Mechanotransduction of mesenchymal melanoma cell invasion into 3D collagen lattices: Filopod-mediated extension–relaxation cycles and force anisotropy

Mesenchymal cell migration in interstitial tissue is a cyclic process of coordinated leading edge protrusion, adhesive interaction with extracellular matrix (ECM) ligands, cell contraction followed by retraction and movement of the cell rear. During migration through 3D tissue, the force fields generated by moving cells are non-isotropic and polarized between leading and trailing edge, however the integration of protrusion formation, cell–substrate adhesion, traction force generation and cell translocation in time and space remain unclear. Using high-resolution 3D confocal reflectance and fluorescence microscopy in GFP/actin expressing melanoma cells, we here employ time-resolved subcellular coregistration of cell morphology, interaction and alignment of actin-rich protrusions engaged with individual collagen fibrils. Using single fibril displacement as sensitive measure for force generated by the leading edge, we show how a dominant protrusion generates extension–retraction cycles transmitted through multiple actin-rich filopods that move along the scaffold in a hand-over-hand manner. The resulting traction force is oscillatory, occurs in parallel to cell elongation and, with maximum elongation reached, is followed by rear retraction and movement of the cell body. Combined live-cell fluorescence and reflection microscopy of the leading edge thus reveals step-wise caterpillar-like extension–retraction cycles that underlie mesenchymal migration in 3D tissue.     

Cell traction force and measurement methods

Cell traction forces (CTFs) are crucial to many biological processes such as inflammation, wound healing, angiogenesis, and metastasis. CTFs are generated by actomyosin interactions and actin polymerization and regulated by intracellular proteins such as alpha-smooth muscle actin (α-SMA) and soluble factors such as transforming growth factor-β (TGF-β). Once transmitted to the extracellular matrix (ECM) through stress fibers via focal adhesions, which are assemblies of ECM proteins, transmembrane receptors, and cytoplasmic structural and signaling proteins (e.g., integrins), CTFs direct many cellular functions, including cell migration, ECM organization, and mechanical signal generation. Various methods have been developed over the years to measure CTFs of both populations of cells and of single cells. At present, cell traction force microscopy (CTFM) is among the most efficient and reliable method for determining CTF field of an entire cell spreading on a two-dimensional (2D) substrate surface. There are currently three CTFM methods, each of which is unique in both how displacement field is extracted from images and how CTFs are subsequently estimated. A detailed review and comparison of these methods are presented. Future research should improve CTFM methods such that they can automatically track dynamic CTFs, thereby providing new insights into cell motility in response to altered biological conditions. In addition, research effort should be devoted to developing novel experimental and theoretical methods for determining CTFs in three-dimensional (3D) matrix, which better reflects physiological conditions than 2D substrate used in current CTFM methods.     

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life from embryonic development and wound healing to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell’s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.     

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is facilitated by actin-rich protrusions called invadopodia that proteolytically degrade the extracellular matrix (ECM). Invadopodia activity has been shown to be dependent on ECM rigidity and cancer cell contractile forces suggesting that rigidity signals can regulate these subcellular structures through actomyosin contractility. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. While some variations between the two assays exist, the protocol presented here provides a method for creating PAAs that can be used in both assays and are easily adaptable to the user’s specific biological and technical needs.     

