Cell migration and invasion assays

The processes of cell migration and invasion are integral to embryonic development and the functioning of adult organisms. Deregulation of these processes contributes to numerous diseases. Ras GTPases and in particular members of the Rho subfamily of GTPases play critical roles in cell migration and invasion. Here, we provide a collection of protocols to assay these functions. We describe two cell migration assays. The monolayer wound healing assay is very easy to implement, whereas the microliter-scale migration assay allows examination of cell behavior on defined extracellular matrices. We also describe two methods that allow the quantification of tumor cell invasion, a versatile transwell Matrigel invasion assay and an organotypic assay that examines the invasion of glioma cells through a rat brain slice.     

An introduction to the wound healing assay using live-cell microscopy

Abstract

The wound healing assay is used in a range of disciplines to study the coordinated movement of a cell population. In this technical review, we describe the workflow of the wound healing assay as monitored by optical microscopy. Although the assay is straightforward, a lack of standardization in its application makes it difficult to compare results and reproduce experiments among researchers. We recommend general guidelines for consistency, including: (1) sample preparation including the creation of the gap, (2) microscope equipment requirements, (3) image acquisition, and (4) the use of image analysis to measure the gap size and its rate of closure over time. We also describe parameters that are specific to the particular research question, such as seeding density and matrix coatings. All of these parameters must be carefully controlled within a given set of experiments in order to achieve accurate and reproducible results.     

长链非编码RNA Dleu2 对宫颈癌细胞增殖、迁移及侵袭的影响

目的:研究长链非编码RNA(long non-coding RNA,lnc RNA)Dleu2对宫颈癌细胞增殖、迁移和侵袭能力的影响。方法:利用高通量Lnc RNA芯片技术检测10例宫颈癌组织及对应的癌旁组织,筛选得到一批表达水平具有显著差异的Lnc RNA,进一步针对可能具有生物学功能的Lnc RNA-Dleu2,利用q-PCR验证其在癌组织样本中的相对低表达。再通过在细胞内过表达Lnc RNA-Dleu2研究其对宫颈癌Hela和Caski细胞系增殖、迁移和侵袭的影响。结果:q-PCR结果验证了Lnc RNA芯片筛选的结果,即相较于癌旁组织和正常宫颈上皮细胞系,Lnc RNA-Dleu2在宫颈癌组织和细胞中均低表达。CCK8和克隆形成实验结果显示,过表达Lnc RNA-Dleu2能显著抑制Hela和Caski细胞增殖能力(P0.01);细胞划痕实验结果显示,过表达Lnc RNA-Dleu2能显著抑制Hela和Caski细胞增殖和迁移能力;Matrigel细胞侵袭实验结果显示,过表达Lnc RNA-Dleu2能显著抑制Hela和Caski细胞的侵袭能力(P0.01)。结论:Lnc RNA-Dleu2在宫颈癌中相对低表达,提高Dleu2表达水平能够抑制宫颈癌细胞系Hela和Caski的增殖、迁移和侵袭能力。     

Single-cell tracking reveals super-spreading brain cancer cells with high persistence

Cell migration is a fundamental characteristic of vital processes such as tissue morphogenesis, wound healing and immune cell homing to lymph nodes and inflamed or infected sites. Therefore, various brain defect diseases, chronic inflammatory diseases as well as tumor formation and metastasis are associated with aberrant or absent cell migration. We embedded multicellular brain cancer spheroids in Matrigel? and utilized single-particle tracking to extract the paths of cells migrating away from the spheroids. We found that – in contrast to local invasion – single cell migration is independent of Matrigel? concentration and is characterized by high directionality and persistence. Furthermore, we identified a subpopulation of super-spreading cells with >200-fold longer persistence times than the majority of cells. These results highlight yet another aspect of cell heterogeneity in tumors.     

Jamming and arrest of cell motion in biological tissues

Collective cell motility is crucial to many biological processes including morphogenesis, wound healing, and cancer invasion. Recently, the biology and biophysics communities have begun to use the term ‘cell jamming’ to describe the collective arrest of cell motion in tissues. Although this term is widely used, the underlying mechanisms are varied. In this review, we highlight three independent mechanisms that can potentially drive arrest of cell motion — crowding, tension-driven rigidity, and reduction of fluctuations — and propose a framework that connects all three. Because multiple mechanisms may be operating simultaneously, this emphasizes that experiments should strive to identify which mechanism dominates in a given situation. We also discuss how specific cell-scale and molecular-scale biological processes, such as cell–cell and cell-substrate interactions, control aspects of these underlying physical mechanisms.     

