Measurement of leukocyte motility and chemotaxis parameters with a linear under-agarose migration assay

The interpretation of quantitative assays for leukocyte chemotactic migration is usually made in terms of measurements such as leading front distance, total migrating cells, and leukotactic index. These quantities allow comparison of cellular migration behavior under specified conditions. They are not useful; however, for comparisons between systems or for correlation with in vivo performance, because they depend upon specific physical aspects of the assay system, such as the geometry, chemoattractant concentration and diffusivity, and observation time. It would be more helpful to measure intrinsic properties of cell movement that could be used for comparison between systems, for correlation with in vivo studies, and to increase our understanding of the cell physiology. In this paper we demonstrate a means of quantitating leukocyte random motility, chemokinesis, and chemotaxis in terms of parameters that do characterize intrinsic cell properties. These parameters are the random motility coefficient and the chemotaxis coefficient, which appear in theoretical models of cell migration. We examine how well such a model describes the leukocyte density profile data observed in a modified under-agarose assay having a linear geometry. Furthermore, we obtain values for the random motility coefficient (and its dependence upon the concentration of the attractant peptide FNLLP) and for the chemotaxis coefficient for leukocytes responding to FNLLP.     

New method for modeling connective-tissue cell migration: improved accuracy on motility parameters

Mathematical models of cell migration based on persistent random walks have been successfully applied to describe the motility of several cell types. However, the migration of slowly moving connective-tissue cells, such as fibroblasts, is difficult to observe experimentally and difficult to describe theoretically. We identify two primary sources of this difficulty. First, cells such as fibroblasts tend to migrate slowly and change shape during migration. This makes accurate determination of cell position difficult. Second, the cell population is considerably heterogeneous with respect to cell speed. Here we develop a method for fitting connective-tissue cell migration data to persistent random walk models, which accounts for these two significant sources of error and enables accurate determination of the cell motility parameters. We demonstrate the usefulness of this method for modeling both isotropic cell motility and biased cell motility, where the migration of a population of cells is influenced by a gradient in a surface-bound adhesive peptide. This method can discern differences in the motility of populations of cells at different points along the peptide gradient and can therefore be used as a tool to quantify the effects of peptide concentration and gradient magnitude on cell migration.     

Migration of isogenic cell lines quantified by dynamic multivariate analysis of single-cell motility

Cell migration is essential in many physiological and pathological processes. To understand this complex behavior, researchers have turned to quantitative, in vitro, image-based measurements to dissect the steps of cellular motility. With the rise of automated microscopy, the bottleneck in these approaches is no longer data acquisition, but data analysis. Using time-lapse microscopy and computer-assisted image analysis, we have developed a novel, quantitative assay that extracts a multivariate profile for cellular mo-tility. This technique measures three dynamic parameters per single cell: speed, surface area, and an in-dex of cell expansion/contraction activity (DECCA). Our assay can be used in combination with a variety of extracellular matrix components, or other soluble agents, to analyze the effects of the microenviron-ment on cellular migration dynamics in vitro. Our application was developed and tested using A431 and HT-1080 cell lines plated on laminin-332 or fibronectin substrates. Our results indicate that HT-1080 cells migrate faster, have a greater surface area, and have a higher DECCA index than A431 cells on both matrices (for all parameters, p < 0.05). Spearman’s correlation coefficients suggest that for these cell lines and matrices, various combinations of the three measurements display low to medium-high levels of correlation. These findings compare well with previous literature. Our approach provides new tools to measure cellular migration dynamics and address questions on the relationship between cell motility and the microenvironment, using only common microscopy techniques, accessible image analysis applica-tions, and a basic desktop computer for image processing.     

The effect of cell sedimentation on measuring chondrocyte population migration using a Boyden chamber