Cellular contractility changes are sufficient to drive epithelial scattering

Epithelial scattering occurs when cells disassemble cell–cell junctions, allowing individual epithelial cells to act in a solitary manner. Epithelial scattering occurs frequently in development, where it accompanies epithelial–mesenchymal transitions and is required for individual cells to migrate and invade. While migration and invasion have received extensive research focus, how cell–cell junctions are detached remains poorly understood. An open debate has been whether disruption of cell–cell interactions occurs by remodeling of cell–cell adhesions, increased traction forces through cell substrate adhesions, or some combination of both processes. Here we seek to examine how changes in adhesion and contractility are coupled to drive detachment of individual epithelial cells during hepatocyte growth factor (HGF)/scatter factor-induced EMT. We find that HGF signaling does not alter the strength of cell–cell adhesion between cells in suspension, suggesting that changes in cell–cell adhesion strength might not accompany epithelial scattering. Instead, cell–substrate adhesion seems to play a bigger role, as cell–substrate adhesions are stronger in cells treated with HGF and since rapid scattering in cells treated with HGF and TGFβ is associated with a dramatic increase in focal adhesions. Increases in the pliability of the substratum, reducing cells ability to generate traction on the substrate, alter cells? ability to scatter. Further consistent with changes in substrate adhesion being required for cell–cell detachment during EMT, scattering is impaired in cells expressing both active and inactive RhoA mutants, though in different ways. In addition to its roles in driving assembly of both stress fibers and focal adhesions, RhoA also generates myosin-based contractility in cells. We therefore sought to examine how RhoA-dependent contractility contributes to cell–cell detachment. Inhibition of Rho kinase or myosin II induces the same effect on cells, namely an inhibition of cell scattering following HGF treatment. Interestingly, restoration of myosin-based contractility in blebbistatin-treated cells results in cell scattering, including global actin rearrangements. Scattering is reminiscent of HGF-induced epithelial scattering without a concomitant increase in cell migration or decrease in adhesion strength. This scattering is dependent on RhoA, as blebbistatin-induced scattering is reduced in cells expressing dominant-negative RhoA mutants. This suggests that induction of myosin-based cellular contractility may be sufficient for cell–cell detachment during epithelial scattering.     

RhoA is down-regulated at cell–cell contacts via p190RhoGAP-B in response to tensional homeostasis

Breast epithelial cells cultured in three-dimensional (3D) collagen gels undergo ductal morphogenesis when the gel is compliant and they can achieve tensional homeostasis. We previously showed that this process requires down-regulation of Rho in compliant collagen gels, but the mechanism remains undefined. In this study, we find that p190RhoGAP-B, but not p190RhoGAP-A, mediates down-regulation of RhoA activity and ductal morphogenesis in T47D cells cultured in compliant 3D collagen gels. In addition, both RhoA and p190RhoGAP-B colocalize with p120-catenin at sites of cell–cell contact. The association between p190RhoGAP-B and p120-catenin is regulated by matrix compliance such that it increases in compliant vs. rigid collagen gels. Furthermore, knockdown of p120-catenin disrupts ductal morphogenesis, disregulates RhoA activity, and results in loss of p190B at cell–cell contacts. Consistent with these findings, using a RhoA-specific FRET biosensor (RhoA-FLARE.sc), we determined spatial RhoA activity to be significantly decreased at cell–cell contacts versus cell–ECM adhesions, and, of importance, spatial RhoA activity is regulated by p190B. This finding suggests that RhoA exists as an inactive pool at cell–cell contacts and is recruited to cell–ECM contacts within stiff matrices. Overall, these results demonstrate that RhoA is down-regulated at cell–cell contacts through p190RhoGAP-B, which is localized to cell–cell contacts by association with p120-catenin that is regulated by tensional homeostasis.     

Atomic force microscope-based single cell force spectroscopy of breast cancer cell lines: An approach for evaluating cellular invasion

