eutopic endometrium and myometrium from women with and without diffuse adenomyosis and with symptomatic FIGO type 2-5 fibroids in the proliferative phase of the menstrual cycle were assessed using RNA sequencing and bioinformatic analysis. Differentially expressed genes (DEG) and potential pathways were validated by quantitative reverse transcription polymerase chain reaction, immunoblotting and Masson staining, using additional clinical samples.ResultsTop biological processes in the endometrium of women with versus without adenomyosis, enriched from DEG, comprised inflammation, extracellular matrix (ECM) organization, collagen degradation and hyaluronan synthesis, which are key in cell migration and cell movement. Top biological processes enriched from DEG in the myometrium of women with versus without adenomyosis revealed ECM organization dysfunction, abnormal sensory pain perception and gamma aminobutyric acid (GABA) synaptic transmission. Dysregulation of prolactin signalling was also enriched in eutopic endometrium and in the myometrium of women with adenomyosis.ConclusionsOverall, our results support the invasive endometrium theory in the pathogenesis of adenomyosis, in which inflammation induces ECM remodelling resulting in a track for subsequent endometrial collective cell migration and onset of adenomyosis. Moreover, abnormal myometrial GABA synaptic transmission may contribute to dysmenorrhoea in women with adenomyosis and is a possible target for novel therapeutic development. Prolactin signalling abnormalities may serve as another opportunity for therapeutic intervention.

Endothelial Cells (ECs) form cohesive cellular lining of the vasculature and play essential roles in both developmental processes and pathological conditions. Collective migration and proliferation of endothelial cells (ECs) are key processes underlying endothelialization of vessels as well as vascular graft, but the complex interplay of mechanical and biochemical signals regulating these processes are still not fully elucidated. While surface topography and biochemical modifications have been used to enhance endothelialization in vitro, thus far such single-modality modifications have met with limited success. As combination therapy that utilizes multiple modalities has shown improvement in addressing various intractable and complex biomedical conditions, here, we explore a combined strategy that utilizes topographical features in conjunction with pharmacological perturbations. We characterized EC behaviors in response to micrometer-scale grating topography in concert with pharmacological perturbations of endothelial adherens junctions (EAJ) regulators. We found that the protein tyrosine phosphatase, PTP1B, serves as a potent regulator of EAJ stability, with PTP1B inhibition synergizing with grating topographies to modulate EAJ rearrangement, thereby augmenting global EC monolayer sheet orientation, proliferation, connectivity, and collective cell migration. Our data delineates the crosstalk between cell-ECM topography sensing and cell-cell junction integrity maintenance and suggests that the combined use of grating topography and PTP1B inhibitor could be a promising strategy for promoting collective EC migration and proliferation.

Gap closure to eliminate physical discontinuities and restore tissue integrity is a fundamental process in normal development and repair of damaged tissues and organs. Here, we demonstrate a nonadhesive gap closure model in which collective cell migration, large-scale actin-network fusion, and purse-string contraction orchestrate to restore the gap. Proliferative pressure drives migrating cells to attach onto the gap front at which a pluricellular actin ring is already assembled. An actin-ring segment switching process then occurs by fusion of actin fibers from the newly attached cells into the actin cable and defusion from the previously lined cells, thereby narrowing the gap. Such actin-cable segment switching occurs favorably at high curvature edges of the gap, yielding size-dependent gap closure. Cellular force microscopies evidence that a persistent rise in the radial component of inward traction force signifies successful actin-cable segment switching. A kinetic model that integrates cell proliferation, actin fiber fusion, and purse-string contraction is formulated to quantitatively account for the gap-closure dynamics. Our data reveal a previously unexplored mechanism in which cells exploit multifaceted strategies in a highly cooperative manner to close nonadhesive gaps.

Collective cell behaviour during embryogenesis and tissue repair requires the coordination of intercellular junctions, cytoskeleton-dependent shape changes controlled by Rho GTPases, and integrin-dependent cell-matrix adhesion. Many different integrins are simultaneously expressed during wound healing, embryonic development, and sprouting angiogenesis, suggesting that there is extensive integrin/integrin cross-talk to regulate cell behaviour. Here, we show that fibronectin-binding β1 and β3 integrins do not act synergistically, but rather antagonize each other during collective cell processes in neuro-epithelial cells, placental trophoblasts, and endothelial cells. Reciprocal β1/β3 antagonism controls RhoA activity in a kindlin-2-dependent manner, balancing cell spreading, contractility, and intercellular adhesion. In this way, reciprocal β1/β3 antagonism controls cell cohesion and cellular plasticity to switch between extreme and opposing states, including epithelial versus mesenchymal-like phenotypes and collective versus individual cell migration. We propose that integrin/integrin antagonism is a universal mechanism to effectuate social cellular interactions, important for tissue morphogenesis, endothelial barrier function, trophoblast invasion, and sprouting angiogenesis.