The Boyden chamber assay provides a convenient method of assessing cell migration and measuring cell motility coefficients at the population level. Previous models of this assay completely ignore cell sedimentation in the suspension, assuming that all cells have already settled on the filter surface before commencing migration within the filter. However, ignoring cell sedimentation could lead to poor data interpretation because the time required for cells to settle through the suspension is close to the incubation period of only a few hours. This study models the Boyden chamber assay by incorporating the cell settling process to account for the cells remaining in the upper well when other cells migrate in the filter. The simulations in this study elucidate the experiments in the literature that test the haptotactic and chemotactic responses of rabbit chondrocytes to type II collagen. This study determines the cell population random motility, as well as the haptotaxis and chemotaxis coefficients, by fitting the experimental data. Results show that the chemotactic motility coefficient is 100 times greater than the haptotactic coefficient, and the equilibrium collagen-receptor dissociation constant is about 10-fold the haptotactic counterpart. Diffusion causes the soluble collagen gradients in the chemotactic case to decline over time, while the coated collagen gradients in the haptotactic assay are likely to remain fixed. As a result, the chemotactic case exhibits a lower number of migrated cells than the haptotactic assay. This study also demonstrates the influences of the dimensionless parameters that control cell behavior in the Boyden assay, providing a reference for future experiment designs.     

HAI-1过表达对SW620细胞体外生长和运动能力的影响

肝细胞生长因子激活因子抑制因子1(hepatocyte growth factor activator inhibitor type 1,HAI-1)能有效抑制肝细胞生长因子激活因子(hepatocyte growth factor activator,HGFA)和丝氨酸蛋白酶Matriptase的活性,并可通过对HGFA和Matriptase活性的调控参与HGF／c—Met信号传导途径。为了解HAI-1在肿瘤细胞的生长和运动中的作用,本研究将人HAI-1基因全长cDNA克隆至pcDNA3．1( )表达载体,并转染人肠癌SW620细胞,用Western blot验证了转染细胞中HAI-1的表达情况,并分别利用生长曲线、软琼脂集落形成、穿膜运动和扩散运动测定等方法检测了HAI-1过表达对SW620细胞生长和运动能力的影响。生长曲线和软琼脂集落形成测定都显示出HAI-1转染细胞与对照组相比差异不十分明显。穿膜运动和扩散运动测定则均显示了HAI-1过表达对细胞运动能力有明显的抑制。因此,HAI-1的过表达虽然在体外对肿瘤细胞生长影响较小,但可以抑制肿瘤细胞的运动迁移能力。     

ATP luminescence-based motility-invasion assay

Directional motility and invasion assays are largely based on the use of Boyden chambers or Transwell culture inserts in which porous membranes separate seeded cells from a chemotactic factor supplied in the medium outside the chamber. The major obstaclefor most currently available assays is that they lack a sensitive, easy, and reliable method of quantifying the nonmotile cell populations. Failure to accountfor all cells within the assay chamber prohibits the determination of percentages of migrated cells. Here we describe an ATP luminescence-based motility-invasion (ALMI) assay that circumvents this problem, enabling investigators to quantify directional cell migration or invasiveness easily. The ALMI assay is based on the detection of ATP in viable cells harvested from inert surfaces that do not generate background signals. We demonstrate how the ALMI assay can be used to assess the effects of various experimental conditions such as growth factor stimulation and ethanol exposure on cell migration. In addition, precoating the membranes with extracellular matrix molecules enabled the measurement of the cell invasion. In conclusion, the ALMI assay provides a reliable and flexible method to quantify cell motility and invasiveness using a luminescence microplate reader.     

Measurement of the chemotaxis coefficient for human neutrophils in the under-agarose migration assay

Clinical and scientific investigations of leukocyte chemotaxis will be greatly aided by an ability to measure quantitative parameters characterizing the intrinsic random motility, chemokinetic, and chemotactic properties of cell populations responding to a given attractant. Quantities typically used at present, such as leading front distances, migrating cell numbers, etc., are unsatisfactory in this regard because their values are affected by many aspects of the assay system unrelated to cell behavioral properties. In this paper we demonstrate the measurement of cell migration parameters that do, in fact, characterize the intrinsic cell chemosensory movement responses using cell density profiles obtained in the linear under-agarose assay. These parameters are the random motility coefficient, mu, and the chemotaxis coefficient, chi, which appear in a theoretical expression for cell population migration. We propose a priori the dependence of chi on attractant concentration, based on an independent experimental correlation of individual cell orientation bias in an attractant gradient with a spatial difference in receptor occupancy. Our under-agarose population migration results are consistent with this proposition, allowing chemotaxis to be reliably characterized by a chemotactic sensitivity constant, chi 0, to which chi is directly proportional. Further, chi 0 has fundamental significance; it represents the reciprocal of the difference in number of bound receptors across cell dimensions required for directional orientation bias. In particular, for the system of human peripheral blood polymorphonuclear neutrophil leukocytes responding to FNLLP, we find that the chemotaxis coefficient is a function of attractant concentration, a following the expression: chi = chi 0NT0 f(a) S(a) Kd/(Kd + a)2 where Kd is the FNLLP-receptor equilibrium dissociation constant and NT0 is the total number of cell surface receptors for FNLLP. f(a) is the fraction of surface receptors remaining after down-regulation, and S(a) is the cell movement speed, both known functions of FNLLP concentration. We find that chi 0NT0 = 0.2 cm; according to a theoretical argument outlined in the Appendix this means that these cells exhibit 75% orientation toward higher attractant concentration when the absolute spatial difference in bound receptors is 0.0025NT0 over 10 micron. (For example, if NT0 = 50,000 this would correspond to a spatial difference of 125 bound receptors over 10 micron.) This result can be compared with estimates obtained from visual studies of individual neutrophils.     

