Dynamics of Single-Cell Protein Covariation during Epithelial-Mesenchymal Transition.

Physiological processes, such as the epithelial-mesenchymal transition (EMT), are mediated by changes in protein interactions. These changes may be better reflected in protein covariation within a cellular cluster than in the temporal dynamics of cluster-average protein abundance. To explore this possibility, we quantified proteins in single human cells undergoing EMT. Covariation analysis of the data revealed that functionally coherent protein clusters dynamically changed their protein-protein correlations without concomitant changes in the cluster-average protein abundance. These dynamics of protein-protein correlations were monotonic in time and delineated protein modules functioning in actin cytoskeleton organization, energy metabolism, and protein transport. These protein modules are defined by protein covariation within the same time point and cluster and, thus, reflect biological regulation masked by the cluster-average protein dynamics. Thus, protein correlation dynamics across single cells offers a window into protein regulation during physiological transitions.

Dynamics of single-cell protein covariation during epithelial-mesenchymal transition.

Physiological processes, such as epithelial-mesenchymal transition (EMT), are mediated by changes in protein interactions. These changes may be better reflected in protein covariation within cellular cluster than in the temporal dynamics of cluster-average protein abundance. To explore this possibility, we quantified proteins in single human cells undergoing EMT. Covariation analysis of the data revealed that functionally coherent protein clusters dynamically changed their protein-protein correlations without concomitant changes in cluster-average protein abundance. These dynamics of protein-protein correlations were monotonic in time and delineated protein modules functioning in actin cytoskeleton organization, energy metabolism and protein transport. These protein modules are defined by protein covariation within the same time point and cluster and thus reflect biological regulation masked by the cluster-average protein dynamics. Thus, protein correlation dynamics across single cells offer a window into protein regulation during physiological transitions.

Multivariate relationships among nucleus and Golgi properties during fibrillar migration are robust to and unchanged by epithelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transition (EMT) and maturation of a fibrillar tumor microenvironment play important roles in breast cancer progression. A better understanding of how these events promote cancer cell migration and invasion could help identify new strategies to curb metastasis. The nucleus and Golgi affect migration in a microenvironment-dependent manner. Nucleus size and mechanics influence the ability of a cell to squeeze through confined tumor microenvironments. Golgi positioning determines front-rear polarity necessary for migration. While the roles of individual attributes of nucleus and Golgi in migration are being clarified, how their manifold features are inter-related and work together remains to be understood at a systems level. Here, to elucidate relationships among nucleus and Golgi properties, we quantified twelve morphological and positional properties of these organelles during fibrillar migration of human mammary epithelial cells. Principal component analysis (PCA) reduced the twelve-dimensional space of measured properties to three principal components that capture 75% of the variations in organelle features. Unexpectedly, nucleus and Golgi properties that co-varied in a PCA model built with data from untreated cells were largely similar to co-variations identified using data from TGFß-treated cells. Thus, while TGFß-mediated EMT significantly alters gene expression and motile phenotype, it did not significantly affect the relationships among nucleus size, aspect ratio and orientation with migration direction and among Golgi size and nucleus-Golgi separation distance. Indeed, in a combined PCA model incorporating data from untreated and TGFß-treated cells, scores of individual cells occupy overlapping regions in principal component space, indicating that TGFß-mediated EMT does not promote a unique "Golgi-nucleus phenotype" during fibrillar migration. These results suggest that migration along spatially-confined fiber-like tracks employs a conserved nucleus-Golgi arrangement that is independent of EMT state.

Golgi Stabilization, Not Its Front-Rear Bias, Is Associated with EMT-Enhanced Fibrillar Migration.