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.

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.

Cell rearrangement caused by collective cell migration (CCM) during free expansion of epithelial monolayers has become a landmark in our current understanding of fundamental biological processes such as tissue development, regeneration, wound healing or cancer invasion. Cell spreading causes formation of mechanical waves which has a feedback effect on cell rearrangement and can lead to the cell jamming state. The mechanical waves describe oscillatory changes in cell velocity, as well as, the rheological parameters that affect them. The velocity oscillations, obtained at a time scale of hours, are in the form of forward and backward flows. Collision of forward and backward flows can induce an increase in the cell compressive stress accompanied with cell packing density which have a feedback impact on cell mobility, tissue viscoelasticity and alters the tissue stiffness. The tissue stiffness depends on the cell packing density and the active/passive (i.e. migrating/resting) state of single cells and can be used as an indicator of cell jamming state transition. Since cell stiffness can be measured it may directly show in which state the multicellular system is. In this work a review of existing modeling approaches is given along with assortment of published experimental findings, in order to invite experimentalists to test given theoretical considerations in multicellular systems.

Upon the initiation of collective cell migration, the cells at the free edge are specified as leader cells; however, the mechanism underlying the leader cell specification remains elusive. Here, we show that lamellipodial extension after the release from mechanical confinement causes sustained extracellular signal-regulated kinase (ERK) activation and underlies the leader cell specification. Live-imaging of Madin-Darby canine kidney (MDCK) cells and mouse epidermis through the use of Förster resonance energy transfer (FRET)-based biosensors showed that leader cells exhibit sustained ERK activation in a hepatocyte growth factor (HGF)-dependent manner. Meanwhile, follower cells exhibit oscillatory ERK activation waves in an epidermal growth factor (EGF) signaling-dependent manner. Lamellipodial extension at the free edge increases the cellular sensitivity to HGF. The HGF-dependent ERK activation, in turn, promotes lamellipodial extension, thereby forming a positive feedback loop between cell extension and ERK activation and specifying the cells at the free edge as the leader cells. Our findings show that the integration of physical and biochemical cues underlies the leader cell specification during collective cell migration., 細胞の集まりからリーダーが生まれる仕組み --出る杭はより出る--. 京都大学プレスリリース. 2022-09-29.

The WAVE regulatory complex (WRC) is the main activator of the Arp2/3 complex, promoting lamellipodial protrusions in migrating cells. The WRC is basally inactive but can be activated by Rac1 and phospholipids, and through phosphorylation. However, the in vivo relevance of the phosphorylation of WAVE proteins remains largely unknown. Here, we identified casein kinase I alpha (CK1α) as a regulator of WAVE, thereby controlling cell shape and cell motility in Drosophila macrophages. CK1α binds and phosphorylates WAVE in vitro. Phosphorylation of WAVE by CK1α appears not to be required for activation but, rather, regulates its stability. Pharmacologic inhibition of CK1α promotes ubiquitin-dependent degradation of WAVE. Consistently, loss of Ck1α but not ck2 function phenocopies the depletion of WAVE. Phosphorylation-deficient mutations in the CK1α consensus sequences within the VCA domain of WAVE can neither rescue mutant lethality nor lamellipodium defects. By contrast, phosphomimetic mutations rescue all cellular and developmental defects. Finally, RNAi-mediated suppression of 26S proteasome or E3 ligase complexes substantially rescues lamellipodia defects in CK1α-depleted macrophages. Therefore, we conclude that basal phosphorylation of WAVE by CK1α protects it from premature ubiquitin-dependent degradation, thus promoting WAVE function in vivo. This article has an associated First Person interview with the first author of the paper., Summary: We identified CK1α as a novel regulator of WAVE controlling cell shape and motility in immune response. Basal phosphorylation of WAVE by CK1α protects it from premature proteasomal degradation.