HAI-1过表达对SW620细胞体外生长和运动能力的影响

肝细胞生长因子激活因子抑制因子1(hepatocyte growth factor activator inhibitor type 1, HAI-1)能有效抑制肝细胞生长因子激活因子(hepatocyte growth factor activator,HGFA)和丝氨酸蛋白酶Matriptase的活性,并可通过对HGFA和Matriptase活性的调控参与HGF/c-Met信号传导途径。为了解HAL-1在肿瘤细胞的生长和运动中的作用,本研究将人HAI-1基因全长cDNA克隆至pcDNA3.1( )表达载体,并转染人肠癌SW620细胞,用Western blot验证了转染细胞中HAI-1的表达情况,并分别利用生长曲线、软琼脂集落形成、穿膜运动和扩散运动测定等方法检测了HAI-1 过表达对SW620细胞生长和运动能力的影响。生长曲线和软琼脂集落形成测定都显示出HAI-1 转染细胞与对照组相比差异不十分明显。穿膜运动和扩散运动测定则均显示了HAI-1过表达对细胞运动能力有明显的抑制。因此,HAI-1的过表达虽然在体外对肿瘤细胞生长影响较小,但可以抑制肿瘤细胞的运动迁移能力。     

Nodal signaling regulates endodermal cell motility and actin dynamics via Rac1 and Prex1

Embryo morphogenesis is driven by dynamic cell behaviors, including migration, that are coordinated with fate specification and differentiation, but how such coordination is achieved remains poorly understood. During zebrafish gastrulation, endodermal cells sequentially exhibit first random, nonpersistent migration followed by oriented, persistent migration and finally collective migration. Using a novel transgenic line that labels the endodermal actin cytoskeleton, we found that these stage-dependent changes in migratory behavior correlated with changes in actin dynamics. The dynamic actin and random motility exhibited during early gastrulation were dependent on both Nodal and Rac1 signaling. We further identified the Rac-specific guanine nucleotide exchange factor Prex1 as a Nodal target and showed that it mediated Nodal-dependent random motility. Reducing Rac1 activity in endodermal cells caused them to bypass the random migration phase and aberrantly contribute to mesodermal tissues. Together, our results reveal a novel role for Nodal signaling in regulating actin dynamics and migration behavior, which are crucial for endodermal morphogenesis and cell fate decisions.     

Redistribution of cytosolic FGFR1 after induced migration of urothelial cells in culture

The distribution of cytosolic fibroblast growth factor receptor 1 (FGFR1) was studied in correlation to cell migration in urothelial cell line g/G. Cell motility was analysed with a new method using consecutive series of photographs of cells relocated on CELLocate coverslips and with image analysis software. The results confirmed that FGF1 stimulated cell motility only when cells were grown on collagen I coating. During the transition from sessile to motile cell phenotype a complete redistribution of cytosolic FGFR1 was revealed. In sessile cells, FGFR1 had a filamentous distribution and its location matched cytokeratin 7. In cells of the migrating phenotype, the distribution of FGFR1 was diffuse, mainly located in cytosol. Our data reveal that the location of cytosolic FGFR1 depends on the motile characteristics of the cell. The results also indicate that attachment of cells to collagen I is crucial for the induction of urothelial cell motility with FGF1.     

