Collective Cell Behavior Research Articles

Epithelial colonies in vitro elongate through collective effects.

Epithelial tissues of the developing embryos elongate by different mechanisms, such as neighbor exchange, cell elongation, and oriented cell division. Since autonomous tissue self-organization is influenced by external cues such as morphogen gradients or neighboring tissues, it is difficult to distinguish intrinsic from directed tissue behavior. The mesoscopic processes leading to the different mechanisms remain elusive. Here, we study the spontaneous elongation behavior of spreading circular epithelial colonies in vitro. By quantifying deformation kinematics at multiple scales, we report that global elongation happens primarily due to cell elongations, and its direction correlates with the anisotropy of the average cell elongation. By imposing an external time-periodic stretch, the axis of this global symmetry breaking can be modified and elongation occurs primarily due to orientated neighbor exchange. These different behaviors are confirmed using a vertex model for collective cell behavior, providing a framework for understanding autonomous tissue elongation and its origins.

An Integrative and Modular Framework to Recapitulate Emergent Behavior in Cell Migration

Cell migration has been a subject of study in a broad variety of biological systems, from morphogenetic events during development to cancer progression. In this work, we describe single-cell movement in a modular framework from which we simulate the collective behavior of glioblastoma cells, the most prevalent and malignant primary brain tumor. We used the U87 cell line, which can be grown as a monolayer or spatially closely packed and organized in 3D structures called spheroids. Our integrative model considers the most relevant mechanisms involved in cell migration: chemotaxis of attractant factor, mechanical interactions and random movement. The effect of each mechanism is integrated into the overall probability of the cells to move in a particular direction, in an automaton-like approach. Our simulations fit and reproduced the emergent behavior of the spheroids in a set of migration assays where single-cell trajectories were tracked. We also predicted the effect of migration inhibition on the colonies from simple experimental characterization of single treated cell tracks. The development of tools that allow complementing molecular knowledge in migratory cell behavior is relevant for understanding essential cellular processes, both physiological (such as organ formation, tissue regeneration among others) and pathological perspectives. Overall, this is a versatile tool that has been proven to predict individual and collective behavior in U87 cells, but that can be applied to a broad variety of scenarios.

Engineered Tools to Study Intercellular Communication.

All multicellular organisms rely on intercellular communication networks to coordinate physiological functions. As members of a dynamic social network, each cell receives, processes, and redistributes biological information to define and maintain tissue homeostasis. Uncovering the molecular programs underlying these processes is critical for prevention of disease and aging and development of therapeutics. The study of intercellular communication requires techniques that reduce the scale and complexity of in vivo biological networks while resolving the molecular heterogeneity in “omic” layers that contribute to cell state and function. Recent advances in microengineering and high‐throughput genomics offer unprecedented spatiotemporal control over cellular interactions and the ability to study intercellular communication in a high‐throughput and mechanistic manner. Herein, this review discusses how salient engineered approaches and sequencing techniques can be applied to understand collective cell behavior and tissue functions.

Bridging from single to collective cell migration: A review of models and links to experiments.

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.

Cell-to-cell diversification in ERBB-RAS-MAPK signal transduction that produces cell-type specific growth factor responses

Growth factors regulate cell fates, including their proliferation, differentiation, survival, and death, according to the cell type. Even when the response to a specific growth factor is deterministic for collective cell behavior, significant levels of fluctuation are often observed between single cells. Statistical analyses of single-cell responses provide insights into the mechanism of cell fate decisions but very little is known about the distributions of the internal states of cells responding to growth factors. Using multi-color immunofluorescent staining, we have here detected the phosphorylation of seven elements in the early response of the ERBB–RAS–MAPK system to two growth factors. Among these seven elements, five were analyzed simultaneously in distinct combinations in the same single cells. Although principle component analysis suggested cell-type and input specific phosphorylation patterns, cell-to-cell fluctuation was large. Mutual information analysis suggested that each cell type uses multitrack (bush-like) signal transduction pathways under conditions in which clear fate changes have been reported. The clustering of single-cell response patterns indicated that the fate change in a cell population correlates with the large entropy of the response, suggesting a bet-hedging strategy is used in decision making. A comparison of true and randomized datasets further indicated that this large variation is not produced by simple reaction noise, but is defined by the properties of the signal-processing network.

