A hybrid computational model for collective cell durotaxis

Collective cell migration is regulated by a complex set of mechanical interactions and cellular mechanisms. Collective migration emerges from mechanisms occurring at single cell level, involving processes like contraction, polymerization and depolymerization, of cell–cell interactions and of cell–substrate adhesion. Here, we present a computational framework which simulates the dynamics of this emergent behavior conditioned by substrates with stiffness gradients. The computational model reproduces the cell’s ability to move toward the stiffer part of the substrate, process known as durotaxis. It combines the continuous formulation of truss elements and a particle-based approach to simulate the dynamics of cell–matrix adhesions and cell–cell interactions. Using this hybrid approach, researchers can quickly create a quantitative model to understand the regulatory role of different mechanical conditions on the dynamics of collective cell migration. Our model shows that durotaxis occurs due to the ability of cells to deform the substrate more in the part of lower stiffness than in the stiffer part. This effect explains why cell collective movement is more effective than single cell movement in stiffness gradient conditions. In addition, we numerically evaluate how gradient stiffness properties, cell monolayer size and force transmission between cells and extracellular matrix are crucial in regulating durotaxis.     

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.     

Extracellular matrix density regulates the formation of tumour spheroids through cell migration

In this work, we show how the mechanical properties of the cellular microenvironment modulate the growth of tumour spheroids. Based on the composition of the extracellular matrix, its stiffness and architecture can significantly vary, subsequently influencing cell movement and tumour growth. However, it is still unclear exactly how both of these processes are regulated by the matrix composition. Here, we present a centre-based computational model that describes how collagen density, which modulates the steric hindrance properties of the matrix, governs individual cell migration and, consequently, leads to the formation of multicellular clusters of varying size. The model was calibrated using previously published experimental data, replicating a set of experiments in which cells were seeded in collagen matrices of different collagen densities, hence producing distinct mechanical properties. At an initial stage, we tracked individual cell trajectories and speeds. Subsequently, the formation of multicellular clusters was also analysed by quantifying their size. Overall, the results showed that our model could accurately replicate what was previously seen experimentally. Specifically, we showed that cells seeded in matrices with low collagen density tended to migrate more. Accordingly, cells strayed away from their original cluster and thus promoted the formation of small structures. In contrast, we also showed that high collagen densities hindered cell migration and produced multicellular clusters with increased volume. In conclusion, this model not only establishes a relation between matrix density and individual cell migration but also showcases how migration, or its inhibition, modulates tumour growth.     

Controlling matrix stiffness and topography for the study of tumor cell migration

Cellular studies have long been performed on the bench top, within Petri dishes and flasks that expose cells to surroundings that differ greatly from their native environment. The complexity of a human tissue is such that to truly replicate a cell’s physiologic microenvironment in vitro is currently impossible. It is nevertheless important to determine how various factors of the microenvironment interact to drive cell behavior, particularly with regard to disease states, such as cancer. Here we focus on two key elements of the cellular microenvironment, matrix stiffness and architecture, in the context of tumor cell behavior. We discuss recent work focusing on the effects of these individual properties on cancer cell migration and describe one technique developed by our lab that could be applied to dissect the effects of specific structural and mechanical cues, and which may lead to useful insights into the potentially synergistic effects of these properties on tumor cell behavior.     

Platelet-derived growth factor in combination with collagen promotes the migration of human skin fibroblasts into a denuded area of a cell monolayer.

Since we have found previously that adult donor skin fibroblasts (TIG-114) migrated more slowly in serum-depleted medium than in medium supplemented with 10% FBS, we tried to identify a factor(s) which promotes fibroblast migration from the edge of a denuded area in a monolayer. In medium supplemented with 10% FBS, the effects of both suramin, a competitor of growth factors at the receptor level, and monensin, an inhibitor of the secretion of extracellular matrix, were examined. Both substances suppressed cell migration, suggesting that growth factors and matrix substances are important for cell migration. Then, we examined the effects of growth factors and extracellular matrix on fibroblast migration in serum-free medium. Platelet-derived growth factor (PDGF), basic fibroblast growth factor, acidic fibroblast growth factor, and transforming growth factor-beta did not stimulate cell migration. Type I collagen, plasma fibronectin, and heparin also did not promote cell migration. However, the combination of PDGF and type I collagen did promote cell migration. Addition of anti-PDGF antibody reduced the stimulatory effect induced by the combination of PDGF and type I collagen. These results suggest that the copresence of growth factors and extracellular matrix regulates fibroblast migration into a denuded area in a monolayer.     