Stress fibers (SFs) are actomyosin bundles commonly found in individually migrating cells in culture. However, whether and how cells use SFs to migrate in vivo or collectively is largely unknown. Studying the collective migration of the follicular epithelial cells in Drosophila, we found that the SFs in these cells show a novel treadmilling behavior that allows them to persist as the cells migrate over multiple cell lengths. Treadmilling SFs grow at their fronts by adding new integrin-based adhesions and actomyosin segments over time. This causes the SFs to have many internal adhesions along their lengths, instead of adhesions only at the ends. The front-forming adhesions remain stationary relative to the substrate and typically disassemble as the cell rear approaches. By contrast, a different type of adhesion forms at the SF’s terminus that slides with the cell’s trailing edge as the actomyosin ahead of it shortens. We further show that SF treadmilling depends on cell movement and identify a developmental switch in the formins that mediate SF assembly, with Dishevelled-associated activator of morphogenesis acting during migratory stages and Diaphanous acting during postmigratory stages. We propose that treadmilling SFs keep each cell on a linear trajectory, thereby promoting the collective motility required for epithelial migration.

In aerobic organisms, oxygen is essential for efficient energy production, and it acts as the last acceptor of the mitochondrial electron transport chain and as regulator of gene expression. However, excessive oxygen can lead to production of deleterious reactive oxygen species. Therefore, the directed migration of single cells or cell clumps from hypoxic areas toward a region of optimal oxygen concentration, named aerotaxis, can be considered an adaptive mechanism that plays a major role in biological and pathological processes. One relevant example is the development of O2 gradients when tumors grow beyond their vascular supply, leading frequently to metastasis. In higher eukaryotic organisms, aerotaxis has only recently begun to be explored, but genetically amenable model organisms suitable to dissect this process remain an unmet need. In this regard, we sought to assess whether Dictyostelium cells, which are an established model for chemotaxis and other motility processes, could sense oxygen gradients and move directionally in their response. By assessing different physical parameters, our findings indicate that both growing and starving Dictyostelium cells under hypoxic conditions migrate directionally toward regions of higher O2 concentration. This migration is characterized by a specific pattern of cell arrangement. A thickened circular front of high cell density (corona) forms in the cell cluster and persistently moves following the oxygen gradient. Cells in the colony center, where hypoxia is more severe, are less motile and display a rounded shape. Aggregation-competent cells forming streams by chemotaxis, when confined under hypoxic conditions, undergo stream or aggregate fragmentation, giving rise to multiple small loose aggregates that coordinately move toward regions of higher O2 concentration. By testing a panel of mutants defective in chemotactic signaling, and a catalase-deficient strain, we found that the latter and the pkbR1null exhibited altered migration patterns. Our results suggest that in Dictyostelium, like in mammalian cells, an intracellular accumulation of hydrogen peroxide favors the migration toward optimal oxygen concentration. Furthermore, differently from chemotaxis, this oxygen-driven migration is a G protein-independent process.

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.

Both tumor cell-intrinsic signals and tumor cell-extrinsic signals from cells within the tumor microenvironment influence tumor cell dissemination and metastasis. The fibrillar collagen receptor tyrosine kinase (RTK) discoidin domain receptor 2 (DDR2) is essential for breast cancer metastasis in mouse models, and high expression of DDR2 in tumor and tumor stromal cells is strongly associated with poorer clinical outcomes. DDR2 tyrosine kinase activity has been hypothesized to be required for the metastatic activity of DDR2; however, inhibition of DDR2 tyrosine kinase activity, along with that of other RTKs, has failed to provide clinically relevant responses in metastatic patients. Here, we show that tyrosine kinase activity-independent action of DDR2 in tumor cells can support Matrigel invasion and in vivo metastasis. Paracrine actions of DDR2 in tumor cells and cancer-associated fibroblasts (CAFs) also support tumor invasion, migration and lung colonization in vivo. These data suggest that tyrosine kinase-independent functions of DDR2 could explain failures of tyrosine kinase inhibitor treatment in metastatic breast cancer patients and highlight the need for alternative therapeutic strategies that inhibit both tyrosine kinase-dependent and -independent actions of RTKs in the treatment of breast cancer. This article has an associated First Person interview with the first author of the paper., Summary: The collagen receptor DDR2, a receptor tyrosine kinase, exhibits tyrosine kinase-independent functions that influence paracrine regulation of breast tumor cell invasion and breast cancer metastasis.