Faraday Waves in a Square Cell Network: The Effects of Varying the Cell Size

We have conducted experiments of the Faraday instability in a network of square cells filled with water for driving frequencies and amplitudes in the intervals 10≤F≤22 Hz and 0.1≤A≤3 mm, respectively. The experiments were aimed at studying the effects of varying the size of the cells on the surface wave patterns. Images of the surface wave patterns were recorded with a high-speed camera. The time series of photographs composing each video was Fourier analyzed, and information about the waveforms was obtained by using a Pearson correlation analysis. For small square cells of side length l=2.5 cm, adjacent cells collaborate synchronously to form regular patterns of liquid bumps over the entire grid, while ordered matrices of oscillons are formed at higher frequencies. As the size of the cells is increased to l=5 cm, collective cell behaviour at lower frequencies is no longer observed. As the frequency is increased, a transition from three triangularly arranged oscillons within each cell to three, or even four, irregularly arranged oscillons is observed. The wave patterns, the waveforms and the energy content necessary to excite Faraday waves are seen to depend on the cell size.

Heterogeneity in Collective Endothelial Cell Behavior is a Driver of Arterio-Venous Remodeling

During vascular development, arteries and veins form in a stepwise process that combines vasculogenesis and sprouting angiogenesis. Despite extensive data on the mechanisms governing blood vessel assembly at the single cell level, little is known about how cell populations migrate in a finely tuned and coordinated manner, and distribute precisely between arteries and veins. Here, we use an endothelial-specific zebrafish reporter, arteriobow, to label small cohorts of arterial cells and trace their progeny from the initial events of vasculogenesis through the process of arterio-venous remodeling. We reveal that the genesis of both arteries and veins relies on the coordination of ten types of collective cell behaviors originating from discrete endothelial cell clusters. Within these behavioral categories, we identify a heterogeneity of collective cell dynamics specific to either arterial or venous remodeling. Using pharmacological blockade, we further show that factors known to control vascular patterning such as cell-intrinsic Notch signaling and cell-extrinsic blood flow, potentially act as regulators by coordinating endothelial cohorts behavior, which in turn instructs the future territory of arterio-venous remodeling.

Spatial heterogeneities shape the collective behavior of signaling amoeboid cells.

In its natural habitat in the forest soil, the cellular slime mold Dictyostelium discoideum is exposed to obstacles. Starving Dictyostelium cells secrete cAMP, which is the key extracellular signaling molecule that promotes the aggregation process required for their long-term survival. Here, we investigated the influence of environmental inhomogeneities on the signaling and pattern formation of Dictyostelium cells. We present experimental data and numerical simulations on the pattern formation of signaling Dictyostelium cells in the presence of periodic arrays of millimeter-sized pillars. We observed concentric cAMP waves that initiated almost synchronously at the pillars and propagated outward. In response to these circular waves, the Dictyostelium cells streamed toward the pillars, forming aggregates arranged in patterns that reflected the periodicity of the lattice of pillars. Our results suggest that, in nature, the excitability threshold and synchronization level of the cells are two key parameters that control the nature of the interaction between cells and spatial heterogeneities in their environment.

Dynamics of nevus development implicate cell cooperation in the growth arrest of transformed melanocytes.

Mutational activation of the BRAF proto-oncogene in melanocytes reliably produces benign nevi (pigmented 'moles'), yet the same change is the most common driver mutation in melanoma. The reason nevi stop growing, and do not progress to melanoma, is widely attributed to a cell-autonomous process of 'oncogene-induced senescence'. Using a mouse model of Braf-driven nevus formation, analyzing both proliferative dynamics and single-cell gene expression, we found no evidence that nevus cells are senescent, either compared with other skin cells, or other melanocytes. We also found that nevus size distributions could not be fit by any simple cell-autonomous model of growth arrest, yet were easily fit by models based on collective cell behavior, for example in which arresting cells release an arrest-promoting factor. We suggest that nevus growth arrest is more likely related to the cell interactions that mediate size control in normal tissues, than to any cell-autonomous, 'oncogene-induced' program of senescence.

Shape-to-graph mapping method for efficient characterization and classification of complex geometries in biological images.

With the ever-increasing quality and quantity of imaging data in biomedical research comes the demand for computational methodologies that enable efficient and reliable automated extraction of the quantitative information contained within these images. One of the challenges in providing such methodology is the need for tailoring algorithms to the specifics of the data, limiting their areas of application. Here we present a broadly applicable approach to quantification and classification of complex shapes and patterns in biological or other multi-component formations. This approach integrates the mapping of all shape boundaries within an image onto a global information-rich graph and machine learning on the multidimensional measures of the graph. We demonstrated the power of this method by (1) extracting subtle structural differences from visually indistinguishable images in our phenotype rescue experiments using the endothelial tube formations assay, (2) training the algorithm to identify biophysical parameters underlying the formation of different multicellular networks in our simulation model of collective cell behavior, and (3) analyzing the response of U2OS cell cultures to a broad array of small molecule perturbations.

Time dependent stress relaxation and recovery in mechanically strained 3D microtissues.

