Abstract Although topological defects in cell monolayers have been recognized as mechanical organizing centers in morphogenetic processes, the mechanism by which cells coordinate their motion at such defects and self-organize into higher-order structures remains elusive. Here, we report the formation of three-dimensional (3D) multicellular mounds in unconfined myoblast monolayers, at well-controlled vortex topological defects. Prior to the onset of bilayering, the vortex structure induces millimeter-scale cell flows converging toward the defect center. As a result, 3D cell mounds form at the defect core, layer-by-layer. These mounds grow by interlayer permeation sustained by the converging cell flows. At late stages, the bell shape of the structured mounds can be modeled with a dynamics driven by these converging flows. Our results therefore highlight the crucial role of integer topological defects in driving large-scale cell flows yielding the formation of highly ordered 3D tissues from a monolayer. We propose that similar mechanisms may be at play in certain morphogenetic and tumorigenic events.

In animals, collective cell migration is critical during development and adult life for repairing organs. It remains, however, poorly understood compared with single-cell migration. The polymerization of branched actin by the RAC1-WAVE-Arp2/3 pathway is established to power membrane protrusions at the front of migrating cells, but also to maintain cell junctions in epithelial monolayers. Here, novel regulators of collective cell migration are identified using a two-pronged approach: candidates are extracted from publicly available RAC1-WAVE-Arp2/3 dependency maps and screened in a second step using CRISPR/Cas9 genetic inactivation. In a wound healing assay, PKN2 knockout (KO) MCF10A cells display decreased collective migration due to destabilization of adherens junctions, whereas MOB4 KO cells display increased collective migration with a loss of migration orientation. Upon wound healing, PKN2 relocalizes to lateral junctions and maintains coordinated migration in the monolayer, whereas MOB4 relocalizes to the front edge of leader and follower cells collectively migrating toward the wound. The role of MOB4 in controlling collective migration requires YAP1, since MOB4 KO cells fail to activate YAP1, and their phenotype is rescued by constitutively active YAP1. Together, these findings reveal two complementary activities required for coordinating cells in collective migration.

Chiral processes that lack mirror symmetry pervade nature from enantioselective molecular interactions to the asymmetric development of organisms. An outstanding challenge at the interface between physics and biology consists in bridging the multiple scales between microscopic and macroscopic chirality. Here, we combine theory, experiments and modern inference algorithms to study a paradigmatic example of dynamic chirality transfer across scales: the generation of tissue-scale flows from subcellular forces. The distinctive properties of our microscopic graph model and the corresponding coarse-grained viscoelasticity are that (i) net cell proliferation is spatially inhomogeneous and (ii) cellular dynamics cannot be expressed as an energy gradient. To overcome the general challenge of inferring microscopic model parameters from noisy high-dimensional data, we develop a nudged automatic differentiation algorithm (NADA) that can handle large fluctuations in cell positions observed in single tissue snapshots. This data-calibrated microscopic model quantitatively captures proliferation-driven tissue flows observed at large scales in our experiments on fibroblastoma cell cultures. Beyond chirality, our inference algorithm can be used to extract interpretable graph models from limited amounts of noisy data of living and inanimate cellular systems such as networks of convection cells and flowing foams.

Vinculin is a mechanotransducer that reinforces links between cell adhesions and linear arrays of actin filaments upon myosin-mediated contractility. Both adhesions to the substratum and neighboring cells, however, are initiated within membrane protrusions that originate from Arp2/3-nucleated branched actin networks. Vinculin has been reported to interact with the Arp2/3 complex, but the role of this interaction remains poorly understood. Here, we compared the phenotypes of vinculin knock-out (KO) cells with those of knock-in (KI-P878A) cells, where the point mutation P878A that impairs the Arp2/3 interaction is introduced in the two vinculin alleles of MCF10A mammary epithelial cells. The interaction of vinculin with Arp2/3 inhibits actin polymerization at membrane protrusions and decreases migration persistence of single cells. In cell monolayers, vinculin recruits Arp2/3 and the vinculin-Arp2/3 interaction participates in cell-cell junction plasticity. Through this interaction, vinculin controls the decision to enter a new cell cycle as a function of cell density.