The loss of apicobasal polarity during the epithelial-to-mesenchymal transition (EMT) is a hallmark of cancer and metastasis. The key feature of this polarity in epithelial cells is the subdivision of the plasma membrane into apical and basolateral domains, with each orchestrating specific intra- and extracellular functions. Epithelial transport and signaling capacities are thought to be determined largely by the quality, quantity, and nanoscale organization of proteins residing in these membrane domains, the apicobasal surfaceomes. Despite its implications for cancer, drug uptake, and infection, our current knowledge of how the polarized surfaceome is organized and maintained is limited. Here, we used chemoproteomic surfaceome scanning to establish proteotype maps of apicobasal surfaceomes and reveal quantitative distributions of, i.e., surface proteases, phosphatases, and tetraspanins as potential key regulators of polarized cell functionality. We show further that the tumor suppressor PTEN regulates polarized surfaceome architecture and uncover a potential role in collective cell migration. Our differential surfaceome analysis provides a molecular framework to elucidate polarized protein networks regulating epithelial functions and PTEN-associated cancer progression., International Journal of Molecular Sciences, 23 (24), ISSN:1422-0067

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.

Collective migration is a key process that is critical during development, as well as in physiological and pathophysiological processes including tissue repair, wound healing and cancer. Studies in genetic model organisms have made important contributions to our current understanding of the mechanisms that shape cells into different tissues during morphogenesis. Recent advances in high-resolution and live-cell-imaging techniques provided new insights into the social behavior of cells based on careful visual observations within the context of a living tissue. In this review, we will compare Drosophila testis nascent myotube migration with established in vivo model systems, elucidate similarities, new features and principles in collective cell migration.

Junctional adhesion molecule (JAM)-A is a cell adhesion receptor localized at epithelial cellcell contacts with enrichment at the tight junctions. Its role during cell-cell contact formation and epithelial barrier formation has intensively been studied. In contrast, its role during collective cell migration is largely unexplored. Here we show that JAM-A regulates collective cell migration of polarized epithelial cells. Depletion of JAM-A in MDCK cells enhances the motility of singly migrating cells but reduces cell motility of cells embedded in a collective by impairing the dynamics of cryptic lamellipodia formation. This activity of JAM-A is observed in cells grown on laminin and collagen I but not on fibronectin or vitronectin. Accordingly, we find that JAM-A exists in a complex with the laminin- and collagen I-binding α3β1 integrin. We also find that JAM-A interacts with CD151, a tetraspanin that forms a stoichiometric complex with α3β1 integrin and that regulates α3β1 integrin activity in different contexts. Mapping experiments indicate that JAM-A associates with both α3β1 integrin and CD151 through its extracellular domain. Similar to depletion of JAM-A, depletion of either α3β1 integrin or CD151 in MDCK cells slows down collective cell migration. Our findings suggest that JAM-A, α3β1 integrin and CD151 exist as a functional complex to regulate collective cell migration of epithelial cells.

The ability to program collective cell migration can allow us to control critical multicellular processes in development, regenerative medicine, and invasive disease. However, while various technologies exist to make individual cells migrate, translating these tools to control myriad, collectively interacting cells within a single tissue poses many challenges. For instance, do cells within the same tissue interpret a global migration ‘command’ differently based on where they are in the tissue? Similarly, since no stimulus is permanent, what are the long-term effects of transient commands on collective cell dynamics? We investigate these questions by bioelectrically programming large epithelial tissues to globally migrate ‘rightward’ via electrotaxis. Tissues clearly developed distinct rear, middle, side, and front responses to a single global migration stimulus. Furthermore, at no point post-stimulation did tissues return to their pre-stimulation behavior, instead equilibrating to a third, new migratory state. These unique dynamics suggested that programmed migration resets tissue mechanical state, which was confirmed by transient chemical disruption of cell-cell junctions, analysis of strain wave propagation patterns, and quantification of cellular crowd dynamics. Overall, this work demonstrates how externally driving the collective migration of a tissue can reprogram baseline cell-cell interactions and collective dynamics, even well beyond the end of the global migratory cue, and emphasizes the importance of considering the supracellular context of tissues and other collectives when attempting to program crowd behaviors.

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.

The collective cell migration is observed in many biological processes such as wound healing, embryogenesis, and cancer metastasis. Despite extensive theoretical and experimental studies on collective cell motion, there is no unified mechanism to explain it. In this work, we experimentally report the collectively growing cell colonies in the sub-marginal region of a freely expanding cell monolayer. These colonies could be responsible for the highly aligned collective cell migration observed in front cell rows. Our results provide a basic framework to understand the physical mechanism responsible for collective cell migration in the freely expanding monolayer.