Characterizing the time-dependent mechanical properties of cells is not only necessary to determine how they deform but also to understand how external forces trigger biochemical-signaling cascades to govern their behavior. At present, mechanical properties are largely assessed by applying local shear or compressive forces on single cells grown in isolation on non-physiological 2D surfaces. In comparison, we developed the microfabricated vacuum actuated stretcher to measure tensile loading of 3D multicellular “microtissue” cultures. Using this approach, we here assessed the time-dependent stress relaxation and recovery responses of microtissues and quantified the spatial viscoelastic deformation following step length changes. Unlike previous results, stress relaxation and recovery in microtissues measured over a range of step amplitudes and pharmacological treatments followed an augmented stretched exponential behavior describing a broad distribution of inter-related timescales. Furthermore, despite the variety of experimental conditions, all responses led to a single linear relationship between the residual elastic stress and the degree of stress relaxation, suggesting that these mechanical properties are coupled through interactions between structural elements and the association of cells with their matrix. Finally, although stress relaxation could be quantitatively and spatially linked to recovery, they differed greatly in their dynamics; while stress recovery acted as a linear process, relaxation time constants changed with an inverse power law with the step size. This assessment of microtissues offers insights into how the collective behavior of cells in a 3D collagen matrix generates the dynamic mechanical properties of tissues, which is necessary to understand how cells deform and sense mechanical forces in vivo.

3D-printed insert-array and 3D-coculture-array for high-throughput screening of cell migration and application to study molecular and cellular influences

Collective cell migration refers to the movement of groups of cells and collective cell behavior and relies on cell-cell communication and cell-environment interactions. Collective cell migration plays a fundamental role in many aspects of cell biology and pathology. Current protocols for studying collective cell migration either use destructive methods or are not convenient for liquid handling. Here we present a novel 3D-printed insert-array and a 3D-coculture-array for collective cell migration study in high-throughput. The fabricated insert-array is comprised of 96 cylinder shaped inserts which can be placed in each well of a 96-well plate generating watertight contact with the bottom of each well. The insert-array has high manufacturing tolerance, and the coefficient of variations of the insert diameter and circularity are 0.67% and 0.03%, respectively. Each insert generates a circular cell-free area within the well without cell damage and provides convenient access for both manual and robotic liquid handling. Using the 3D-printed insert-array, we studied the migration of human umbilical vein endothelial cells (HUVECs) under the molecular influences of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) and under the cellular influences of human mesenchymal stem cells (hMSCs) using the 3D-coculture-array. Our results show that the migration of HUVECs was dose-dependent on the VEGF and bFGF with different correlation patterns. They also generated a synergic pro-migration effect. When cocultured with hMSCs, the migration rate increased significantly while dependent on the number of hMSCs. The effects were partially blocked by VEGF inhibitor which suggests that VEGF secreted from hMSCs plays an important role in cell-to-cell communication during cell migration. The 3D-coculture-array can be manufactured at very low cost and shows higher biomolecule transport efficiency than the commercially available transwell. The calculated Z-factor is 0.66, which classifies our system as a perfect high-throughput assay. In summary, our newly developed insert-array and 3D-coculture-array provide a versatile platform to study collective cell migration in high-throughput as well as the molecular and cellular influences upon it.

Formation and Developmental Specification of the Odontogenic and Osteogenic Mesenchymes.

Within the mandible, the odontogenic and osteogenic mesenchymes develop in a close proximity and form at about the same time. They both originate from the cranial neural crest. These two condensing ecto-mesenchymes are soon separated from each other by a very loose interstitial mesenchyme, whose cells do not express markers suggesting a neural crest origin. The two condensations give rise to mineralized tissues while the loose interstitial mesenchyme, remains as a soft tissue. This is crucial for proper anchorage of mammalian teeth. The situation in all three regions of the mesenchyme was compared with regard to cell heterogeneity. As the development progresses, the early phenotypic differences and the complexity in cell heterogeneity increases. The differences reported here and their evolution during development progressively specifies each of the three compartments. The aim of this review was to discuss the mechanisms underlying condensation in both the odontogenic and osteogenic compartments as well as the progressive differentiation of all three mesenchymes during development. Very early, they show physical and structural differences including cell density, shape and organization as well as the secretion of three distinct matrices, two of which will mineralize. Based on these data, this review highlights the consecutive differences in cell-cell and cell-matrix interactions, which support the cohesion as well as mechanosensing and mechanotransduction. These are involved in the conversion of mechanical energy into biochemical signals, cytoskeletal rearrangements cell differentiation, or collective cell behavior.

Effect of Geometric Curvature on Collective Cell Migration in Tortuous Microchannel Devices.

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.

Reciprocal integrin/integrin antagonism through kindlin-2 and Rho GTPases regulates cell cohesion and collective migration

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.

