Estrogen promotes reversible epithelial-to-mesenchymal-like transition and collective motility in MCF-7 breast cancer cells

The role of estrogen in the motility and invasion of breast cancer cells is controversial. Although estrogen receptor (ER)-positive breast tumors are considered less aggressive and more differentiated they still undergo metastasis. In many types of epithelial cancers, the ability to undergo metastasis has been associated with a loss of epithelial features and acquisition of mesenchymal properties leading to migration of individual cells, a process known as epithelial-to-mesenchymal transition (EMT). In this report, we show that a subset of ER-positive breast cancer cells can acquire mesenchymal-like features and motility in a reversible manner. In MCF-7 breast cancer cells estrogen-promoted acquisition of mesenchymal-like features while antiestrogens, such as tamoxifen, prevented this transition. Moreover, pharmacological inhibition of Src family kinases decreased the ability of estrogen to promote epithelial-to-mesenchymal-like transition. In addition to mesenchymal-like motility, a subset of estrogen-treated cells also moved as cell clusters (collective motility). While membrane localization of E-cadherin/beta-catenin was decreased in fibroblast-like cells, enhanced levels of E-cadherin/beta-catenin were detected in motile cell clusters. Thus, during tumor progression, estrogen may foster motility and invasion of ER-positive breast cancer by promoting simultaneously reversible EMT-like changes and collective motility. These studies suggest that antiestrogen therapy and Src family kinase inhibitors may decrease development of metastases in ER-positive breast cancer by blocking estrogen-dependent migration of human breast cancer cells.     

miR-449a对人乳腺癌细胞MCF-7增殖及迁移能力的影响

目的：通过敲低微小RNA （microRNA,miRNA）-449a的方法研究miR-449a对人乳腺癌细胞MCF-7的增殖和迁移能力的影响。方法：采用miRNA芯片在乳腺癌细胞MCF-7和人正常乳腺细胞MCF-10A筛选具有表达差异的miRNA;化学合成法制备miR-449a的抑制剂（inhibitor）,转染后经real-time PCR验证表达的变化;细胞增殖CCK-8实验对转染后细胞增殖能力进行检测;划痕实验检测细胞转移能力,transwell小室实验检测细胞侵袭的改变;蛋白免疫印迹法（Western blot）实验对MCF-7细胞增殖和迁移相关的β-catenin和E-cadherin蛋白进行检测;通过生物信息学软件预测miR-449a潜在靶基因为Notch 1,荧光素酶实验检测Notch 1是miR-449a的靶基因。结果：分别收集MCF-7和MCF-10A细胞,芯片结果显示miR-449a在MCF-7细胞的表达水平显著高于MCF-10A;本研究将细胞分为未处理组（Mock组）,阴性对照组（negative control组,NC组）和处理组,通过收集不同组MCF-7细胞进行试验,CCK-8结果显示miR-449a下调后MCF-7细胞增殖能力显著降低;划痕实验结果显示miR-449a表达降低导致MCF-7细胞转移能力降低;transwell实验结果显示MCF-7细胞侵袭受到抑制;Western blot结果发现miR-449a敲低后β-catenin表达降低,E-cadherin表达增加;荧光素酶试验结果显示,miR-449a能够显著降低Notch 1-3'-UTR质粒的荧光素活性（P<0.01）。结论：在乳腺癌细胞MCF-7中敲低miR-449a能够显著抑制癌细胞增殖和迁移,而这一变化可能通过降低Notch 1蛋白表达实现的。     

Down-regulation of TSG101 by small interfering RNA inhibits the proliferation of breast cancer cells through the MAPK/ERK signal pathway