Activity driven glassy dynamics is ubiquitous in collective cell migration,intracellular transport, dynamics in bacterial and ants colonies as well as artificially driven synthetic systems such as vibrated granular materials, etc. Active glasses are hitherto assumed to be qualitatively similar to their equilibrium counterparts at a suitably defined effective temperature, ff. Combining large-scale simulations with analytical mode-coupling theory for such systems, we show that, in fact, an active glass is qualitatively different from an equilibrium glassy system. Although the relaxation dynamics can be similar to an equilibrium system at a ff, effects of activity on the dynamic heterogeneity (DH), which has emerged as a cornerstone of glassy dynamics, is quite nontrivial and complex. In particular, active glasses show dramatic growth of DH, and systems with similar relaxation time and ff can have widely varying DH. Comparison of our non-equilibrium extended mode-coupling theory for such systems with simulation results show that the theory captures the basic characteristics of such systems. Our study raises fundamental questions on the supposedly central role of DH in controlling the relaxation dynamics in a glassy system and can have important implications even for the equilibrium glassy dynamics.

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.

Cell motion is crucial in human health and development. Cells may migrate individually or in highly coordinated groups. Cell motion results from complex intra- and extra-cellular mechanochemical interactions. Computational models have become a powerful tool to shed light on the mechanisms that regulate cell migration. The phase-field method is an emerging modeling technique that permits a simple and direct formulation of the moving cell problem and the interaction between the cell and its environment. This paper intends to be a comprehensive review of phase-field models of individual and collective cell migration. We describe a numerical implementation, based on isogeometric analysis, which successfully deals with the challenges associated with phase-field problems. We present numerical simulations that illustrate the unique capabilities of the phase-field approach for cell migration. In particular, we show 2D and 3D simulations of individual cell migration in confined and fibrous environments that highlight the mechanochemical interplay between the cell and the extracellular environment. We also show 2D simulations of cell co-attraction in non-confluent multicellular systems, in which the use of the phase-field method is crucial to capture the dynamics of the multicellular system.

Gelatin-based hydrogels have received particular attention for tissue-engineering applications given their biocompatibility, ease of tuning their physical properties through chemical modifications, and incorporation of antibacterial activity. While several studies have focused on the detailed quantification of biomechanical properties of these gels, considerably less attention has been paid to understanding how adhesivity of these gels impacts single as well as collective cell migration, which directly determines the efficacy of wound healing. In this study, we address this question by quantifying fibroblast motility and antibacterial activity of silver nanoparticle (AgNP)-entrapped methacrylated gelatin (GelMA) hydrogels. Using 5 and 15% GelMA soft gels cross-linked with 1 min UV exposure, we first show that cells spread more and migrate faster on 15% GelMA gels. Next, we show that ∼10 nm AgNPs entrapped in 15% GelMA gels get released over a time-scale greater than 72 h and exhibit antibacterial activity against both Gram-positive and Gram-negative bacteria at concentrations nontoxic to cells. Finally, using a polydimethylsiloxane (PDMS) device for simulating wound healing, we show that closure of ∼800 μm gaps on GelMA gels is significantly faster compared with other conditions. Together, our findings illustrate the potential of AgNP-entrapped soft GelMA gels as scaffolds for achieving accelerated wound healing of deep dermal wounds by enabling fast fibroblast migration and minimization of microbial infections.

Directed collective cell migration governs cell orientation during tissue morphogenesis, wound healing, and tumor metastasis. Unfortunately, current methods for initiating collective cell migration, such as scratching, laser ablation, and stencils, either introduce uncontrolled cell-injury, involve multiple fabrication processes, or have utility limited to cells with strong cell–cell junctions. Using three-dimensional (3D) bioprinted gelatin methacryloyl (GelMA) micropatterns on temperature-responsive poly(N-isopropylacrylamide) (PNIPAm) coated interfaces, we demonstrate that directed injury-free collective cell migration could occur in parallel and perpendicular directions. After seeding cells, we created cell-free spaces between two 3D bioprinted GelMA micropatterns by lowering the temperature of PNIPAm interfaces to promote the cell detachment. Unlike conventional collective cell migration methods initiated by stencils, we observed well-organized cell migration in parallel and perpendicular to 3D bioprinted micropatterns as a function of the distance between 3D bioprinted micropatterns. We further established the utility of controlled collective cell migration for directed functional myotube formation using 3D bioprinted fingerprintlike micropatterns as well as iris musclelike concentric circular patterns. Our platform is unique for myoblast alignment and myotube formation because it does not require anisotropic guidance cues. Together, our findings establish how to achieve controlled collective cell migration, even at the macroscale, for tissue engineering and regeneration.