A Novel Role for Lh3 Dependent ECM Modifications during Neural Crest Cell Migration in Zebrafish

During vertebrate development, trunk neural crest cells delaminate along the entire length of the dorsal neural tube and initially migrate as a non-segmented sheet. As they enter the somites, neural crest cells rearrange into spatially restricted segmental streams. Extracellular matrix components are likely to play critical roles in this transition from a sheet-like to a stream-like mode of migration, yet the extracellular matrix components and their modifying enzymes critical for this transition are largely unknown. Here, we identified the glycosyltransferase Lh3, known to modify extracellular matrix components, and its presumptive substrate Collagen18A1, to provide extrinsic signals critical for neural crest cells to transition from a sheet-like migration behavior to migrating as a segmental stream. Using live cell imaging we show that in lh3 null mutants, neural crest cells fail to transition from a sheet to a stream, and that they consequently enter the somites as multiple streams, or stall shortly after entering the somites. Moreover, we demonstrate that transgenic expression of lh3 in a small subset of somitic cells adjacent to where neural crest cells switch from sheet to stream migration restores segmental neural crest cell migration. Finally, we show that knockdown of the presumptive Lh3 substrate Collagen18A1 recapitulates the neural crest cell migration defects observed in lh3 mutants, consistent with the notion that Lh3 exerts its effect on neural crest cell migration by regulating post-translational modifications of Collagen18A1. Together these data suggest that Lh3–Collagen18A1 dependent ECM modifications regulate the transition of trunk neural crest cells from a non-segmental sheet like migration mode to a segmental stream migration mode.     

Mechano-sensing and cell migration: a 3D model approach

Cell migration is essential for tissue development in different physiological and pathological conditions. It is a complex process orchestrated by chemistry, biological factors, microstructure and surrounding mechanical properties. Focusing on the mechanical interactions, cells do not only exert forces on the matrix that surrounds them, but they also sense and react to mechanical cues in a process called mechano-sensing. Here, we hypothesize the involvement of mechano-sensing in the regulation of directional cell migration through a three-dimensional (3D) matrix. For this purpose, we develop a 3D numerical model of individual cell migration, which incorporates the mechano-sensing process of the cell as the main mechanism regulating its movement. Consistent with this hypothesis, we found that factors, such as substrate stiffness, boundary conditions and external forces, regulate specific and distinct cell movements.     

Serum osteopontin, an enhancer of tumor metastasis to bone, promotes B16 melanoma cell migration

Tumor malignancy is associated with several features such as proliferation ability and frequency of metastasis. Since tumor metastasis shortens patients' lifetime, establishment of therapy for anti-metastasis is very important. Osteopontin (OPN), which abundantly expressed in bone matrix, is involved in cell adhesion, migration, extracellular matrix (ECM) invasion and cell proliferation via interaction with its receptor, that is, alphavbeta3 integrin. OPN is believed to be a positive regulator of tumor metastasis in vivo. However, how OPN regulates metastasis is largely unknown. Here, we explore the role of OPN in cell migration. Serum from wild-type mice induced cell migration of B16 melanoma cells, while serum from OPN-deficient mouse suppressed this event. The presence of recombinant OPN significantly enhanced cell migration compared to albumin containing medium. OPN-induced cell migration was suppressed by inhibiting the ERK/MAPK pathway indicating that OPN-induced cell migration depends on this pathway. Overexpression of OPN in these cancer cells per se promoted cell proliferation and tended to increase B16 cell migration suggesting that OPN promotes bone metastasis by playing dual roles both in host microenvironment and in tumor cell itself. In conclusion, the elevated OPN expression in host tissue and tumor cell itself promotes tumor cell migration reading to tumor metastasis, suggesting that neutralization of OPN-induced signal might be effective in suppression of tumor metastasis.     