Differential effects of matrix and growth factors on endothelial and fibroblast motility: application of a modified cell migration assay

Cell migration is crucial in virtually every biological process and strongly depends on the nature of the surrounding matrix. An assay that enables real-time studies on the effects of defined matrix components and growth factors on cell migration is not available. We have set up a novel, quantitative migration assay, which enables unharmed cells to migrate along a defined matrix. Here, we used this so-called barrier-assay to define the contribution of fibronectin (FN) and Collagen-I (Col-I) to vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and lysophosphatidic acid (LPA)-induced cell migration of endothelial cells (EC) and fibroblasts. In EC, both FN and Col-I stimulated migration, but FN-induced motility was random, while net movement was inhibited. Addition of bFGF and VEGF overcame the effect of FN, with VEGF causing directional movement. In contrast, in 3T3 fibroblasts, FN stimulated motility and this effect was enhanced by bFGF. This motility was more efficient and morphologically completely different compared to LPA stimulation. Strikingly, directional migration of EC was not paralleled by higher amounts of stable microtubules (MT) or an increased reorientation of the microtubule-organizing centre (MTOC). For EC, the FN effect appeared concentration dependent; high FN was able to induce migration, while for fibroblasts both low and high concentrations of FN induced motility. Besides showing distinct responses of the different cells to the same factors, these results address contradictive reports on FN and show that the interplay between matrix components and growth factors determines both pattern and regulation of cell migration. J. Cell. Biochem. 99: 1536-1552, 2006. (c) 2006 Wiley-Liss, Inc.     

Shc and FAK differentially regulate cell motility and directionality modulated by PTEN.

Cell migration is modulated by regulatory molecules such as growth factors, oncogenes, and the tumor suppressor PTEN. We previously described inhibition of cell migration by PTEN and restoration of motility by focal adhesion kinase (FAK) and p130 Crk-associated substrate (p130(Cas)). We now report a novel pathway regulating random cell motility involving Shc and mitogen-activated protein (MAP) kinase, which is downmodulated by PTEN and additive to a FAK pathway regulating directional migration. Overexpression of Shc or constitutively activated MEK1 in PTEN- reconstituted U87-MG cells stimulated integrin- mediated MAP kinase activation and cell migration. Conversely, overexpression of dominant negative Shc inhibited cell migration; Akt appeared uninvolved. PTEN directly dephosphorylated Shc. The migration induced by FAK or p130(Cas) was directionally persistent and involved extensive organization of actin microfilaments and focal adhesions. In contrast, Shc or MEK1 induced a random type of motility associated with less actin cytoskeletal and focal adhesion organization. These results identify two distinct, additive pathways regulating cell migration that are downregulated by tumor suppressor PTEN: one involves Shc, a MAP kinase pathway, and random migration, whereas the other involves FAK, p130(Cas), more extensive actin cytoskeletal organization, focal contacts, and directionally persistent cell motility. Integration of these pathways provides an intracellular mechanism for regulating the speed and the directionality of cell migration.     

Nectin-like molecule-5/Tage4 enhances cell migration in an integrin-dependent, Nectin-3-independent manner

Cell migration plays roles in invasion of transformed cells and scattering of embryonic mesenchymal cells into surrounding tissues. We have found that Ig-like Necl-5/Tage4 is up-regulated in NIH3T3 cells transformed by an oncogenic Ras (V12Ras-NIH3T3 cells) and heterophilically trans-interacts with a Ca(2+)-independent Ig-like cell adhesion molecule nectin-3, eventually enhancing their intercellular motility. We show here that Necl-5 furthermore enhances cell migration in a nectin-3-independent manner. Studies using L fibroblasts expressing various mutants of Necl-5, NIH3T3 cells, and V12Ras-NIH3T3 cells have revealed that Necl-5 enhances serum- and platelet-derived growth factor-induced cell migration. The extracellular region of Necl-5 is necessary for directional cell migration, but not for random cell motility. The cytoplasmic region of Necl-5 is necessary for both directional and random cell movement. Necl-5 colocalizes with integrin alpha(V)beta(3) at leading edges of migrating cells. Analyses using an inhibitor or an activator of integrin alpha(V)beta(3) or a dominant negative mutant of Necl-5 have shown the functional association of Necl-5 with integrin alpha(V)beta(3) in cell motility. Cdc42 and Rac small G proteins are activated by the action of Necl-5 and required for the serum-induced, Necl-5-enhanced cell motility. These results indicate that Necl-5 regulates serum- and platelet-derived growth factor-induced cell migration in an integrin-dependent, nectin-3-independent manner, when cells do not contact other cells. We furthermore show here that enhanced motility and metastasis of V12Ras-NIH3T3 cells are at least partly the result of up-regulated Necl-5.     