Epithelial-to-mesenchymal transition (EMT) and maturation of collagen fibrils in the tumor microenvironment play a significant role in cancer cell invasion and metastasis. Confinement along fiber-like tracks enhances cell migration. To what extent and in what manner EMT further promotes migration in a microenvironment already conducive to migration is poorly understood. Here, we show that TGFß-mediated EMT significantly enhances migration on fiber-like micropatterned tracks of collagen, doubling migration speed and tripling persistence relative to untreated mammary epithelial cells. Thus, cell-intrinsic EMT and extrinsic fibrillar tracks have nonredundant effects on motility. To better understand EMT-enhanced fibrillar migration, we investigated the regulation of Golgi positioning, which is involved in front-rear polarization and persistent cell migration. Confinement along fiber-like tracks has been reported to favor posterior Golgi positioning, whereas anterior positioning is observed during 2-day wound healing. Although EMT also regulates cell polarity, little is known about its effect on Golgi positioning. Here, we show that EMT induces a 2:1 rearward bias in Golgi positioning; however, positional bias explains less than 2% of single-cell variability in migration speed and persistence. Meanwhile, EMT significantly stabilizes Golgi positioning. Cells that enhance migration in response to TGFß maintain Golgi position for 2- to 4-fold longer than nonresponsive counterparts irrespective of whether the Golgi is ahead or behind the nucleus. In fact, 28% of TGFß-responsive cells exhibit a fully committed Golgi phenotype with the organelle either in the anterior or posterior position for over 90% of the time. Furthermore, single-cell differences in Golgi stability capture up to 18% of variations in migration speed. These results suggest a hypothesis that the Golgi may be part of a core physical scaffold that affects how cell-generated forces are distributed during migration. A stable scaffold would be expected to more consistently and therefore more productively distribute forces over time, leading to efficient migration.

Label-Free Automated Cell Tracking: Analysis of the Role of E-cadherin Expression in Collective Electrotaxis.

Collective cell migration plays an important role in wound healing, organogenesis, and the progression of metastatic disease. Analysis of collective migration typically involves laborious and time-consuming manual tracking of individual cells within cell clusters over several dozen or hundreds of frames. Herein, we develop a label-free, automated algorithm to identify and track individual epithelial cells within a free-moving cluster. We use this algorithm to analyze the effects of partial E-cadherin knockdown on collective migration of MCF-10A breast epithelial cells directed by an electric field. Our data show that E-cadherin knockdown in free-moving cell clusters diminishes electrotactic potential, with empty vector MCF-10A cells showing 16% higher directedness than cells with E-cadherin knockdown. Decreased electrotaxis is also observed in isolated cells at intermediate electric fields, suggesting an adhesion-independent role of E-cadherin in regulating electrotaxis. In additional support of an adhesion-independent role of E-cadherin, isolated cells with reduced E-cadherin expression reoriented within an applied electric field 60% more quickly than control. These results have implications for the role of E-cadherin expression in electrotaxis and demonstrate proof-of-concept of an automated algorithm that is broadly applicable to the analysis of collective migration in a wide range of physiological and pathophysiological contexts.

Positive Quantitative Relationship between EMT and Contact-Initiated Sliding on Fiber-like Tracks.

Epithelial-mesenchymal transition (EMT) is a complex process by which cells acquire invasive properties that enable escape from the primary tumor. Complete EMT, however, is not required for metastasis: circulating tumor cells exhibit hybrid epithelial-mesenchymal states, and genetic perturbations promoting partial EMT induce metastasis in vivo. An open question is whether and to what extent intermediate stages of EMT promote invasiveness. Here, we investigate this question, building on recent observation of a new invasive property. Migrating cancer cell lines and cells transduced with prometastatic genes slide around other cells on spatially confined, fiberlike micropatterns. We show here that low-dosage/short-duration exposure to transforming growth factor beta (TGFß) induces partial EMT and enables sliding on narrower (26 µm) micropatterns than untreated counterparts (41 µm). High-dosage/long-duration exposure induces more complete EMT, including disrupted cell-cell contacts and reduced E-cadherin expression, and promotes sliding on the narrowest (15 µm) micropatterns. These results identify a direct and quantitative relationship between EMT and cell sliding and show that EMT-associated invasive sliding is progressive, with cells that undergo partial EMT exhibiting intermediate sliding behavior and cells that transition more completely through EMT displaying maximal sliding. Our findings suggest a model in which fiber maturation and EMT work synergistically to promote invasiveness during cancer progression.

Regulators of Metastasis Modulate the Migratory Response to Cell Contact under Spatial Confinement.