c-Src-mediated epithelial cell migration and invasion regulated by PDZ binding site

c-Src tyrosine kinase controls proliferation, cell adhesion, and cell migration and is highly regulated. A novel regulatory mechanism to control c-Src function that has recently been identified involves the C-terminal amino acid sequence Gly-Glu-Asn-Leu (GENL) of c-Src as ligand for PDZ domains. Herein, we determined the biological relevance of this c-Src regulation in human breast epithelial cells. The intact GENL sequence maintained c-Src in an inactive state in starved cells and restricted c-Src functions that might lead to metastatic transformation under normal growth conditions. c-Src with a C-terminal Leu/Ala mutation in GENL (Src-A) promoted the activation and translocation of cortactin and focal adhesion kinase and increased the motility and persistence of cell migration on the basement membrane. Src-A promoted increased extracellular proteolytic activity, and in acinar cultures, it led to the escape of cells through the basement membrane into the surrounding matrix. We ascribe the regulatory function of C-terminal Leu to the role of GENL in modulating c-Src activity downstream of cell matrix adhesion. We propose that the C terminus of c-Src via its GENL sequence presents a mechanism that restricts c-Src in epithelia and prevents progression toward an invasive phenotype.     

Laminin-211 in skeletal muscle function

A chain is no stronger than its weakest link is an old idiom that holds true for muscle biology. As the name implies, skeletal muscle’s main function is to move the bones. However, for a muscle to transmit force and withstand the stress that contractions give rise to, it relies on a chain of proteins attaching the cytoskeleton of the muscle fiber to the surrounding extracellular matrix. The importance of this attachment is illustrated by a large number of muscular dystrophies caused by interruption of the cytoskeletal-extracellular matrix interaction. One of the major components of the extracellular matrix is laminin, a heterotrimeric glycoprotein and a major constituent of the basement membrane. It has become increasingly apparent that laminins are involved in a multitude of biological functions, including cell adhesion, differentiation, proliferation, migration and survival. This review will focus on the importance of laminin-211 for normal skeletal muscle function.     

Integrin α9β1

Integrins are transmembrane heterodimeric receptors responsible for transducing and modulating signals between the extracellular matrix and cytoskeleton, ultimately influencing cell functions such as adhesion and migration. Integrin α9β1 is classified within a two member sub-family of integrins highlighted in part by its specialized role in cell migration. The importance of this role is demonstrated by its regulation of numerous biological functions including lymphatic valve morphogenesis, lymphangiogenesis, angiogenesis and hematopoietic homeostasis. Compared to other integrins the signaling mechanisms that transduce α9β1-induced cell migration are not well described. We have recently shown that Src tyrosine kinase plays a key proximal role to control α9β1 signaling. Specifically it activates inducible nitric oxide synthase (iNOS) and in turn nitric oxide (NO) production as a means to transduce cell migration. Furthermore, we have also described a role for FAK, Erk and Rac1 in α9β1 signal transduction. Here we provide an over view of known integrin α9β1 signaling pathways and highlight its roles in diverse biological conditions.     

Cohort migration of carcinoma cells: differentiated colorectal carcinoma cells move as coherent cell clusters or sheets.