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.     

Planar Cell Polarity Signaling in Collective Cell Movements During Morphogenesis and Disease

Collective and directed cell movements are crucial for diverse developmental processes in the animal kingdom, but they are also involved in wound repair and disease. During these processes groups of cells are oriented within the tissue plane, which is referred to as planar cell polarity (PCP). This requires a tight regulation that is in part conducted by the PCP pathway. Although this pathway was initially characterized in flies, subsequent studies in vertebrates revealed a set of conserved core factors but also effector molecules and signal modulators, which build the fundamental PCP machinery. The PCP pathway in Drosophila regulates several developmental processes involving collective cell movements such as border cell migration during oogenesis, ommatidial rotation during eye development, and embryonic dorsal closure. During vertebrate embryogenesis, PCP signaling also controls collective and directed cell movements including convergent extension during gastrulation, neural tube closure, neural crest cell migration, or heart morphogenesis. Similarly, PCP signaling is linked to processes such as wound repair, and cancer invasion and metastasis in adults. As a consequence, disruption of PCP signaling leads to pathological conditions. In this review, we will summarize recent findings about the role of PCP signaling in collective cell movements in flies and vertebrates. In addition, we will focus on how studies in Drosophila have been relevant to our understanding of the PCP molecular machinery and will describe several developmental defects and human disorders in which PCP signaling is compromised. Therefore, new discoveries about the contribution of this pathway to collective cell movements could provide new potential diagnostic and therapeutic targets for these disorders.     

A simple method for using silicone elastomer masks for quantitative analysis of cell migration without cellular damage or substrate disruption

Cell migration is fundamental to many biological processes, including development, normal tissue remodeling, wound healing, and many pathologies. However, cell migration is a complex process, and understanding its regulation in health and disease requires the ability to manipulate and measure this process quantitatively under controlled conditions. This report describes a simple in vitro assay for quantitative analysis of cell migration in two-dimensional cultures that is an inexpensive alternative to the classic “scratch” assay. The method described utilizes flexible silicone masks fabricated in the lab according to the research demands of the specific experiment to create a cell-free area for cells to invade, followed by quantitative analysis based on widely available microscopic imaging tools. This experimental approach has the important advantage of visualizing cell migration in the absence of the cellular damage and disruption of the substrate that occurs when the “wound” is created in the scratch assay. This approach allows the researcher to study the intrinsic migratory characteristics of cells in the absence of potentially confounding contributions from cellular responses to injury and disruption of cell–substrate interactions. This assay has been used with vascular smooth muscle cells, fibroblasts, and epithelial cell types, but should be applicable to the study of practically any type of cultured cell. Furthermore, this method can be easily adapted for use with fluorescence microscopy, molecular biological, or pharmacological manipulations to explore the molecular mechanisms of cell migration, live cell imaging, fluorescence microscopy, and correlative immunolabeling.     

Chk1反义寡核苷酸联合顺铂抑制卵巢癌侵袭转移的分子机制

目的:探究Chk1反义寡核苷酸(CHK1-ASODN)单独或联合顺铂(DDP)对卵巢癌细胞系SKOV-3侵袭转移能力的影响,并阐明其可能的分子机制。方法:体外培养人卵巢癌细胞系SKOV-3,CHK1-ASODN单独或联合DDP处理48 h后,划痕实验检测细胞迁移能力;Transwell实验检测细胞侵袭能力;显微镜下观察细胞上皮或间质表型特征;Western blot及实时定量PCR技术分别检测上皮间质转化(EMT)特异性标志物(E-cadherin、N-cadherin)以及EMT关键调控分子ZEB1的蛋白及m RNA的表达水平。结果:与对照组相比较,CHK1-ASODN单独或联合DDP均能显著抑制SKOV-3细胞的迁移及侵袭(P0.05);细胞表现为间质化表型;E-cadherin的表达显著升高(P0.05),而N-cadherin的表达则显著降低(P0.05);ZEB1的表达显著降低(P0.05)。结论:CHK1-ASODN单独或联合DDP下调ZEB1的表达进而逆转EMT可能是其抑制卵巢癌侵袭转移的重要机制之一。     