Collagen is a promising material for corneal tissue engineering. The surface topography of collagen is critical for cornea reconstruction and regeneration. Herein, we fabricated collagen films with microgrooved surface to investigate the effect of collagen film topography on corneal cell migration and antifibrosis. We found that the patterned films with microscale grooves could greatly affect the orientation, proliferation, migration, and gene expression of rabbit corneal epithelial cells and keratocytes. Compared with the natural cornea, the optimized collagen films with microgroove pattern show similar swelling performance, optical clarity, and biodegradability, which could efficiently promote the epithelial cell migration, accelerate wound healing process, and inhibit the fibrosis of keratocytes. These results suggest that collagen films with microgroove pattern are promising in corneal tissue engineering.

Collective invasion drives multicellular cancer cells to spread to surrounding normal tissues. To fully comprehend metastasis, the methodology of analysis of individual cell migration in tissue should be well developed. Extracting and classifying cells with similar migratory characteristics in a colony would facilitate an understanding of complex cell migration patterns. Here, we used electrospun fibers as the extracellular matrix for the in vitro modeling of collective cell migration, clustering of mesenchymal and epithelial cells based on trajectories, and analysis of collective migration patterns based on trajectory similarity. We normalized the trajectories to eliminate the effect of cell location on clustering and used uniform manifold approximation and projection to perform dimensionality reduction on the time-series data before clustering. When the clustering results were superimposed on the trajectories before normalization, the results still exhibited positional similarity, thereby demonstrating that this method can identify cells with similar migration patterns. The same cluster contained both mesenchymal and epithelial cells, and this result was related to cell location and cell division. These data highlight the reliability of this method in identifying consistent migration patterns during collective cell migration. This provides new insights into the epithelial–mesenchymal interactions that affect migration patterns.

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.

Confluent cell monolayers and epithelia tissues show remarkable patterns and correlations in structural arrangements and actively-driven collective flows. We simulate these properties using multiphase field models. The models are based on cell deformations and cell-cell interactions and we investigate the influence of microscopic details to incorporate active forces on emerging phenomena. We compare four different approaches, one in which the activity is determined by a random orientation, one where the activity is related to the deformation of the cells and two models with subcellular details to resolve the mechanochemical interactions underlying cell migration. The models are compared with respect to generic features, such as solid-to-liquid phase transitions, cell shape variability, emerging nematic properties, as well as vorticity correlations and flow patterns in large confluent monolayers and confinements. All results are compared with experimental data for a large variety of cell cultures. The appearing qualitative differences of the models show the importance of microscopic details and provide a route towards predictive simulations of patterns and correlations in cell colonies., Comment: 12 pages

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.

SUMMARYCoordination of cell proliferation and migration is fundamental for life, and its dysregulation has catastrophic consequences, as cancer. How cell cycle progression affects migration, and vice-versa, remains largely unknown. We address these questions by combining in silico modelling and in vivo experimentation in the zebrafish Trunk Neural Crest (TNC). TNC migrate collectively, forming chains with a leader cell directing the movement of trailing followers. We show that the acquisition of migratory identity is autonomously controlled by Notch signalling in TNC. High Notch activity defines leaders, while low Notch determines followers. Moreover, cell cycle progression is required for TNC migration and is regulated by Notch. Cells with low Notch activity stay longer in G1 and become followers, while leaders with high Notch activity quickly undergo G1/S transition and remain in S-phase longer. We propose that migratory behaviours are defined through the interaction of Notch signalling and cell cycle progression.

A key regulator of collective cell migrations, which drive development and cancer metastasis, is substrate stiffness. Increased substrate stiffness promotes migration and is controlled by Myosin. Using Drosophila border cell migration as a model of collective cell migration, we identify, for the first time, that the actin bundling protein Fascin limits Myosin activity in vivo. Loss of Fascin results in: increased activated Myosin on the border cells and their substrate, the nurse cells; decreased border cell Myosin dynamics; and increased nurse cell stiffness as measured by atomic force microscopy. Reducing Myosin restores on-time border cell migration in fascin mutant follicles. Further, Fascin’s actin bundling activity is required to limit Myosin activation. Surprisingly, we find that Fascin regulates Myosin activity in the border cells to control nurse cell stiffness to promote migration. Thus, these data shift the paradigm from a substrate stiffness-centric model of regulating migration, to uncover that collectively migrating cells play a critical role in controlling the mechanical properties of their substrate in order to promote their own migration. This understudied means of mechanical regulation of migration is likely conserved across contexts and organisms, as Fascin and Myosin are common regulators of cell migration.