The adhesiveness of cancerous cells to their neighboring cells significantly contributes to tumor progression and metastasis. The single-cell force spectroscopy (SCFS) approach was implemented to survey the cell–cell adhesion force between cancerous cells in three cancerous breast cell lines (MCF-7, T47D, and MDA-MB-231). The gene expression levels of two dominant cell adhesion markers (E-cadherin and N-cadherin) were quantified by real-time PCR. Additionally, the local stiffness of the cell bodies was measured by atomic force microscopy (AFM), and the actin cytoskeletal organization was examined by confocal microscopy. Results indicated that the adhesion force between cells was conversely correlated with their invasion potential. The highest adhesion force was observed in the MCF-7 cells. A reduction in cell–cell adhesion, which is required for the detachment of cells from the main tumor during metastasis, is partly due to the loss of E-cadherin expression and the enhanced expression of N-cadherins. The reduced adhesion was accompanied by the softening of cells, as described by the rearrangement of actin filaments through confocal microscopy observations. The softening of the cell body and the reduced cellular adhesiveness are two adaptive mechanisms through which malignant cells achieve the increased deformability, motility, and strong metastasis potential necessary for passage through endothelial junctions and positioning in host tissue. This study presented application of SCFS to survey cell phenotype transformation during cancer progression. The results can be implemented as a platform for further investigations that target the manipulation of cellular adhesiveness and stiffness as a therapeutic choice.     

XLMR protein related to neurite extension (Xpn/KIAA2022) regulates cell–cell and cell–matrix adhesion and migration

X-linked mental retardation (XLMR) is a common cause of moderate to severe intellectual disability in males. XLMR protein related to neurite extension (Xpn, also known as KIAA2022) has been implicated as a gene responsible for XLMR in humans. Although Xpn is highly expressed in the developing brain and is involved in neurite outgrowth in PC12 cells and neurons, little is known about the functional role of Xpn. Here, we show that Xpn regulates cell–cell and cell–matrix adhesion and migration in PC12 cells. Xpn knockdown enhanced cell–cell and cell–matrix adhesion mediated by N-cadherin and β1-integrin, respectively. N-Cadherin and β1-integrin expression at the mRNA and protein levels was significantly increased in Xpn knockdown PC12 cells. Furthermore, overexpressed Xpn protein was strongly expressed in the nuclei of PC12 and 293T cells. Finally, depletion of Xpn perturbed cellular migration by enhancing N-cadherin and β1-integrin expression in a PC12 cell wound healing assay. We conclude that Xpn regulates cell–cell and cell–matrix adhesion and cellular migration by regulating the expression of adhesion molecules.     

A Highlights from MBoC Selection: Spatial distribution of cell–cell and cell–ECM adhesions regulates force balance while main-taining E-cadherin molecular tension in cell pairs

Mechanical linkage between cell–cell and cell–extracellular matrix (ECM) adhesions regulates cell shape changes during embryonic development and tissue homoeostasis. We examined how the force balance between cell–cell and cell–ECM adhesions changes with cell spread area and aspect ratio in pairs of MDCK cells. We used ECM micropatterning to drive different cytoskeleton strain energy states and cell-generated traction forces and used a Förster resonance energy transfer tension biosensor to ask whether changes in forces across cell–cell junctions correlated with E-cadherin molecular tension. We found that continuous peripheral ECM adhesions resulted in increased cell–cell and cell–ECM forces with increasing spread area. In contrast, confining ECM adhesions to the distal ends of cell–cell pairs resulted in shorter junction lengths and constant cell–cell forces. Of interest, each cell within a cell pair generated higher strain energies than isolated single cells of the same spread area. Surprisingly, E-cadherin molecular tension remained constant regardless of changes in cell–cell forces and was evenly distributed along cell–cell junctions independent of cell spread area and total traction forces. Taken together, our results showed that cell pairs maintained constant E-cadherin molecular tension and regulated total forces relative to cell spread area and shape but independently of total focal adhesion area.     

Alteration of Cellular Behavior and Response to PI3K Pathway Inhibition by Culture in 3D Collagen Gels

Most investigations into cancer cell drug response are performed with cells cultured on flat (2D) tissue culture plastic. Emerging research has shown that the presence of a three-dimensional (3D) extracellular matrix (ECM) is critical for normal cell behavior including migration, adhesion, signaling, proliferation and apoptosis. In this study we investigate differences between cancer cell signaling in 2D culture and a 3D ECM, employing real-time, live cell tracking to directly observe U2OS human osteosarcoma and MCF7 human breast cancer cells embedded in type 1 collagen gels. The activation of the important PI3K signaling pathway under these different growth conditions is studied, and the response to inhibition of both PI3K and mTOR with PI103 investigated. Cells grown in 3D gels show reduced proliferation and migration as well as reduced PI3K pathway activation when compared to cells grown in 2D. Our results quantitatively demonstrate that a collagen ECM can protect U2OS cells from PI103. Overall, our data suggests that 3D gels may provide a better medium for investigation of anti-cancer drugs than 2D monolayers, therefore allowing better understanding of cellular response and behavior in native like environments.     