Microfluidic devices for studying chemotaxis and electrotaxis

Directed cell migration plays important roles in physiological processes such as host defense, wound healing, cancer metastasis and embryogenesis. Many organisms are capable of directional migration, which can be guided by diverse cellular factors including chemical and electrical cues. Recently, microfluidic devices that consist of small channels with micrometer dimensions are being developed for cell migration studies. These devices can precisely configure and flexibly manipulate chemical concentration gradients and electric fields, and thus can be used to study the complex guiding mechanisms for cell migration. In this paper we highlight recent applications of microfluidic devices for cell migration research, with a focus on electric field-directed cell migration, to provide important and timely updates of this rapidly developing research field.     

A novel functional gene selection method provides a systematic view of cell migration

Cell migration is a central process that is essential for embryonic development, wound repair, inflammatory response, homeostasis and tumor metastasis. A method of genome-wide selection based on the gain-of-function has been devised to identify novel cell migration-promoting genes in cultured cells. After the introduction of the retroviral mouse brain cDNA library into NIH3T3 mouse fibroblast cells, migration-promoted cells were selected by a three-dimensional migration assay using cell culture inserts. After five rounds of enrichment, cDNAs were retrieved from the cells that passed the selection processes. Cell migration-promoting activity was confirmed by independent migration assays for the retrieved cDNAs. Multiple cell migration-promoting genes were successfully isolated by this method. The genes identified can be used to gain a systematic view of cell migration. The gain-of-function selection method described here can be combined with RNAi-mediated loss-of-function screen or selection to be a more powerful tool for the systems biology research of cell migration.     

Development of pipette tip gap closure migration assay (s-ARU method) for studying semi-adherent cell lines

This work presents a pipette tip gap closure migration assay prototype tool (semi-adherent relative upsurge—s-ARU—method) to study cell migration or wound healing in semi-adherent cell lines, such as lymph node carcinoma of the prostate (LNCaP). Basically, it consists of a 6-well cover plate modification, where pipette tips with the filter are shortened and fixed vertically to the inner surface of the cover plate, with their heights adjusted to touch the bottom of the well center. This provides a barrier for the inoculated cells to grow on, creating a cell-free gap. Such a uniform gap formed can be used to study migration assay for both adherent as well as semi-adherent cells. After performing time studies, effective measurement of gap area can be carried out conveniently through image analysis software. Here, the prototype was tested for LNCaP cells, treated with testosterone and flutamide as well as with bacteriophages T4 and M13. A scratch assay using PC3 adherent cells was also performed for comparison. It was observed that s-ARU method is suitable for studying LNCaP cells migration assay, as observed from our results with testosterone, flutamide, and bacteriophages (T4 and M13). Our method is a low-cost handmade prototype, which can be an alternative to the other migration assay protocol(s) for both adherent and semi-adherent cell cultures in oncological research along with other biological research applications.     

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.     

Quantum dot-based cell motility assay

Motility and migration are measurable characteristics of cells that are classically associated with the invasive potential of cancer cells, but in vitro assays of invasiveness have been less than perfect. We previously developed an assay to monitor cell motility and migration using water-soluble CdSe/ZnS nanocrystals; cells engulf the fluorescent nanocrystals as they crawl across them and leave behind a fluorescent-free trail. We show here that semiconductor nanocrystals can also be used as a sensitive two-dimensional in vitro invasion assay. We used this assay to compare the behavior of seven different adherent human cell lines, including breast epithelial MCF 10A, breast tumor MDA-MB-231, MDA-MB-435S, MCF 7, colon tumor SW480, lung tumor NCI H1299, and bone tumor Saos-2, and observed two distinct behaviors of cancer cells that can be used to further categorize these cells. Some cancer cell lines demonstrate fibroblastic behaviors and leave long fluorescent-free trails as they migrate across the dish, whereas other cancer cells leave clear zones of varying sizes around their periphery. This assay uses fluorescence detection, requires no processing, and can be used in live cell studies. These features contribute to the increased sensitivity of this assay and make it a powerful new tool for discriminating between non-invasive and invasive cancer cell lines.     