Many dynamic physiological processes, including embryo development and wound healing, involve collective cell migration. During this process, leader cells emerge in self-organization manner to guide other cells migration. The mechanisms of their formation, however, are not clear. We hypothesized that the physical forces among cells, and between cell and substrate influence the formation of leader cells. Here, we presented a computational model of cell monolayer migration based on the Glazier-Graner-Hogeweg (GGH) model. Our results indicated that the adhesion forces among cells and the cell-substrate adhesion forces regulate the formation of leader cells. Increasing cellular mobility also affected the characters of leader cells. In summary, our results provided evidence for that physical forces among cells and between cell and substrate, along with mobility of individual cells, affect the self-organization process of leader cell formation during collective cell migration.

The collective cell migration is thought to be a dynamic and interactive behavior of cell cohorts which is essential for diverse physiological developments in living organisms. Recent studies revealed that topographical properties of the environment regulate the migration modes of cell cohorts, such as diffusion versus contraction relaxation transport and the appearance of vortices in larger available space. However, conventional in vitro assays fail to observe the change in cells behavior in response to the structural changes. Here, we have developed a method to fabricate the flexible three-dimensional structures of capillary microtunnels to examine the behavior of vascular endothelial cells (ECs). The microtunnels with altering diameters were formed inside gelatin-gel by spot heating a portion of gelatin by irradiating the µm-sized absorption at the tip of the microneedle with a focused permeable 1064 nm infrared laser. The ECs moved and spread two-dimensionally on the inner surface of capillary microtunnels as monolayer instead of filling the capillary. In contrast to the 3D straight topographical constraint, which exhibited width dependence migration velocity, leading ECs altered its migration velocity accordingly to the change in supply of the cells behind the leading ECs, caused by the progression through the diameter altering structure. Our findings provide insights into the collective migration properties in 3D confinement structures as fluid-like behavior with conservation of cell numbers.

Integrin-mediated substrate-rigidity sensing triggers microtubule acetylation, modulating mechanosensitive cellular responses and focal adhesion dynamics, subsequently promoting actomyosin organization and collective cell migration.

Although collective cell migration (CCM) is a highly coordinated migratory mode, perturbations in the form of jamming state transitions and vice versa often occur even in 2D. These perturbations are involved in various biological processes, such as embryogenesis, wound healing, and cancer invasion. CCM induces accumulation of cell residual stress, which has a feedback impact to cell packing density. Density-mediated change of cell mobility influences the state of viscoelasticity of multicellular systems and on that base the jamming state transition. Although a good comprehension of how cells collectively migrate by following molecular rules has been generated, the impact of cellular rearrangements on cell viscoelasticity remains less understood, thus considering that the density-driven evolution of viscoelasticity caused by reduction of cell mobility could result in a powerful tool in order to address the contribution of cell jamming state transition in CCM and help to understand this important but still a controversial topic. In this work a review of existing literature in CCM modeling is given along with an assortment of published experimental findings, in order to invite experimentalists to test the given theoretical considerations in multicellular systems. In addition, five viscoelastic states gained within three regimes: (1) convective regime, (2) conductive regime, and (3) damped-conductive regime, which were discussed with special emphasis of jamming and unjamming states.

Fluorescence lifetime imaging (FLIM) is a powerful tool to quantify local changes in cell metabolism without loss of spatial resolution. Here, we adopted FLIM to characterize spatio-temporal metabolic changes occurring during collective cell migration. Using an established in vitro system, we measured biomechanical and metabolic changes during migration of epithelial cell monolayers. We developed a custom image analysis pipeline that combines machine learning segmentation and curve fitting analysis to analyze FLIM data. Our findings – which were validated via separate measurements of cytoplasmic redox ratio, glucose uptake, and mitochondrial membrane potential – are consistent with a glycolytic shift during collective cell migration.