The breast tumor microenvironment (TMEN) is a unique niche where protein fibers help to promote invasion and metastasis. Cells migrating along these fibers are constantly interacting with each other. How cells respond to these interactions has important implications. Cancer cells that circumnavigate or slide around other cells on protein fibers take a less tortuous path out of the primary tumor; conversely, cells that turn back upon encountering other cells invade less efficiently. The contact response of migrating cancer cells in a fibrillar TMEN is poorly understood. Here, using high-aspect ratio micropatterns as a model fibrillar platform, we show that metastatic cells overcome spatial constraints to slide effectively on narrow fiber-like dimensions, whereas nontransformed MCF-10A mammary epithelial cells require much wider micropatterns to achieve moderate levels of sliding. Downregulating the cell-cell adhesion protein, E-cadherin, enables MCF-10A cells to slide on narrower micropatterns; meanwhile, introducing exogenous E-cadherin in metastatic MDA-MB-231 cells increases the micropattern dimension at which they slide. We propose the characteristic fibrillar dimension (CFD) at which effective sliding is achieved as a metric of sliding ability under spatial confinement. Using this metric, we show that metastasis-promoting genetic perturbations enhance cell sliding and reduce CFD. Activation of ErbB2 combined with downregulation of the tumor suppressor and cell polarity regulator, PARD3, reduced the CFD, in agreement with their cooperative role in inducing metastasis in vivo. The CFD was further reduced by a combination of ErbB2 activation and transforming growth factor ß stimulation, which is known to enhance invasive behavior. These findings demonstrate that sliding is a quantitative property and a decrease in CFD is an effective metric to understand how multiple genetic hits interact to change cell behavior in fibrillar environments. This quantitative framework sheds insights into how genetic perturbations conspire with fibrillar maturation in the TMEN to drive the invasive behavior of cancer cells.

Collective migration exhibits greater sensitivity but slower dynamics of alignment to applied electric fields.

During development and disease, cells migrate collectively in response to gradients in physical, chemical and electrical cues. Despite its physiological significance and potential therapeutic applications, electrotactic collective cell movement is relatively less well understood. Here, we analyze the combined effect of intercellular interactions and electric fields on the directional migration of non-transformed mammary epithelial cells, MCF-10A. Our data show that clustered cells exhibit greater sensitivity to applied electric fields but align more slowly than isolated cells. Clustered cells achieve half-maximal directedness with an electric field that is 50% weaker than that required by isolated cells; however, clustered cells take ∼2-4 fold longer to align. This trade-off in greater sensitivity and slower dynamics correlates with the slower speed and intrinsic directedness of collective movement even in the absence of an electric field. Whereas isolated cells exhibit a persistent random walk, the trajectories of clustered cells are more ballistic as evidenced by the superlinear dependence of their mean square displacement on time. Thus, intrinsically-directed, slower clustered cells take longer to redirect and align with an electric field. These findings help to define the operating space and the engineering trade-offs for using electric fields to affect cell movement in biomedical applications.

Cell chemotaxis on paper for diagnostics.

Microfluidic chemotaxis platforms have historically been utilized to probe phenomena such as neutrophil migration and are beginning to be developed for diagnostic applications; however, current microfluidic chemotaxis systems require specialized engineering equipment such as syringe pumps and long time frames (hours) to develop a chemokine gradient, and cell chemotaxis typically requires multiple additional hours. The paperfluidic device described in this work is a low-cost, sharp (2 mm wide), quasi-stable (at least 20 min) and rapidly generated (<1 s) chemokine gradient system capable of examining cell migration response over short time frames (20 min) that can be easily assembled. A proof-of-concept experiment on human pan-T cells showed significant (p ≪ 0.01) directed migration to the chemokine gradient over the control condition. This new technique for cell migration studies provides a foundational step in designing microfluidic chemotactic platforms for point-of-care diagnostics.

FGF signaling regulates Wnt ligand expression to control vulval cell lineage polarity in C. elegans.

The interpretation of extracellular cues leading to the polarization of intracellular components and asymmetric cell divisions is a fundamental part of metazoan organogenesis. The Caenorhabditis elegans vulva, with its invariant cell lineage and interaction of multiple cell signaling pathways, provides an excellent model for the study of cell polarity within an organized epithelial tissue. Here, we show that the fibroblast growth factor (FGF) pathway acts in concert with the Frizzled homolog LIN-17 to influence the localization of SYS-1, a component of the Wnt/ß-catenin asymmetry pathway, indirectly through the regulation of cwn-1. The source of the FGF ligand is the primary vulval precursor cell (VPC) P6.p, which controls the orientation of the neighboring secondary VPC P7.p by signaling through the sex myoblasts (SMs), activating the FGF pathway. The Wnt CWN-1 is expressed in the posterior body wall muscle of the worm as well as in the SMs, making it the only Wnt expressed on the posterior and anterior sides of P7.p at the time of the polarity decision. Both sources of cwn-1 act instructively to influence P7.p polarity in the direction of the highest Wnt signal. Using single molecule fluorescence in situ hybridization, we show that the FGF pathway regulates the expression of cwn-1 in the SMs. These results demonstrate an interaction between FGF and Wnt in C. elegans development and vulval cell lineage polarity, and highlight the promiscuous nature of Wnts and the importance of Wnt gradient directionality within C. elegans.