We designed to investigate the effects of down-regulating the tumor susceptibility gene 101 (TSG101) on the proliferation and apoptosis of the human breast cancer MCF-7 cell line, and the role of the MAPK/ERK signal pathway in this process. The siRNA against TSG101 was transfected into the breast cancer MCF-7 cell line using Lipofectamine 2000. After TSG101 knockdown, the proliferation of MCF-7 cells was measured by the MTT assay. The cell cycle distribution and apoptosis were examined by using flow cytometry while cell migration was measured using a transwell assay. The protein level of p-ERK was further assessed by immunofluorescence and western blotting. Our results are as following, the MCF-7 cells transfected with TSG101 siRNA proliferated significantly slower and exhibited significantly increased rates of apoptosis compared to the control cells. In the TSG101 siRNA transfected cells, the percentage of cells in the G?/G? and S phase of the cell cycle was significantly higher and lower, respectively, compared to the control cells. Moreover, the migration ability of TSG101 siRNA transfected cells was lower than the control groups. Lastly, the level of p-ERK protein in TSG101 siRNA transfected cells was significantly decreased compared with the control cells. In conclusion, TSG101 knockdown in breast cancer cells induces apoptosis and inhibits proliferation. The TSG101 depleted cells are arrested at the G?/S transition of the cell cycle. The migration of breast cancer cells is also impaired by TSG101 siRNA. TSG101 may play a biological role through modulation of the MAPK/ERK signaling pathway in breast cancer.     

RNA甲基化酶WTAP对乳腺癌MCF-7和耐阿霉素MCF-7/ADR细胞迁移的影响

为了深入探讨乳腺癌治疗耐药的发生机制并寻找乳腺癌治疗耐药的潜在治疗手段,我们以乳腺癌MCF-7和耐阿霉素MCF-7/ADR细胞为研究对象,研究RNA甲基化酶WTAP对其迁移的影响。MTT试验发现阿霉素对MCF-7细胞活力的抑制作用大于对MCF-7/ADR细胞的抑制作用。qPCR和western-blot试验发现WTAP在耐阿霉素细胞MCF-7/ADR中高表达,同时transwell试验发现在MCF-7细胞中增加WTAP的表达对MCF-7细胞的迁移没有影响,而在MCF-7/ADR细胞中敲低WTAP的表达会抑制MCF-7/ADR细胞的迁移。western-blot试验进一步证明了这一作用是通过抑制上皮间质转化(EMT)来发挥的。这一发现有助于为乳腺癌的临床治疗提供新的理论依据并为乳腺癌的治疗提供新的策略。     

Cathepsin G-induced malignant progression of MCF-7 cells involves suppression of PAF signaling through induced expression of PAFAH1B2

Breast cancer is primarily classified into ductal and lobular types, as well as into noninvasive and invasive cancer. Invasive cancer involves lymphatic and hematogenous metastasis. In breast cancer patients with distant metastases, a neutrophil-derived serine protease; cathepsin G (Cat G), is highly expressed in breast cancer cells. Cat G induces cell migration and multicellular aggregation of MCF-7 human breast cancer cells; however, the mechanism is not clear. Recently, platelet-activating factor (PAF)-acetylhydrolase (PAF-AH), the enzyme responsible for PAF degradation, was reported to be overexpressed in some tumor types, including pancreatic and breast cancers. In this study, we investigated whether PAF-AH is involved in Cat G-induced aggregation and migration of MCF-7 cells. We first showed that Cat G increased PAF-AH activity and elevated PAFAH1B2 expression in MCF-7 cells. The elevated expression of PAFAH1B2 was also observed in human breast cancer tissue specimens by immunohistochemical analysis. Furthermore, knockdown of PAFAH1B2 in MCF-7 cells suppressed the cell migration and aggregation induced by low concentrations, but not high concentrations, of Cat G. Carbamoyl PAF (cPAF), a nonhydrolyzable PAF analog, completely suppressed Cat G-induced migration of MCF-7 cells. In addition, PAF receptor (PAFR) inhibition induced cell migration of MCF-7 cells even in the absence of Cat G, suggesting that Cat G suppresses the activation of PAFR through enhanced PAF degradation due to elevated expression of PAFAH1B2 and thereby induces malignant phenotypes in MCF-7 cells. Our findings may lead to a novel therapeutic modality for treating breast cancer by modulating the activity of Cat G/PAF signaling.     