Cell migration—The role of integrin glycosylation

Background

Cell migration is an essential process in organ homeostasis, in inflammation, and also in metastasis, the main cause of death from cancer. The extracellular matrix (ECM) serves as the molecular scaffold for cell adhesion and migration; in the first phase of migration, adhesion of cells to the ECM is critical. Engagement of integrin receptors with ECM ligands gives rise to the formation of complex multiprotein structures which link the ECM to the cytoplasmic actin skeleton. Both ECM proteins and the adhesion receptors are glycoproteins, and it is well accepted that N-glycans modulate their conformation and activity, thereby affecting cell–ECM interactions. Likely targets for glycosylation are the integrins, whose ability to form functional dimers depends upon the presence of N-linked oligosaccharides. Cell migratory behavior may depend on the level of expression of adhesion proteins, and their N-glycosylation that affect receptor-ligand binding.

Scope of review

The mechanism underlying the effect of integrin glycosylation on migration is still unknown, but results gained from integrins with artificial or mutated N-glycosylation sites provide evidence that integrin function can be regulated by changes in glycosylation.

General significance

A better understanding of the molecular mechanism of cell migration processes could lead to novel diagnostic and therapeutic approaches and applications. For this, the proteins and oligosaccharides involved in these events need to be characterized.     

Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

Delineation of a cell’s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell’s ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue.     

Cell jamming: Collective invasion of mesenchymal tumor cells imposed by tissue confinement

Background

Cancer invasion is a multi-step process which coordinates interactions between tumor cells with mechanotransduction towards the surrounding matrix, resulting in distinct cancer invasion strategies. Defined by context, mesenchymal tumors, including melanoma and fibrosarcoma, develop either single-cell or collective invasion modes, however, the mechanical and molecular programs underlying such plasticity of mesenchymal invasion programs remain unclear.

Methods

To test how tissue anatomy determines invasion mode, spheroids of MV3 melanoma and HT1080 fibrosarcoma cells were embedded into 3D collagen matrices of varying density and stiffness and analyzed for migration type and efficacy with matrix metalloproteinase (MMP)-dependent collagen degradation enabled or pharmacologically inhibited.

Results

With increasing collagen density and dependent on proteolytic collagen breakdown and track clearance, but independent of matrix stiffness, cells switched from single-cell to collective invasion modes. Conversion to collective invasion included gain of cell-to-cell junctions, supracellular polarization and joint guidance along migration tracks.

Conclusions

The density of the extracellulair matrix (ECM) determines the invasion mode of mesenchymal tumor cells. Whereas fibrillar, high porosity ECM enables single-cell dissemination, dense matrix induces cell–cell interaction, leader–follower cell behavior and collective migration as an obligate protease-dependent process.

General significance

These findings establish plasticity of cancer invasion programs in response to ECM porosity and confinement, thereby recapitulating invasion patterns of mesenchymal tumors in vivo. The conversion to collective invasion with increasing ECM confinement supports the concept of cell jamming as a guiding principle for melanoma and fibrosarcoma cells into dense tissue.This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols. Download video file.^{(68M, mov)}     

Automated tracking of unmarked cells migrating in three-dimensional matrices applied to anti-cancer drug screening