Engineering cell-cell signaling.

Juxtacrine cell-cell signaling mediated by the direct interaction of adjoining mammalian cells is arguably the mode of cell communication that is most recalcitrant to engineering. Overcoming this challenge is crucial for progress in biomedical applications, such as tissue engineering, regenerative medicine, immune system engineering and therapeutic design. Here, we describe the significant advances that have been made in developing synthetic platforms (materials and devices) and synthetic cells (cell surface engineering and synthetic gene circuits) to modulate juxtacrine cell-cell signaling. In addition, significant progress has been made in elucidating design rules and strategies to modulate juxtacrine signaling on the basis of quantitative, engineering analysis of the mechanical and regulatory role of juxtacrine signals in the context of other cues and physical constraints in the microenvironment. These advances in engineering juxtacrine signaling lay a strong foundation for an integrative approach to utilize synthetic cells, advanced 'chassis' and predictive modeling to engineer the form and function of living tissues.

Short-lived, transitory cell-cell interactions foster migration-dependent aggregation.

During embryonic development, motile cells aggregate into cohesive groups, which give rise to tissues and organs. The role of cell migration in regulating aggregation is unclear. The current paradigm for aggregation is based on an equilibrium model of differential cell adhesivity to neighboring cells versus the underlying substratum. In many biological contexts, however, dynamics is critical. Here, we provide evidence that multicellular aggregation dynamics involves both local adhesive interactions and transport by cell migration. Using time-lapse video microscopy, we quantified the duration of cell-cell contacts among migrating cells that collided and adhered to another cell. This lifetime of cell-cell interactions exhibited a monotonic decreasing dependence on substratum adhesivity. Parallel quantitative measurements of cell migration speed revealed that across the tested range of adhesive substrata, the mean time needed for cells to migrate and encounter another cell was greater than the mean adhesion lifetime, suggesting that aggregation dynamics may depend on cell motility instead of the local differential adhesivity of cells. Consistent with this hypothesis, aggregate size exhibited a biphasic dependence on substratum adhesivity, matching the trend we observed for cell migration speed. Our findings suggest a new role for cell motility, alongside differential adhesion, in regulating developmental aggregation events and motivate new design principles for tuning aggregation dynamics in tissue engineering applications.

Modular design of micropattern geometry achieves combinatorial enhancements in cell motility.

Basic micropattern shapes, such as stripes and teardrops, affect individual facets of cell motility, such as migration speed and directional bias, respectively. Here, we test the idea that these individual effects on cell motility can be brought together to achieve multidimensional improvements in cell behavior through the modular reconstruction of the simpler "building block" micropatterns. While a modular design strategy is conceptually appealing, current evidence suggests that combining environmental cues, especially molecular cues, such as growth factors and matrix proteins, elicits a highly nonlinear, synergistic cell response. Here, we show that, unlike molecular cues, combining stripe and teardrop geometric cues into a hybrid, spear-shaped micropattern yields combinatorial benefits in cell speed, persistence, and directional bias. Furthermore, cell migration speed and persistence are enhanced in a predictable, additive manner on the modular spear-shaped design. Meanwhile, the spear micropattern also improved the directional bias of cell movement compared to the standard teardrop geometry, revealing that combining geometric features can also lead to unexpected synergistic effects in certain aspects of cell motility. Our findings demonstrate that the modular design of hybrid micropatterns from simpler building block shapes achieves combinatorial improvements in cell motility. These findings have implications for engineering biomaterials that effectively mix and match micropatterns to modulate and direct cell motility in applications, such as tissue engineering and lab-on-a-chip devices.

Matrix stiffening sensitizes epithelial cells to EGF and enables the loss of contact inhibition of proliferation.