DKK-1通过上调nm23表达抑制乳腺癌细胞迁移

Dickkopf-1(DKK-1)作为Wnt/β-连环蛋白(Wnt/β-catenin)经典信号传导通路的拮抗剂而受到关注.为了进一步阐明DKK-1在乳腺癌细胞迁移中的作用及其分子机制,应用我们建立的乳腺癌细胞MCF-7高转移倾向亚克隆LM-MCF-7细胞株,比较了DKK-1在不同转移能力的乳腺癌细胞株中表达水平及其与细胞迁移能力的关系.结果显示,DKK-1在LM-MCF-7细胞中表达明显下调;"伤口愈合"实验结果表明,在MCF-7细胞中,RNA干扰DKK-1可导致细胞迁移能力增强;相反,在LM-MCF-7细胞中过表达DKK-1则可抑制细胞的迁移.进一步研究结果显示,DKK-1为肿瘤转移抑制因子nm23的上游激活因子.因此,我们的研究结果表明,DKK-1表达水平下调导致nm23表达水平下调,解除了对乳腺癌细胞迁移的抑制作用,是LM-MCF-7乳腺癌细胞具有高迁移能力的原因之一;反之,与LM-MCF-7相比,DKK-1在MCF-7细胞中高表达,其通过上调nm23可抑制乳腺癌细胞迁移.这一发现对进一步揭示乳腺癌细胞转移的分子机制具有的重要意义.     

Effect of fetal bovine serum on erythropoietin receptor expression and viability of breast cancer cells

Erythropoietin receptors (EPORs) are present not only in erythrocyte precursors but also in non-hematopoietic cells including cancer cells. In this study, we determined the effect of fetal bovine serum (FBS) in culture medium on the EPOR expression and viability of the estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells. Using flow cytometry, we showed that the inclusion of 10% FBS in the medium increased the EPOR expressions and viabilities of MDA-MB-231 and MCF-7 cells. The MDA-MB-231 showed greater EPOR expression than MCF-7 cells, suggesting that the presence of ERs on cells is associated with poor expression of EPOR. Culture medium containing 10% FBS also caused increased number of breast cancer cells entering the synthesis phase of the cell cycle. The study also showed that rHuEPO treatment did not affect viability of breast cancer cells. In conclusion, it was shown that the inclusion of FBS in culture medium increased expression of EPOR in breast cancer cells and rHuEPO treatment had no effect on the proliferation of these cancer cells.     

Erythropoietin promotes MCF-7 breast cancer cell migration by an ERK/mitogen-activated protein kinase-dependent pathway and is primarily responsible for the increase in migration observed in hypoxia

Recent studies indicate that cancer cells express erythropoietin receptor (EpoR). In this study, we have shown that erythropoietin (Epo) activates the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), and promotes migration in MCF-7 breast cancer cells. Epo-stimulated MCF-7 cell migration was blocked by the MEK inhibitor PD098059 and by dominant negative MEK-1, indicating an essential role for ERK. When MCF-7 cells were exposed to hypoxia (1.0% O(2)) for 3 h, the Epo mRNA level increased 2.4 +/- 0.5-fold, the basal level of ERK activation increased, and cell migration increased 2.0 +/- 0.1-fold. Soluble EpoR and Epo-neutralizing antibody significantly inhibited hypoxia-induced MCF-7 cell migration, suggesting a major role for autocrine EpoR cell signaling. MCF-7 cell migration under hypoxic conditions was also inhibited by PD098059. These experiments identify a novel pathway by which exogenously administered Epo, and Epo that is produced locally by cancer cells under hypoxic conditions, may stimulate cancer cell migration.     

Lysophosphatidic acid regulates the motility of MCF10CA1a breast cancer cell sheets via two opposing signaling pathways

Aberrant cell migration leads to the dispersal of malignant cells. The ubiquitous lipid mediator lysophosphatidic acid (LPA) modulates cell migration and is implicated in tumor progression. Yet, the signaling cascades that regulate LPA's effect on cell motility remain unclear. Using time-lapse imaging and quantitative analyses, we studied the role of signaling cascades that act downstream of LPA on the motility of MCF10CA1a breast cancer cells. We found that LPA alters cell motility via two major signaling pathways. The Rho/ROCK signaling cascade is the predominant pathway that increases E-Cadherin containing cell–cell adhesions and cortical arrangement of actomyosin to promote slow, directional, spatially coherent and temporally consistent movement. In contrast, Gα_i/o- and Gα_q/11-dependent signaling cascades lessen directionality and support the independent movement of cells. The net effect of LPA on breast cancer cell migration therefore results from the integrated signaling activity of the Rho/ROCK and Gα_i/o- and Gα_q/11-dependent pathways, thus allowing for a dynamic migratory response to changes in the cellular or microenvironmental context.     