In oncology, combating the spread of tumor cells is a clinical need which currently remains unsatisfied. Identifying anti-migratory compounds usually requires in vitro screening of a large number of molecules. Efficient and realistic (i.e., preferably 3D) in vitro tests are thus required in order to quantify the anti-migratory effects of anti-cancer drugs. To remain compatible with high-throughput screening, we focus on assays where unlabeled cells are migrating in 3D transparent gels and are observed under time-lapse 3D phase-contrast microscopy. In this context, we present a method for automatically tracking cells that combines a template matching preprocessing step with a mean-shift process. The preprocessing step consists in performing a correlation of a cell template with each observed volume in order to provide a phase-contrast artifact-free volume where the cells appear as correlation peaks surrounded by smooth gradients. This transformation enables the cells to be efficiently tracked by a mean-shift process. Robustness and efficiency of this approach are qualitatively and quantitatively shown in various experiments. Finally, we successfully applied our method to the quantitative characterization of the anti-migratory impact of cytochalasin-D on cancer cells. In conclusion, our method can efficiently be used for drug screening aiming to evidence drug-induced effects on cell migration in 3D transparent environments, such as matrix gels.     

The Role of Vinculin in the Regulation of the Mechanical Properties of Cells

Vinculin couples as a focal adhesion protein the extracellular matrix (ECM) through integrins to the actomyosin cytoskeleton. During the last years vinculin has become the focus of cell mechanical measurements and a key protein regulating the transmission of contractile forces. In earlier reports vinculin has been described as an inhibitor of cell migration on planar substrates, because knock-out of vinculin in F9 mouse embryonic carcinoma cells and mouse embryonic fibroblasts showed increased cell motility on 2D substrates. The role of vinculin in cell invasion through a 3D extracellular matrix is still fragmentarily investigated. This review presents vinculin in its role as a regulator of cellular mechanical functions. Contractile force generation is reduced when vinculin is absent, or enhanced when vinculin is present. Moreover, the generation of contractile forces is a prerequisite for cell invasion through a dense 3D ECM, where the pore-size is smaller than the diameter of the cell nucleus (<2 μm). Measurements of cell’s biophysical properties will be presented. In summary, vinculin’s leading role among focal adhesion proteins in regulating the mechanical properties of cells will be discussed.     

Lymphangiogenesis in post-natal tissue remodeling: lymphatic endothelial cell connection with its environment

The main physiological function of the lymphatic vasculature is to maintain tissue fluid homeostasis. Lymphangiogenesis or de novo lymphatic formation is closely associated with tissue inflammation in adults (i.e. wound healing, allograft rejection, tumor metastasis). Until recently, research on lymphangiogenesis focused mainly on growth factor/growth factor-receptor pathways governing this process. One of the lymphatic vessel features is the incomplete or absence of basement membrane. This close association of endothelial cells with the underlying interstitial matrix suggests that cell–matrix interactions play an important role in lymphangiogenesis and lymphatic functions. However, the exploration of interaction between extracellular matrix (ECM) components and lymphatic endothelial cells is in its infancy. Herein, we describe ECM–cell and cell–cell interactions on lymphatic system function and their modification occurring in pathologies including cancer metastasis.     

Coupling traction force patterns and actomyosin wave dynamics reveals mechanics of cell motion

Motile cells can use and switch between different modes of migration. Here, we use traction force microscopy and fluorescent labeling of actin and myosin to quantify and correlate traction force patterns and cytoskeletal distributions in Dictyostelium discoideum cells that move and switch between keratocyte‐like fan‐shaped, oscillatory, and amoeboid modes. We find that the wave dynamics of the cytoskeletal components critically determine the traction force pattern, cell morphology, and migration mode. Furthermore, we find that fan‐shaped cells can exhibit two different propulsion mechanisms, each with a distinct traction force pattern. Finally, the traction force patterns can be recapitulated using a computational model, which uses the experimentally determined spatiotemporal distributions of actin and myosin forces and a viscous cytoskeletal network. Our results suggest that cell motion can be generated by friction between the flow of this network and the substrate.