Cells on the move: a dialogue between polarization and motility

Throughout evolution, both prokaryotic and eukaryotic cells have developed a variety of biochemical mechanisms to define the direction and proximity of extracellular stimuli. This process is essential for the cell to reply properly to the environmental cues that determine cell migration, proliferation, and differentiation. Chemotaxis is the cellular response to chemical attractants that direct cell migration, a process that plays a central role in many physiological situations, such as host immune responses, angiogenesis, wound healing, embryogenesis, and neuronal patterning, among others. In addition, cell migration takes part in pathological states, including inflammation and tumor metastasis. Indeed, tumor progression to invasion and metastasis depends on the active motility of the invading cancer cells and the endothelial cell bed during tumor neovascularization. Cell migration switches "off" and "on," based on quantitative differences in molecular components such as adhesion receptors, cytoskeletal linking proteins, and extracellular matrix ligands, and by regulating the affinity of membrane-bound chemoattractant receptors. A clear understanding of how cells sense chemoattractants is, therefore, of pivotal importance in the biology of the normal cell as well as in prevention of malignant cell invasion. Here we offer a perspective on cell migration that emphasizes the relationship between cell polarization and cell movement and the importance of the equilibrium between the signals that drive each process for the control of tumor cell invasion.     

重楼皂苷Ⅶ抑制肺癌H460细胞增殖和迁移能力研究

为研究重楼皂苷Ⅶ(polyphyllin Ⅶ)抑制人肺癌H460细胞增殖、迁移能力和诱导凋亡的作用和机制。本实验采用MTT法检测重楼皂苷Ⅶ处理后H460细胞生长抑制率,Hoechst 33258染色观察细胞形态,细胞集落形成实验考察细胞的增殖能力,划痕实验和Transwell小室实验研究H460细胞迁移和侵袭能力的改变,并通过western blot法检测在重楼皂苷Ⅶ处理前后细胞蛋白表达变化情况。结果发现,重楼皂苷Ⅶ可显著抑制H460细胞增殖,影响其集落形成,并使细胞形态发生变化,抑制细胞体外的迁移和侵袭能力,并可诱导凋亡的发生。重楼皂苷Ⅶ处理后,H460细胞中基质金属蛋白酶MMP-2和MMP-9显著降低,凋亡相关蛋白剪切型caspase-3、Bax表达增加,ICAD、Bcl-2表达降低,提示重楼皂苷Ⅶ体外可能通过调节基质金属蛋白酶抑制H460细胞迁移和侵袭,同时诱导凋亡的发生。     

Quantitative analysis of random migration of cells using time-lapse video microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion (1, 2). During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues (3) provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment. Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration (2). Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration. Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function (4), hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO(2) supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.     

CrkI and p130(Cas) complex regulates the migration and invasion of prostate cancer cells

Prostate cancer metastasis is often associated with poor prognosis. The molecular coupling of the adaptor protein Crk to the docking protein p130(Cas) serves as a switch that regulates cell migration in several invasive cancer cells and Ack appears to act upstream of CrkII to modulate the cell motility. However, the precise role of Ack, Crk and p130(Cas) complex in prostate cancer migration remains unknown. In this study we examined the expression of Crk and p130(Cas) in prostate cancer cell lines, and found that CrkI and p130(Cas) protein level was higher in highly invasive PC-3M and PC-3 cell lines than in moderately invasive DU-145 cells. Upon shRNA mediated knockdown of CrkI and p130(Cas) in PC-3M cells, cell migration and invasion were significantly inhibited as analyzed by wound healing assay and transwell invasion assay. Furthermore, co-immunoprecipitation assay showed that p130(Cas) interacted with CrkI in PC-3M cells and the stability of p130(Cas) and CrkI depended on each other. AckI interacted with both CrkI and p130(Cas) and the interaction of AckI with CrkI seemed to be independent of p130(Cas) . Taken together, our results demonstrate the high expression of CrkI and p130(Cas) in invasive prostate cancer cells and the important role of CrkI/p130(Cas) complex in the migration and invasion of prostate cancer cells. These data suggest that CrkI/p130(Cas) could be exploited as potential molecular therapeutic target for prostate cancer metastasis.