Background The technical development of imaging techniques in life sciences has enabled the three-dimensional recording of living samples at increasing temporal resolutions. Dynamic 3D data sets of developing organisms allow for time-resolved quantitative analyses of morphogenetic changes in three dimensions, but require efficient and automatable analysis pipelines to tackle the resulting Terabytes of image data. Particle image velocimetry (PIV) is a robust and segmentation-free technique that is suitable for quantifying collective cellular migration on data sets with different labeling schemes. This paper presents the implementation of an efficient 3D PIV package using the Julia programming language—quickPIV. Our software is focused on optimizing CPU performance and ensuring the robustness of the PIV analyses on biological data. Results QuickPIV is three times faster than the Python implementation hosted in openPIV, both in 2D and 3D. Our software is also faster than the fastest 2D PIV package in openPIV, written in C++. The accuracy evaluation of our software on synthetic data agrees with the expected accuracies described in the literature. Additionally, by applying quickPIV to three data sets of the embryogenesis of Tribolium castaneum, we obtained vector fields that recapitulate the migration movements of gastrulation, both in nuclear and actin-labeled embryos. We show normalized squared error cross-correlation to be especially accurate in detecting translations in non-segmentable biological image data. Conclusions The presented software addresses the need for a fast and open-source 3D PIV package in biological research. Currently, quickPIV offers efficient 2D and 3D PIV analyses featuring zero-normalized and normalized squared error cross-correlations, sub-pixel/voxel approximation, and multi-pass. Post-processing options include filtering and averaging of the resulting vector fields, extraction of velocity, divergence and collectiveness maps, simulation of pseudo-trajectories, and unit conversion. In addition, our software includes functions to visualize the 3D vector fields in Paraview.

Physiological and pathological morphogenetic events involve a wide array of collective movements, suggesting that these multicellular arrangements confer biochemical and biomechanical properties that contribute to tissue scale organization. The cardiopharyngeal progenitors of the tunicate Ciona provide the simplest possible model of collective cell migration. They form cohesive bilateral cell pairs, leader-trailer polarized along the migration path as they migrate between the ventral epidermis and trunk endoderm. Here, circumventing difficulties in quantifying cellular mechanics in live embryos, we use the Cellular Potts Model to computationally probe the distributions of forces consistent with the shapes and collective polarity of migrating cell pairs. Combining computational modeling, confocal microscopy, and molecular perturbations, we first determine that cardiopharyngeal progenitors display hallmarks of supracellular organization, with differential distributions of protrusive forces, cell-matrix adhesion, and myosin-based retraction forces along the leader-trailer axis. Combined 4D simulations and experimental observations suggest that cell-cell communication helps establish a hierarchy that contributes to aligning collective polarity with the direction of migration, as observed with three or more cells bothin silicoandin vivo. Our approach reveals emerging properties of the migrating collective. Specifically, cell pairs are more persistent, thus migrating over longer distances, and presumably with higher accuracy. Finally, simulations suggest that polarized cell pairs literally join forces to deform the trunk endoderm, as they migrate through the extracellular space. We thus propose that the polarized supracellular organization of cardiopharyngeal progenitors confers emergent physical properties that determine mechanical interactions with their environment during morphogenesis.

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, pre-existing 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 pre-existing 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 to demonstrate accelerated wound closure in vitro. These results are in keeping with those from diverse, non-cellular systems, and confirm that endogenous collectivity should be considered as a key, quantitative design variable when optimizing external control of collective 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.

Cell migration can be defined as the spatial translocation of a cell in time and it has been shown to be essential all along the life of unicellular and multicellular livings. In order to efficiently move toward their target tissues, individually or collectively migrating cells require to respond and integrate molecular and mechanical signals from their environment. Comparatively, the function of chemical cues in cell migration is well understood, while the role of tissue mechanics on cell migration is just starting to be elucidated and is currently under intense research. Recent data suggest that the elastic properties of the environment, as well as the dynamic tuning of the viscoelasticity within a migratory cluster of cells, are essential for efficient collective cell migration (CCM) in vivo. In this chapter we discuss why viscoelasticity is a parameter to be accounted when studying cell migration. Our aim is to provide evidence on how tuning the viscoelastic properties of migratory cells and those contained in their environments can ensure correct embryogenesis, wound healing, and metastasis. For this we provide background on viscoelasticity and useful definitions for both, biologists and physicists. Then we discuss the mechanisms by which cells internalize mechanical inputs and we attempt to correlate environmental viscoelasticity with cellular and molecular responses of collectively migrating clusters. Finally, we link these new cellular and molecular signatures to modifications of the cluster’s viscoelasticity—as part of a feedback loop that allows cell collectives to adapt to the challenging geometries of their microenvironment in order to efficiently migrate. Along this chapter we do provide evidence from both, single and collectively migrating cells but we mostly focus on CCM.