Active migration of tumor cells is usually assessed as single cell locomotion in vitro using Boyden chamber-type assays. In vivo, however, carcinoma cells, malignant cells of epithelial origin, frequently invade the surrounding tissue as coherent clusters or nests of cells. We have called this type of movement "cohort migration". In our work, the invasion front of colon carcinomas consisted of compact tumor glands, partially resolved glands or markedly resolved glands with scattered tumor cell clusters or single cells lying ahead. In the former two types, which constituted about a half of all cases, cohort migration seems to be the predominant mechanism, whereas both cohort migration and single cell locomotion may be involved in the last one. In this light, it is very advantageous to investigate the mechanisms involved in the cohort migration. In this review, we present a two-dimensional motility assay as a cohort migration model, in which human colorectal carcinoma cells move outwards from the cell islands mainly as localized coherent sheets of cells when stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor/scatter factor (HGF/SF). Within the migrating cell sheets, wide intercellular gaps occur at the lower portion of the cells to allow the cells to extend leading lamellae forward while close cell-cell contacts remain at the upper portion of the cells. This localized modulation of cell-cell adhesion at the lower portion of the cells is associated with increased tyrosine phosphorylation of the E-cadherin-catenin complex in TPA-induced cohort migration and with reduced alpha-catenin complexed with E-cadherin in HGF/SF-induced cohort migration. Furthermore, fibronectin deposited by migrating cells is essential for their movement, and on the gelatin-coated substrate even degradation and remodeling of the substrate by matrix metalloproteinases are also needed. Thus, in cohort migration it is likely that cells are released from cell-cell adhesion only at the lower portion of the cells via modulation of E-cadherin-catenin-based mechanism, and this change allows the cells to extend leading lamellae onto the extracellular matrix substrate remodeled by deposition of fibronectin and organized digestion.     

A mechanistic protrusive-based model for 3D cell migration

Cell migration is essential for a variety of biological processes, such as embryogenesis, wound healing, and the immune response. After more than a century of research—mainly on flat surfaces—, there are still many unknowns about cell motility. In particular, regarding how cells migrate within 3D matrices, which more accurately replicate in vivo conditions. We present a novel in silico model of 3D mesenchymal cell migration regulated by the chemical and mechanical profile of the surrounding environment. This in silico model considers cell’s adhesive and nuclear phenotypes, the effects of the steric hindrance of the matrix, and cells ability to degradate the ECM. These factors are crucial when investigating the increasing difficulty that migrating cells find to squeeze their nuclei through dense matrices, which may act as physical barriers. Our results agree with previous in vitro observations where fibroblasts cultured in collagen-based hydrogels did not durotax toward regions with higher collagen concentrations. Instead, they exhibited an adurotactic behavior, following a more random trajectory. Overall, cell’s migratory response in 3D domains depends on its phenotype, and the properties of the surrounding environment, that is, 3D cell motion is strongly dependent on the context.     

CXCL12/CXCR4 signalling in neuronal cell migration

The chemokine CXCL12 (or SDF-1) and its receptor CXCR4 have originally been described as regulators of cell interactions in the immune system. However, over the past years it has become clear that this receptor/ligand pair is an important component of the machinery that controls cell migration in different regions of the developing nervous system. Here we will review some of these functions of the CXCL12/CXCR4 system, focusing on migration events in the cerebellum and the cortex. Furthermore, we will discuss these findings in light of the recently discovered second receptor for CXCL12, CXCR7, and the original functional properties of this molecule that have been described in zebrafish.     

What are the key mechanical mechanisms governing integrin-mediated cell migration in three-dimensional fiber networks?

Cell migration plays a vital role in numerous processes such as development, wound healing, or cancer. It is well known that numerous complex mechanisms are involved in cell migration. However, so far it remains poorly understood what are the key mechanisms required to produce the main characteristics of this behavior. The reason is a methodological one. In experimental studies, specific factors and mechanisms can be promoted or inhibited. However, while doing so, there can always be others in the background which play key roles but which have simply remained unattended so far. This makes it very difficult to validate any hypothesis about a minimal set of factors and mechanisms required to produce cell migration. To overcome this natural limitation of experimental studies, we developed a computational model where cells and extracellular matrix fibers are represented by discrete mechanical objects on the micrometer scale. In this model, we had exact control of the mechanisms by which cells and matrix fibers interacted with each other. This enabled us to identify the key mechanisms required to produce physiologically realistic cell migration (including advanced phenomena such as durotaxis and a biphasic relation between migration efficiency and matrix stiffness). We found that two main mechanisms are required to this end: a catch-slip bond of individual integrins and cytoskeletal actin-myosin contraction. Notably, more advanced phenomena such as cell polarization or details of mechanosensing were not necessary to qualitatively reproduce the main characteristics of cell migration observed in experiments.