Cell migration or cytokinesis and proliferation? – Revisiting the “go or grow” hypothesis in cancer cells in vitro

The mortality of patients with solid tumors is mostly due to metastasis that relies on the interplay between migration and proliferation. The “go or grow” hypothesis postulates that migration and proliferation spatiotemporally excludes each other.We evaluated this hypothesis on 35 cell lines (12 mesothelioma, 13 melanoma and 10 lung cancer) on both the individual cell and population levels. Following three-day-long videomicroscopy, migration, proliferation and cytokinesis-length were quantified. We found a significantly higher migration in mesothelioma cells compared to melanoma and lung cancer while tumor types did not differ in mean proliferation or duration of cytokinesis. Strikingly, we found in melanoma and lung cancer a significant positive correlation between mean proliferation and migration. Furthermore, non-dividing melanoma and lung cancer cells displayed slower migration. In contrast, in mesothelioma there were no such correlations. Interestingly, negative correlation was found between cytokinesis-length and migration in melanoma. FAK activation was higher in melanoma cells with high motility.We demonstrate that the cancer cells studied do not defer proliferation for migration. Of note, tumor cells from various organ systems may differently regulate migration and proliferation. Furthermore, our data is in line with the observation of pathologists that highly proliferative tumors are often highly invasive.     

Migration inhibition of an epithelial cell line by s-Con A and the effect on its invasiveness

In an attempt to delineate the role of tumor-cell motility in the process of invasion, we compared the migration of NBT II in a two-dimensional migration assay with its migration in a three-dimensional invasion assay. Both systems were maintained with and without succinylated concanavalin A (s-Con A) dissolved in the culture medium. This lectin has a reversible inhibitory effect on the migration of cells in vitro. The migration of NBT-II aggregates, seeded in flasks containing 200 micrograms/ml s-Con A or without s-Con A, was studied by time-lapse photomicrography. In the presence of s-Con A, migration was immediately stopped. When the treated medium was replaced by culture medium to which 10 mM alpha-methyl-mannose was added, the inhibition of migration was abolished. The invasive capacity of NBT II in the presence or absence of s-Con A was studied by confronting precultured fragments of 9- to 11-day-old embryonic chick heart (0.4 mm in diameter) with NBT-II aggregates (0.2 mm) made in the presence or absence of s-Con A. Light microscopy showed no difference in the extent of invasion. To demonstrate the presence of s-Con A in the invading tumor cells, immunoperoxidase staining for Con A was done. The treated cultures stained positively while the controls were negative. The data presented here question the correlation between tumor-cell motility in two-dimensional system and the invasive behavior of these cells in three dimensions, and implies that the ability or inability of cells to migrate on plastic does not necessarily reflect their invasiveness in vitro.     

Plectin deficiency in liver cancer cells promotes cell migration and sensitivity to sorafenib treatment

Plectin involved in activation of kinases in cell signaling pathway and plays important role in cell morphology and migration. Plectin knockdown promotes cell migration by activating focal adhesion kinase and Rac1-GTPase activity in liver cells. Sorafenib is a multi-targeting tyrosine kinase inhibitor that improves patient survival on hepatocellular carcinoma. The aim of this study is to investigate the correlation between the expression of plectin and cell migration as well as the sensitivity of hepatoma cell lines exposing to sorafenib. Hepatoma cell lines PLC/PRF/5 and HepG2 were used to examine the level of plectin expression and cell migration in comparison with Chang liver cell line. In addition, sensitivity of the 3 cell lines to sorafenib treatment was also measured. Expression of plectin was lower in PLC/PRF/5 and HepG2 hepatoma cells than that of Chang liver cells whereas HepG2 and PLC/PRF/5 cells exhibit higher rate of cell migration in trans-well migration assay. Immunohistofluorecent staining on E-cadherin revealed the highest rate of collective cell migration in HepG2 cells and the lowest was found in Chang liver cells. Likewise, HepG2 cell line was most sensitive to sorafenib treatment and Chang liver cells exhibited the least sensitivity. The drug sensitivity to sorafenib treatment showed inverse correlation with the expression of plectin. We suggest that plectin deficiency and increased E-cadherin in hepatoma cells were associated with higher rates of cell motility, collective cell migration as well as higher drug sensitivity to sorafenib treatment.     