Role of PTHrp and PTHrp-engaged pathways in MCF-7 cells migration/invasion.

In this paper the effect of N-terminal parathyroid hormone-related protein (PTHrp) and PTHrp-engaged pathways on MCF-7 breast cancer cell migration/invasivity and matrix metalloproteinases (MMPs) production were investigated. We found that: a) migration is not affected by PTHrp and Forskolin (FK)-activated PKA, while Phorbol Myristate Acetate (PMA)-activated PKC strongly stimulates MCF-7 cells motility. b) MMPs production was unaffected by PTHrp, but FK reduced membrane-type (MT)-1 MMP expression. Conversely, PMA induced a marked increase of MT1-MMP and MMP-9. c) Chemical activation of PKC is not sufficient, by itself, to confer invasive ability to MCF-7 cells, unless they were provided with additional factors, supplied by fibroblasts. d) Matrix invasion likely occurs through an activation cascade, involving at least three components: pro-MMP-9 and MT-1 MMP (supplied by PMA-stimulated MCF-7 cells) and pro MMP-2 (supplied by fibroblasts). e) The selective chemical inhibition of the adenylylciclase (AC)/PKA and phospholipase C (PLC)/PKC pathways confirmed that MCF-7 cells invasivity is not affected by exogenous PTHrp, which can only modulate their growth. However, the PTHrp responsibility in breast cancer invasion cannot be completely excluded. Indeed, fibroblasts are known to respond to PTHrp (which is a normal product of MCF-7 as well as other breast cancer cells) with enhanced release of MMP-2. On the basis of the documented requirement of fibroblast-derived MMP-2 for MCF-7 cell invasivity, a novel humoral fibroblast-breast cancer cell interaction, mediated by PTHrp, can be recognised.     

Distinct mechanisms mediate the initial and sustained phases of cell migration in epidermal growth factor receptor-overexpressing cells

Elevated levels of epidermal growth factor receptor (EGFR) are predictive of increased invasion and metastasis in many human cancers. In the present study, we have shown that two distinct pathways regulate cell migration in EGFR-overexpressing invasive cells such as MDA 468 breast cancer cells: mitogen-activated protein kinase (MAPK or ERK 1 and 2) pathways play a major role in early stages to cell migration; and protein kinase C delta isoforms (PKC-delta) play a significant role in later stages of sustained cell migration. Inhibition of MAPK activity with MAP kinase kinase (MEK) inhibitor PD98059 blocks early stages of cell migration (up to 4 h); however, cells revert back to enhanced cell migration after 4 h. While inhibition of PKC-delta activity with rottlerin or dominant-negative PKC-delta expression blocks sustained cell migration after 4 h and up to 12 h, the combination of MAPK and PKC inhibitors completely blocked transforming growth factor alpha (TGF-alpha)-induced cell migration in EGFR-overexpressing breast cancer cells. However, inhibition of MAPK activity completely blocked cell migration in low EGFR-expressing non-invasive breast cancer cells such as MCF-7 cells. Forced overexpression of EGFR in MCF-7 cells (EGFR/MCF-7 cells) resulted in cell migration patterns seen in MDA 468 cells, that is, MAPK pathways play a major role in early stages to cell migration, and PKC-delta plays a major role in later stages of sustained cell migration. The above data demonstrate that EGFR-overexpressing invasive cells have the ability to compensate the loss of MAPK-mediated signaling through activation of PKC-delta signaling for cell migration, which plays a major role in invasion and metastasis. In addition, data suggest that inhibition of MAPK and PKC-delta signaling pathways should abrogate cell migration and invasion in EGFR-overexpressing human breast cancer cells.     