Directed collective cell migration is central to morphogenesis, wound healing and cancer progression. Although the molecular anisotropy of the microenvironment guides this migration, its impact on cell flow patterns remains unexplored. Here we show that subcellular microgrooves elicit a polar mode of collective migration in bidirectional lanes, whose widths reach hundreds of micrometres. This directed form of flocking is observed in vitro with a confluent monolayer of human bronchial epithelial cells whose dynamics is chaotic on featureless substrates. A hydrodynamic theory of active polar fluids and corresponding numerical simulations account for this disorder-to-laning transition and further predict that anisotropic friction associated with the grooves lowers the transition threshold, which we confirm experimentally. Therefore, the microscopic anisotropy of the environment both directs collective cell motion along the substrate easy axis and shapes cell migration patterns. The flow patterns induced by this collective contact guidance are different from those resulting from supracellular confinement, demonstrating that collective migration is impacted by the different length scales of the microenvironment. Our findings offer a strategy for directing cells to specific geometries and functions in tissue engineering applications such as organoid morphogenesis. Supracellular cues play a key role in directing collective cell migration in processes such as wound healing and cancer invasion. New findings emphasize the importance of all length scales of the microenvironment in shaping cell migration patterns.

Hydrostatic skeletons such as the Hydra's consist of two stacked layers of muscle cells perpendicularly oriented. In vivo, these bilayers first assemble, and then the muscle fibers of both layers develop and organize with this crisscross orientation. In the present work, we identify an alternative mechanism of crisscross bilayering of myoblasts in vitro, which results from the prior local organization of these active cells in the initial monolayer. The myoblast sheet can be described as a contractile active nematic in which, as expected, most of the +1/2 topological defects associated with this nematic order self-propel. However, as a result of the production of extracellular matrix (ECM) by the cells, a subpopulation of these comet-like defects does not show any self-propulsion. Perpendicular bilayering occurs at these stationary defects. Cells located at the head of these defects converge toward their core where they accumulate until they start migrating on top of the tail of the first layer, while the tail cells migrate in the opposite direction under the head. Since the cells keep their initial orientations, the two stacked layers end up perpendicularly oriented. This concerted process leading to a crisscross bilayering is mediated by the secretion of ECM.

Smooth muscle cells (SMCs) are mural cells that play a vital contractile function in many tissues. Abnormalities in SMC organization are associated with many diseases including atherosclerosis, asthma, and uterine fibroids. Various studies have reported that SMCs cultured on flat surfaces can spontaneously form three-dimensional clusters whose organization resembles that encountered in some of these pathological settings. Remarkably, how these structures form remains unknown. Here we combine in vitro experiments and physical modeling to show that three-dimensional clusters initiate when cellular contractile forces induce a hole in a flat SMC sheet, a process that can be modeled as the brittle fracture of a viscoelastic material. The subsequent evolution of the nascent cluster can be modeled as an active dewetting process with cluster shape evolution driven by a balance between cluster surface tension, arising from both cell contractility and adhesion, and cluster viscous dissipation. The description of the physical mechanisms governing the spontaneous emergence of these intriguing three-dimensional clusters may offer insight into SMC-related disorders.

Collectively migrating cells in living organisms are often guided by their local environment, including physical barriers and internal interfaces. Well-controlled in vitro experiments have shown that, when confined in adhesive stripes, monolayers of moderately active spindle-shaped cells self-organize at well-defined angle to the stripes' longitudinal direction and spontaneously give rise to a simple shear flow, where the average cellular orientation smoothly varies across the system. However, the impact of physical boundaries on highly active, chaotic, multicellular systems is currently unknown, despite its potential relevance. In this work, we show that human fibrosarcoma cells (HT1080) close to an interface exhibit a spontaneous edge current with broken left-right symmetry, while in the bulk the cell flow remains chaotic. These localized edge currents result from an interplay between nematic order, microscopic chirality, and topological defects. Using a combination of in vitro experiments, numerical simulations, and theoretical work, we demonstrate the presence of a self-organized layer of thorn 1/2 defects anchored at the boundary and oriented at a well-defined angle close to, but smaller than, 90 degrees with respect to the boundary direction. These self-organized defects act as local sources of chiral active stress generating the directed edge flows. Our work therefore highlights the impact of topology on the emergence of collective cell flows at boundaries. It also demonstrates the role of chirality in the emergence of edge flows. Since chirality and boundaries are common properties of multicellular systems, this work suggests a new possible mechanism for collective cellular flows.