Anchorage to a compliant extracellular matrix (ECM) and contact with neighboring cells impose important constraints on the proliferation of epithelial cells. How anchorage and contact dependence are inter-related and how cells weigh these adhesive cues alongside soluble growth factors to make a net cell cycle decision remain unclear. Here, we show that a moderate 4.5-fold stiffening of the matrix reduces the threshold amount of epidermal growth factor (EGF) needed to over-ride contact inhibition by over 100-fold. At EGF doses in the range of the dissociation constant (K(d)) for ligand binding, epithelial cells on soft matrices are contact inhibited with DNA synthesis restricted to the periphery of cell clusters. By contrast, on stiff substrates, even EGF doses at sub-K(d) levels over-ride contact inhibition, leading to proliferation throughout the cluster. Thus, matrix stiffening significantly sensitizes cells to EGF, enabling contact-independent spatially uniform proliferation. Contact inhibition on soft substrates requires E-cadherin, and the loss of contact inhibition upon matrix stiffening is accompanied by the disruption of cell-cell contacts, changes in the localization of the EGF receptor and ZO-1, and selective attenuation of ERK, but not Akt, signaling. We propose a quantitative framework for the epigenetic priming (via ECM stiffening) of a classical oncogenic pathway (EGF) with implications for the regulation of tissue growth during morphogenesis and cancer progression.

Intercellular mechanotransduction during multicellular morphodynamics.

Multicellular structures are held together by cell adhesions. Forces that act upon these adhesions play an integral role in dynamically re-shaping multicellular structures during development and disease. Here, we describe different modes by which mechanical forces are transduced in a multicellular context: (i) indirect mechanosensing through compliant substratum, (ii) cytoskeletal 'tug-of-war' between cell-matrix and cell-cell adhesions, (iii) cortical contractility contributing to line tension, (iv) stresses associated with cell proliferation, and (v) forces mediating collective migration. These modes of mechanotransduction are recurring motifs as they play a key role in shaping multicellular structures in a wide range of biological contexts. Tissue morphodynamics may ultimately be understood as different spatio-temporal combinations of a select few multicellular transformations, which in turn are driven by these mechanotransduction motifs that operate at the bicellular to multicellular length scale.

Quantitative effect of scaffold abundance on signal propagation.

Protein scaffolds bring together multiple components of a signalling pathway, thereby promoting signal propagation along a common physical 'backbone'. Scaffolds play a prominent role in natural signalling pathways and provide a promising platform for synthetic circuits. To better understand how scaffolding quantitatively affects signal transmission, we conducted an in vivo sensitivity analysis of the yeast mating pathway to a broad range of perturbations in the abundance of the scaffold Ste5. Our measurements show that signal throughput exhibits a biphasic dependence on scaffold concentration and that altering the amount of scaffold binding partners reshapes this biphasic dependence. Unexpectedly, the wild-type level of Ste5 is approximately 10-fold below the optimum needed to maximize signal throughput. This sub-optimal configuration may be a tradeoff as increasing Ste5 expression promotes baseline activation of the mating pathway. Furthermore, operating at a sub-optimal level of Ste5 may provide regulatory flexibility as tuning Ste5 expression up or down directly modulates the downstream phenotypic response. Our quantitative analysis reveals performance tradeoffs in scaffold-based modules and defines engineering challenges for implementing molecular scaffolds in synthetic pathways.

Tunable interplay between epidermal growth factor and cell-cell contact governs the spatial dynamics of epithelial growth.

Contact-inhibition of proliferation constrains epithelial tissue growth, and the loss of contact-inhibition is a hallmark of cancer cells. In most physiological scenarios, cell-cell contact inhibits proliferation in the presence of other growth-promoting cues, such as soluble growth factors (GFs). How cells quantitatively reconcile the opposing effects of cell-cell contact and GFs, such as epidermal growth factor (EGF), remains unclear. Here, using quantitative analysis of single cells within multicellular clusters, we show that contact is not a "master switch" that overrides EGF. Only when EGF recedes below a threshold level, contact inhibits proliferation, causing spatial patterns in cell cycle activity within epithelial cell clusters. Furthermore, we demonstrate that the onset of contact-inhibition and the timing of spatial patterns in proliferation may be reengineered. Using micropatterned surfaces to amplify cell-cell interactions, we induce contact-inhibition at a higher threshold level of EGF. Using a complementary molecular genetics approach to enhance cell-cell interactions by overexpressing E-cadherin also increases the threshold level of EGF at which contact-inhibition is triggered. These results lead us to propose a state diagram in which epithelial cells transition from a contact-uninhibited state to a contact-inhibited state at a critical threshold level of EGF, a property that may be tuned by modulating the extent of cell-cell contacts. This quantitative model of contact-inhibition has direct implications for how tissue size may be determined and deregulated during development and tumor formation, respectively, and provides design principles for engineering epithelial tissue growth in applications such as tissue engineering.