Pyruvate Carboxylase Is Up-Regulated in Breast Cancer and Essential to Support Growth and Invasion of MDA-MB-231 Cells

Pyruvate carboxylase (PC) is an anaplerotic enzyme that catalyzes the carboxylation of pyruvate to oxaloacetate, which is crucial for replenishing tricarboxylic acid cycle intermediates when they are used for biosynthetic purposes. We examined the expression of PC by immunohistochemistry of paraffin-embedded breast tissue sections of 57 breast cancer patients with different stages of cancer progression. PC was expressed in the cancerous areas of breast tissue at higher levels than in the non-cancerous areas. We also found statistical association between the levels of PC expression and tumor size and tumor stage (P < 0.05). The involvement of PC with these two parameters was further studied in four breast cancer cell lines with different metastatic potentials; i.e., MCF-7, SKBR3 (low metastasis), MDA-MB-435 (moderate metastasis) and MDA-MB-231 (high metastasis). The abundance of both PC mRNA and protein in MDA-MB-231 and MDA-MB-435 cells was 2-3-fold higher than that in MCF-7 and SKBR3 cells. siRNA-mediated knockdown of PC expression in MDA-MB-231 and MDA-MB-435 cells resulted in a 50% reduction of cell proliferation, migration and in vitro invasion ability, under both glutamine-dependent and glutamine-depleted conditions. Overexpression of PC in MCF-7 cells resulted in a 2-fold increase in their proliferation rate, migration and invasion abilities. Taken together the above results suggest that anaplerosis via PC is important for breast cancer cells to support their growth and motility.     

Biological Screening of Polyphenol Derivatives for Anti-Proliferative,Anti-Apoptotic and Anti-Migrative Activities in Human Breast Cancer Cell Lines MCF-7

Breast cancer is known as the most common type of invasive cancer in women. It is well-known that phenolic compounds play an important role in the treatment of this disease. This study hypothesized that isoeugenol based two polyphenolic compounds 1 and 2 exerts its anti-proliferative effects through the induction of apoptosis and cell migration arrest on human breast cancer cell. Based on this hypothesis, the study aimed to investigate the anti-proliferative, anti-migrative effects of these compounds and their possible basic molecular mechanisms of action in MCF-7 cell lines. As a result, isoeugenol-based compounds 1 and 2 showed anti-proliferative, anti-apoptotic and anti-migrative effects in MCF-7 breast cancer cells. This result was supported by molecular analyzes and it was determined that there were changes in the expression of some gene regions involved in apoptosis and migration. Additionally, it was a remarkable result that cell viability inhibition did not occur in healthy breast tissue cells and no cytotoxic effect was observed. The existence of such a differentiation between cancer cells and healthy cells significantly increases the potential of these compounds to be used as chemotherapeutic drug active ingredients without side effects.     

Epithelial-Stromal Interactions in Human Breast Cancer: Effects on Adhesion,Plasma Membrane Fluidity and Migration Speed and Directness

Interactions occurring between malignant cells and the stromal microenvironment heavily influence tumor progression. We investigated whether this cross-talk affects some molecular and functional aspects specifically correlated with the invasive phenotype of breast tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration) by co-culturing mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer-associated fibroblasts, CAFs) in 2D or 3D (nodules) cultures. Confocal immunofluorescence analysis of the epithelial adhesion molecule E-cadherin on frozen nodule sections demonstrated that NFs and CAFs, respectively, induced or inhibited its expression in MCF-7 cells. An increase in the mesenchymal adhesion protein N-cadherin was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of cancer cells. CAFs, in turn, promoted N-cadherin up-regulation in MDA-MB-231 cells and its de novo expression in MCF-7 cells. Beyond promotion of “cadherin switching”, another sign of the CAF-triggered epithelial-mesenchymal transition (EMT) was the induction of vimentin expression in MCF-7 cells. Plasma membrane labeling of monolayer cultures with the fluorescent probe Laurdan showed an enhancement of the membrane fluidity in cancer cells co-cultured with NFs or CAFs. An increase in lipid packing density of fibroblast membranes was promoted by MCF-7 cells. Time-lapsed cell tracking analysis of mammary cancer cells co-cultured with NFs or CAFs revealed an enhancement of tumor cell migration velocity, even with a marked increase in the directness induced by CAFs.Our results demonstrate a reciprocal influence of mammary cancer and fibroblasts on various adhesiveness/invasiveness features. Notably, CAFs'' ability to promote EMT, reduction of cell adhesion, increase in membrane fluidity, and migration velocity and directness in mammary cancer cells can be viewed as an overall progression- and invasion-promoting effect.     