Lens-free microscopes are simple setups, as they do not require any lens, but in return, they do not give directly an image of the sample but a hologram of it.Consequently, the final image must be reconstructed, but this reconstruction is often degraded because of the well-known phase wrapping phase.However, Convolutional Neural Networks (CNN) have been recently used to solve the phase wrapping problem in different field as digital holography.In this paper, we try this approach with lens-free microscopy in order to reconstruct large and thick supracellular structures.

Although fibroblast heterogeneity is recognized in primary tumors, both its characterization in and its impact on metastases remain unknown. Here, combining flow cytometry, immunohistochemistry and RNA-sequencing on breast cancer samples, we identify four Cancer-Associated Fibroblast (CAF) subpopulations in metastatic lymph nodes (LN). Two myofibroblastic subsets, CAF-S1 and CAF-S4, accumulate in LN and correlate with cancer cell invasion. By developing functional assays on primary cultures, we demonstrate that these subsets promote metastasis through distinct functions. While CAF-S1 stimulate cancer cell migration and initiate an epithelial-to-mesenchymal transition through CXCL12 and TGFβ pathways, highly contractile CAF-S4 induce cancer cell invasion in 3-dimensions via NOTCH signaling. Patients with high levels of CAFs, particularly CAF-S4, in LN at diagnosis are prone to develop late distant metastases. Our findings suggest that CAF subset accumulation in LN is a prognostic marker, suggesting that CAF subsets could be examined in axillary LN at diagnosis.

During metastatic dissemination, streams of cells collectively migrate through a network of narrow channels within the extracellular matrix, before entering into the blood stream. This strategy is believed to outperform other migration modes, based on the observation that individual cancer cells can take advantage of confinement to switch to an adhesion-independent form of locomotion. Yet, the physical origin of this behaviour has remained elusive and the mechanisms behind the emergence of coherent flows in populations of invading cells under confinement are presently unknown. Here we demonstrate that human fibrosarcoma cells (HT1080) confined in narrow stripe-shaped regions undergo collective migration by virtue of a novel type of topological edge currents, resulting from the interplay between liquid crystalline (nematic) order, microscopic chirality and topological defects. Thanks to a combination of in vitro experiments and theory of active hydrodynamics, we show that, while heterogeneous and chaotic in the bulk of the channel, the spontaneous flow arising in confined populations of HT1080 cells is rectified along the edges, leading to long-ranged collective cell migration, with broken chiral symmetry. These edge currents are fuelled by layers of +1/2 topological defects, orthogonally anchored at the channel walls and acting as local sources of chiral active stress. Our work highlights the profound correlation between confinement and collective migration in multicellular systems and suggests a possible mechanism for the emergence of directed motion in metastatic cancer.

Activity and autonomous motion are fundamental in living and engineering systems. This has stimulated the new field of ‘active matter’ in recent years, which focuses on the physical aspects of propulsion mechanisms, and on motility-induced emergent collective behavior of a larger number of identical agents. The scale of agents ranges from nanomotors and microswimmers, to cells, fish, birds, and people. Inspired by biological microswimmers, various designs of autonomous synthetic nano- and micromachines have been proposed. Such machines provide the basis for multifunctional, highly responsive, intelligent (artificial) active materials, which exhibit emergent behavior and the ability to perform tasks in response to external stimuli. A major challenge for understanding and designing active matter is their inherent nonequilibrium nature due to persistent energy consumption, which invalidates equilibrium concepts such as free energy, detailed balance, and time-reversal symmetry. Unraveling, predicting, and controlling the behavior of active matter is a truly interdisciplinary endeavor at the interface of biology, chemistry, ecology, engineering, mathematics, and physics. The vast complexity of phenomena and mechanisms involved in the self-organization and dynamics of motile active matter comprises a major challenge. Hence, to advance, and eventually reach a comprehensive understanding, this important research area requires a concerted, synergetic approach of the various disciplines. The 2020 motile active matter roadmap of Journal of Physics: Condensed Matter addresses the current state of the art of the field and provides guidance for both students as well as established scientists in their efforts to advance this fascinating area.