     

Cytoplasmic tail-dependent internalization of membrane-type 1 matrix metalloproteinase is important for its invasion-promoting activity.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral membrane proteinase that degrades the pericellular extracellular matrix (ECM) and is expressed in many migratory cells, including invasive cancer cells. MT1-MMP has been shown to localize at the migration edge and to promote cell migration; however, it is not clear how the enzyme is regulated during the migration process. Here, we report that MT1-MMP is internalized from the surface and that this event depends on the sequence of its cytoplasmic tail. Di-leucine (Leu571-572 and Leu578-579) and tyrosine573 residues are important for the internalization, and the mu2 subunit of adaptor protein 2, a component of clathrin-coated pits for membrane protein internalization, was found to bind to the LLY573 sequence. MT1-MMP was internalized predominantly at the adherent edge and was found to colocalize with clathrin-coated vesicles. The mutations that disturb internalization caused accumulation of the enzyme at the adherent edge, though the net proteolytic activity was not affected much. Interestingly, whereas expression of MT1-MMP enhances cell migration and invasion, the internalization-defective mutants failed to promote either activity. These data indicate that dynamic turnover of MT1-MMP at the migration edge by internalization is important for proper enzyme function during cell migration and invasion.     

Competing tumor cell migration mechanisms caused by interstitial fluid flow

In the seminal work by Swartz and collaborators (Shields et al., 2007) it was discovered that autologously secreted or activated (ECM-bound) chemokine forms local pericellular diffusion gradients skewed by fluid convection, and the cells subsequently chemotact up the flow-directed gradient. However, in (Polacheck et al., 2011) Kamm and collaborators found that there is a competing downstream and upstream migration transport mechanism. Their study showed that both mechanisms are present at the same time and the relative strength of these two stimuli governs the directional bias in migration for a cell population and is a function of cell density, interstitial flow rate, and CCR7 receptor availability. The main objective of this work is to give a possible explanation of these two different concurrent cell migration mechanisms by means of a theoretical model. Relying on multiphase modelling, separate momentum balance equations are formulated, respectively, for the cell phase and the interstitial fluid (IF) phase. In order to represent proteolytic activity and autologous chemotaxis a non-moving ECM component is included, as well as proteases secreted by the cancer cells and chemokine that can be released from ECM. The cell and IF momentum balance equations include cell-ECM and fluid-ECM resistance force terms (i.e., classical Darcy’s equation terms), but also a cell-fluid interaction term that can account for a more indirect effect that fluid-generated stress may have on cancer cells. We illustrate how the cancer cells can work through this term and effectively avoid being pushed in the flow direction, and even create upstream migration by controlling its magnitude and sign. We think of this as the mathematical interpretation of the experimental observation by Kamm and collaborators that the fluid generated matrix adhesion tension on the upstream side of cells activates integrin adhesion complexes, resulting in activation of focal adhesion (FA) proteins. The model predicts that generally the strength of the upstream migration mechanism is sensitive to the cell volume fraction: a lower density of cells is subject to a weaker upstream migration effect; a higher density of cancer cells can more effectively generate upstream migration. This behavior is a result of the nonlinear coupling between cell-ECM, fluid-ECM, and cell-fluid interaction terms that naturally are involved in the mathematical expression for the net cell velocity.     

Misshapen decreases integrin levels to promote epithelial motility and planar polarity in Drosophila

Complex organ shapes arise from the coordinate actions of individual cells. The Drosophila egg chamber is an organ-like structure that lengthens along its anterior–posterior axis as it grows. This morphogenesis depends on an unusual form of planar polarity in the organ’s outer epithelial layer, the follicle cells. Interestingly, this epithelium also undergoes a directed migration that causes the egg chamber to rotate around its anterior–posterior axis. However, the functional relationship between planar polarity and migration in this tissue is unknown. We have previously reported that mutations in the Misshapen kinase disrupt follicle cell planar polarity. Here we show that Misshapen’s primary role in this system is to promote individual cell motility. Misshapen decreases integrin levels at the basal surface, which may facilitate detachment of each cell’s trailing edge. These data provide mechanistic insight into Misshapen’s conserved role in cell migration and suggest that follicle cell planar polarity may be an emergent property of individual cell migratory behaviors within the epithelium.