Topology control of human fibroblast cells monolayer by liquid crystal elastomer.

Eukaryotic cells in living tissues form dynamic patterns with spatially varying orientational order that affects important physiological processes such as apoptosis and cell migration. The challenge is how to impart a predesigned map of orientational order onto a growing tissue. Here, we demonstrate an approach to produce cell monolayers of human dermal fibroblasts with predesigned orientational patterns and topological defects using a photoaligned liquid crystal elastomer (LCE) that swells anisotropically in an aqueous medium. The patterns inscribed into the LCE are replicated by the tissue monolayer and cause a strong spatial variation of cells phenotype, their surface density, and number density fluctuations. Unbinding dynamics of defect pairs intrinsic to active matter is suppressed by anisotropic surface anchoring allowing the estimation of the elastic characteristics of the tissues. The demonstrated patterned LCE approach has potential to control the collective behavior of cells in living tissues, cell differentiation, and tissue morphogenesis.

A mammalian Wnt5a-Ror2-Vangl2 axis controls the cytoskeleton and confers cellular properties required for alveologenesis.

Alveolar formation increases the surface area for gas-exchange and is key to the physiological function of the lung. Alveolar epithelial cells, myofibroblasts and endothelial cells undergo coordinated morphogenesis to generate epithelial folds (secondary septa) to form alveoli. A mechanistic understanding of alveologenesis remains incomplete. We found that the planar cell polarity (PCP) pathway is required in alveolar epithelial cells and myofibroblasts for alveologenesis in mammals. Our studies uncovered a Wnt5a-Ror2-Vangl2 cascade that endows cellular properties and novel mechanisms of alveologenesis. This includes PDGF secretion from alveolar type I and type II cells, cell shape changes of type I cells and migration of myofibroblasts. All these cellular properties are conferred by changes in the cytoskeleton and represent a new facet of PCP function. These results extend our current model of PCP signaling from polarizing a field of epithelial cells to conferring new properties at subcellular levels to regulate collective cell behavior.

P120catenin tuning of VE-cadherin endocytosis controls collective cell behavior during angiogenesis.

How cells can stick together while moving through a complex environment is not fully understood. In this issue, Grimsley-Myers et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.201909081) demonstrate that, in the case of blood vessel formation, the balance between the maintenance of endothelial integrity and the dynamics of cell-cell contacts required for collective migration relies on VE-cadherin endocytosis.

Identifying density-dependent interactions in collective cell behaviour.

Scratch assays are routinely used to study collective cell behaviour in vitro. Typical experimental protocols do not vary the initial density of cells, and typical mathematical modelling approaches describe cell motility and proliferation based on assumptions of linear diffusion and logistic growth. Jin et al. (Jin et al. 2016 J. Theor. Biol.390, 136-145 (doi:10.1016/j.jtbi.2015.10.040)) find that the behaviour of cells in scratch assays is density-dependent, and show that standard modelling approaches cannot simultaneously describe data initiated across a range of initial densities. To address this limitation, we calibrate an individual-based model to scratch assay data across a large range of initial densities. Our model allows proliferation, motility, and a direction bias to depend on interactions between neighbouring cells. By considering a hierarchy of models where we systematically and sequentially remove interactions, we perform model selection analysis to identify the minimum interactions required for the model to simultaneously describe data across all initial densities. The calibrated model is able to match the experimental data across all densities using a single parameter distribution, and captures details about the spatial structure of cells. Our results provide strong evidence to suggest that motility is density-dependent in these experiments. On the other hand, we do not see the effect of crowding on proliferation in these experiments. These results are significant as they are precisely the opposite of the assumptions in standard continuum models, such as the Fisher-Kolmogorov equation and its generalizations.

Cell-free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices.

Thanks to the synthetic biology, the laborious and restrictive procedure for producing a target protein in living microorganisms by biotechnological approaches can now experience a robust, pliant yet efficient alternative. The new system combined with lab-on-chip microfluidic devices and nanotechnology offers a tremendous potential envisioning novel cell-free formats such as DNA brushes, hydrogels, vesicular particles, droplets, as well as solid surfaces. Acting as robust microreactors/microcompartments/minimal cells, the new platforms can be tuned to perform various tasks in a parallel and integrated manner encompassing gene expression, protein synthesis, purification, detection, and finally enabling cell-cell signaling to bring a collective cell behavior, such as directing differentiation process, characteristics of higher order entities, and beyond. In this review, we issue an update on recent cell-free protein synthesis (CFPS) formats. Furthermore, the latest advances and applications of CFPS for synthetic biology and biotechnology are highlighted. In the end, contemporary challenges and future opportunities of CFPS systems are discussed.