Fibroblast growth factor 8 increases breast cancer cell growth by promoting cell cycle progression and by protecting against cell death

Fibroblast growth factor 8 (FGF-8) is expressed in a large proportion of breast cancers, whereas its level in normal mammary gland epithelium is low. Previous studies have shown that FGF-8b stimulates breast cancer cell growth in vitro and in vivo. To explore the mechanisms by which FGF-8b promotes growth, we studied its effects on cell cycle regulatory proteins and signalling pathways in mouse S115 and human MCF-7 breast cancer cells. We also studied the effect of FGF-8b on cell survival. FGF-8b induced cell cycle progression and up-regulated particularly cyclin D1 mRNA and protein in S115 cells. Silencing cyclin D1 with siRNA inhibited most but not all FGF-8b-induced proliferation. Inhibition of the FGF-8b-activated ERK/MAPK pathway decreased FGF-8b-stimulated proliferation. Blocking the constitutively active PI3K/Akt and p38 MAPK pathways also lowered FGF-8b-induced cyclin D1 expression and proliferation. Corresponding results were obtained in MCF-7 cells. In S115 and MCF-7 mouse tumours, FGF-8b increased cyclin D1 and Ki67 levels. Moreover, FGF-8b opposed staurosporine-induced S115 cell death which effect was blocked by inhibiting the PI3K/Akt pathway but not the ERK/MAPK pathway. In conclusion, our results suggest that FGF-8b increases breast cancer cell growth both by stimulating cell cycle progression and by protecting against cell death.     

Rab35 is required for Wnt5a/Dvl2-induced Rac1 activation and cell migration in MCF-7 breast cancer cells

The small GTPases regulate many major biological processes in both tumorigenesis and tumor progression such as cell survival, actin cytoskeleton organization, cell polarity and movement. Wnt5a, a non-canonical Wnt family member, is implicated in the activation of small GTPases in breast cancer. We previously demonstrated that Wnt5a signaling stimulates the migration of breast cancer cells MDA-MB-231 via activating RhoA. However, we found here that RhoA activation was not enhanced by Wnt5a in breast cancer cells MCF-7. The conflicting results prompted us to further probe novel small GTPases in response to Wnt5a and investigate the mechanisms whereby cell migration is regulated. We showed here that Wnt5a dose dependently activated Dvl2, Rab35 and Rac1 and subsequently promoted the migration of MCF-7 cells, which was, however, abolished by knocking down Wnt5a expression via small interfering RNA (siRNA) transfection. Dvl2 siRNA significantly decreased background and Wnt5a-induced Rab35/Rac1 activation and, consequently, cell migration. Rab35 short hairpin RNA (shRNA) remarkably inhibited background and Wnt5a-induced Rac1 activation and cell migration. Additionally, blockade of Rac1 activation with Rac1 siRNA suppressed background and Wnt5a-induced cell migration. Co-immunoprecipitation and immunofluorescence assays showed that Dvl2 bound to Rab35 in mammalian cells. Taken together, we demonstrated that Wnt5a promotes breast cancer cell migration via the Dvl2/Rab35/Rac1 signaling pathway. These findings implicate Wnt5a signaling in regulating small GTPases, which could be targeted for manipulating breast cancer cell migration.     

Automated time-lapse microscopy and high-resolution tracking of cell migration

We describe a novel fully automated high-throughput time-lapse microscopy system and evaluate its performance for precisely tracking the motility of several glioma and osteoblastic cell lines. Use of this system revealed cell motility behavior not discernable with conventional techniques by collecting data (1) from closely spaced time points (minutes), (2) over long periods (hours to days), (3) from multiple areas of interest, (4) in parallel under several different experimental conditions. Quantitation of true individual and average cell velocity and path length was obtained with high spatial and temporal resolution in “scratch” or “wound healing” assays. This revealed unique motility dynamics of drug-treated and adhesion molecule-transfected cells and, thus, this is a considerable improvement over current methods of measurement and analysis. Several fluorescent vital labeling methods commonly used for end-point analyses (GFP expression, DiO lipophilic dye, and Qtracker nanocrystals) were found to be useful for time-lapse studies under specific conditions that are described. To illustrate one application, fluorescently labeled tumor cells were seeded onto cell monolayers expressing ectopic adhesion molecules, and this resulted in consistently reduced tumor cell migration velocities. These highly quantitative time-lapse analysis methods will promote the creation of new cell motility assays and increase the resolution and accuracy of existing assays.Joseph S. Fotos and Vivek P. Patel contributed equally to this work