Interplay between shear stress and adhesion on neutrophil locomotion

Leukocyte locomotion over the lumen of inflamed endothelial cells is a critical step, following firm adhesion, in the inflammatory response. Once firmly adherent, the cell will spread and will either undergo diapedesis through individual vascular endothelial cells or will migrate to tight junctions before extravasating to the site of injury or infection. Little is known about the mechanisms of neutrophil spreading or locomotion, or how motility is affected by the physical environment. We performed a systematic study to investigate the effect of the type of adhesive ligand and shear stress on neutrophil motility by employing a parallel-plate flow chamber with reconstituted protein surfaces of E-selectin, E-selectin/PECAM-1, and E-selectin/ICAM-1. We find that the level and type of adhesive ligand and the shear rate are intertwined in affecting several metrics of migration, such as the migration velocity, random motility, index of migration, and the percentage of cells moving in the direction of flow. On surfaces with high levels of PECAM-1, there is a near doubling in random motility at a shear rate of 180 s(-1) compared to the motility in the absence of flow. On surfaces with ICAM-1, neutrophil random motility exhibits a weaker response to shear rate, decreasing slightly when shear rate is increased from static conditions to 180 s(-1), and is only slightly higher at 1000 s(-1) than in the absence of flow. The random motility increases with increasing surface concentrations of E-selectin and PECAM-1 under static and flow conditions. Our findings illustrate that the endothelium may regulate neutrophil migration in postcapillary venules through the presentation of various adhesion ligands at sites of inflammation.     

Stimulation of endothelial cell migration in culture by ladsin,a laminin-5-like cell adhesion protein

Summary Ladsin is a laminin-like cell-adhesive scatter factor with potent cell motility-stimulating ability and was purified from serum-free conditioned medium of a malignant human gastric adenocarcinoma cell line STKM-1. To test its possible role in tumor angiogenesis, we investigated its effect on primary culture of endothelial cells (human umbilical vein endothelial cells) and endothelial cell line ECV304 in this study. Cell adhesion and motility effects of ladsin were observed in both types of endothelial cells. In cell-attachment assay, ladsin interacted with integrin α3β1 that was expressed on the endothelial cell surface. In Boyden chambers, ladsin stimulated both directed and random migration of ECV304 cells. Ladsin induced repair of artificial wounds generated in ECV304 cell monolayers by stimulating cell migration. Ladsin did not affect the growth rate of ECV304 cells at a low cell density but significantly increased the saturation cell density. These results suggest that ladsin may be involved in the adhesion and migration of endothelial cells under some physiological and pathological conditions.     

Real-Time Motion Analysis Reveals Cell Directionality as an Indicator of Breast Cancer Progression

Cancer cells alter their migratory properties during tumor progression to invade surrounding tissues and metastasize to distant sites. However, it remains unclear how migratory behaviors differ between tumor cells of different malignancy and whether these migratory behaviors can be utilized to assess the malignant potential of tumor cells. Here, we analyzed the migratory behaviors of cell lines representing different stages of breast cancer progression using conventional migration assays or time-lapse imaging and particle image velocimetry (PIV) to capture migration dynamics. We find that the number of migrating cells in transwell assays, and the distance and speed of migration in unconstrained 2D assays, show no correlation with malignant potential. However, the directionality of cell motion during 2D migration nicely distinguishes benign and tumorigenic cell lines, with tumorigenic cell lines harboring less directed, more random motion. Furthermore, the migratory behaviors of epithelial sheets observed under basal conditions and in response to stimulation with epidermal growth factor (EGF) or lysophosphatitic acid (LPA) are distinct for each cell line with regard to cell speed, directionality, and spatiotemporal motion patterns. Surprisingly, treatment with LPA promotes a more cohesive, directional sheet movement in lung colony forming MCF10CA1a cells compared to basal conditions or EGF stimulation, implying that the LPA signaling pathway may alter the invasive potential of MCF10CA1a cells. Together, our findings identify cell directionality as a promising indicator for assessing the tumorigenic potential of breast cancer cell lines and show that LPA induces more cohesive motility in a subset of metastatic breast cancer cells.