Bone is the most frequent metastasis site for breast cancer. As well as dramatically increasing disease burden, bone metastases are also an indicator of poor prognosis. One of the main challenges in investigating bone metastasis in breast cancer is engineering in vitro models that replicate the features of in vivo bone environments. Such in vitro models ideally enable the biology of the metastatic cells to mimic their in vivo behavior as closely as possible. Here, taking benefit of cutting-edge technologies both in microfabrication and cancer cell biology, we have developed an in vitro breast cancer bone-metastasis model. To do so we first 3D printed a bone scaffold that reproduces the trabecular architecture and that can be conditioned with osteoblast-like cells, a collagen matrix, and mineralized calcium. We thus demonstrated that this device offers an adequate soil to seed primary breast cancer bone metastatic cells. In particular, patient-derived xenografts being considered as a better approach than cell lines to achieve clinically relevant results, we demonstrate the ability of this biomimetic bone niche model to host patient-derived xenografted metastatic breast cancer cells. These patient-derived xenograft cells show a long-term survival in the bone model and maintain their cycling propensity, and exhibit the same modulated drug response as in vivo. This experimental system enables access to the idiosyncratic features of the bone microenvironment and cancer bone metastasis, which has implications for drug testing.

Cells' ability to apply contractile forces to their environment and to sense its mechanical properties (e.g., rigidity) are among their most fundamental features.Yet, the interrelations between contractility and mechanosensing, in particular, whether contractile force generation depends on mechanosensing, are not understood.We use theory and extensive experiments to study the time evolution of cellular contractile forces and show that they are generated by time-dependent actomyosin contractile displacements that are independent of the environment's rigidity.Consequently, contractile forces are nonmechanosensitive.We further show that the force-generating displacements are directly related to the evolution of the actomyosin network, most notably to the time-dependent concentration of F-actin.The emerging picture of force generation and mechanosensitivity offers a unified framework for understanding contractility.

Transformed isolated cells are usually extruded from normal epithelia and subsequently eliminated. However, multicellular tumors outcompete healthy cells, highlighting the importance of collective effects. Here, we investigate this situation in vitro by controlling in space and time the activity of the Src oncoprotein within a normal Madin–Darby Canine Kidney (MDCK) epithelial cell monolayer. Using an optogenetics approach with cells expressing a synthetic light-sensitive version of Src (optoSrc), we reversibly trigger the oncogenic activity by exposing monolayers to well-defined light patterns. We show that small populations of activated optoSrc cells embedded in the non-transformed monolayer collectively extrude as a tridimensional aggregate and remain alive, while the surrounding normal cells migrate towards the exposed area. This phenomenon requires an interface between normal and transformed cells and is partially reversible. Traction forces show that Src-activated cells either actively extrude or are pushed out by the surrounding cells in a non-autonomous way.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Activity and autonomous motion are fundamental in living and engineering systems. This has stimulated the new field of active matter in recent years, which focuses on the physical aspects of propulsion mechanisms, and on motility-induced emergent collective behavior of a larger number of identical agents. The scale of agents ranges from nanomotors and microswimmers, to cells, fish, birds, and people. Inspired by biological microswimmers, various designs of autonomous synthetic nano- and micromachines have been proposed. Such machines provide the basis for multifunctional, highly responsive, intelligent (artificial) active materials, which exhibit emergent behavior and the ability to perform tasks in response to external stimuli. A major challenge for understanding and designing active matter is their inherent nonequilibrium nature due to persistent energy consumption, which invalidates equilibrium concepts such as free energy, detailed balance, and time-reversal symmetry. Unraveling, predicting, and controlling the behavior of active matter is a truly interdisciplinary endeavor at the interface of biology, chemistry, ecology, engineering, mathematics, and physics. The vast complexity of phenomena and mechanisms involved in the self-organization and dynamics of motile active matter comprises a major challenge. Hence, to advance, and eventually reach a comprehensive understanding, this important research area requires a concerted, synergetic approach of the various disciplines.