Analyzing the Roles of Rho GTPases in Cancer Cell Adhesion to Endothelial Cells Under Flow Conditions.

Adhesion between cancer cells and endothelial cells, lining the blood vessels, is an important event in tumor progression and metastasis formation. The expression of Rho GTPases is frequently altered in cancers, and they are known to regulate cell migration through their effects on adhesion and cytoskeletal dynamics. Several different types of assays are used to investigate how cancer cells attach to and cross the endothelium. Here, we describe an in vitro technique to study the effects of Rho GTPases on human cancer cell adhesion to endothelial cells under shear stress coupled to live cell imaging.

Probing Intravascular Adhesion and Extravasation of Tumor Cells with Microfluidics.

Cancer metastasis is a multistep process during which tumor cells leave the primary tumor mass and form distant secondary colonies that are lethal. Circulating tumor cells (CTCs) are transported by body fluids to reach distant organs, where they will extravasate and either remain dormant or form new tumor foci. Development of methods to study the behavior of CTCs at the late stages of the intravascular journey is thus required to dissect the molecular mechanisms at play. Using recently developed microfluidics approaches, we have demonstrated that CTCs arrest intravascularly, through a two-step process: (a) CTCs stop using low energy and rapidly activated adhesion receptors to form transient metastable adhesions and (b) CTCs stabilize their adhesions to the endothelial layer with high energy and slowly activated adhesion receptors. In this methods chapter, we describe these easy-to-implement quantitative methods using commercially available microfluidic channels. We detail the use of fast live imaging combined to fine-tuned perfusion to measure the adhesion potential of CTC depending on flow velocities. We document how rapidly engaged early metastable adhesion can be discriminated from slower activated stable adhesion using microfluidics. Finally, CTC extravasation potential can be assessed within this setup using long-term cell culture under flow. Altogether, this experimental pipeline can be adapted to probe the adhesion (to the endothelial layer) and extravasation potential of any circulating cell.

3D Printed Multiplexed Competitive Migration Assays with Spatially Programmable Release Sources.

Here, a 3D printed multiplexed competitive migration assay is reported for characterizing a chemotactic response in the presence of multiple spatially distributed chemoattractants. The utility of the assay is demonstrated by examining the chemotactic response of human glioblastoma cells to spatially opposing chemotactic gradients of epidermal growth factor (EGF) and bradykinin (BK). Competitive migration assays involving spatially opposing gradients of EGF and BK that are optimized in the absence of the second chemoattractant show that 46% more glioblastoma cells migrate toward EGF sources. The migration velocities of human glioblastoma cells toward EGF and BK sources are reduced by 7.6 ± 2.2% and 11.6 ± 6.3% relative to those found in the absence of the spatially opposing chemoattractant. This work provides new insight to the chemotactic response associated with glioblastoma-vasculature interactions and a versatile, user-friendly platform for characterizing the chemotactic response of cells in the presence of multiple spatially distributed chemoattractants.

In vitro humanized 3D microfluidic chip for testing personalized immunotherapeutics for head and neck cancer patients.

OBJECTIVES: Immunotherapy and personalized medicine therapeutics are emerging as promising approaches in the management of head and neck squamous cell carcinoma (HNSCC). In spite of that, there is yet no assay that could predict individual response to immunotherapy. METHODS: We manufactured an in vitro 3D microfluidic chip to test the efficacy of immunotherapy. The assay was first tested using a tongue cancer cell line (HSC-3) embedded in a human tumour-derived matrix "Myogel/fibrin" and immune cells from three healthy donors. Next, the chips were used with freshly isolated cancer cells, patients' serum and immune cells. Chips were loaded with different immune checkpoint inhibitors, PD-L1 antibody and IDO 1 inhibitor. Migration of immune cells towards cancer cells and the cancer cell proliferation rate were evaluated. RESULTS: Immune cell migration towards HSC-3 cells was cancer cell density dependent. IDO 1 inhibitor induced immune cells to migrate towards cancer cells both in HSC-3 and in two HNSCC patient samples. Efficacy of PD-L1 antibody and IDO 1 inhibitor was patient dependent. CONCLUSION: We introduced the first humanized in vitro microfluidic chip assay to test immunotherapeutic drugs against HNSCC patient samples. This assay could be used to predict the efficacy of immunotherapeutic drugs for individual patients.

Three-Dimensional Printing of Cell Exclusion Spacers (CES) for Use in Motility Assays.

PURPOSE: Cell migration/invasion assays are widely used in commercial drug discovery screening. 3D printing enables the creation of diverse geometric restrictive barrier designs for use in cell motility studies, permitting on-demand assays. Here, the utility of 3D printed cell exclusion spacers (CES) was validated as a cell motility assay. METHODS: A novel CES fit was fabricated using 3D printing and customized to the size and contour of 12 cell culture plates including 6 well plates of basal human brain vascular endothelial (D3) cell migration cells compared with 6 well plates with D3 cells challenged with 1uM cytochalasin D (Cyto-D), an F-actin anti-motility drug. Control and Cyto-D treated cells were monitored over 3 days under optical microscopy. RESULTS: Day 3 cell migration distance for untreated D3 cells was 1515.943µm ± 10.346µm compared to 356.909µm ± 38.562µm for the Cyt-D treated D3 cells (p < 0.0001). By day 3, untreated D3 cells reached confluency and completely filled the original voided spacer regions, while the Cyt-D treated D3 cells remained significantly less motile. CONCLUSIONS: Cell migration distances were significantly reduced by Cyto-D, supporting the use of 3D printing for cell exclusion assays. 3D printed CES have great potential for studying cell motility, migration/invasion, and complex multi-cell interactions.

A PDMS Device Coupled with Culture Dish for In Vitro Cell Migration Assay.

Cell migration and invasion are important factors during tumor progression and metastasis. Wound-healing assay and the Boyden chamber assay are efficient tools to investigate tumor development because both of them could be applied to measure cell migration rate. Therefore, a simple and integrated polydimethylsiloxane (PDMS) device was developed for cell migration assay, which could perform quantitative evaluation of cell migration behaviors, especially for the wound-healing assay. The integrated device was composed of three units, which included cell culture dish, PDMS chamber, and wound generation mold. The PDMS chamber was integrated with cell culture chamber and could perform six experiments under different conditions of stimuli simultaneously. To verify the function of this device, it was utilized to explore the tumor cell migration behaviors under different concentrations of fetal bovine serum (FBS) and transforming growth factor (TGF-ß) at different time points. This device has the unique capability to create the "wound" area in parallel during cell migration assay and provides a simple and efficient platform for investigating cell migration assay in biomedical application.

Studying the Role of AMPK in Angiogenesis.

The role of AMPK in angiogenesis can be studied using in vitro and in vivo assays. The endothelial spheroid assay is a robust three-dimensional in vitro test, which allows investigation of tubular morphogenesis by integrating cell-cell as well as cell-matrix interactions. The Matrigel plug assay validates the process of angiogenesis in vivo and allows studies in genetically modified mice. Here, we give a detailed description of both assays and their application in AMPK research.

Assessing the Influence of a Protease in Cell Migration Using the Barrier-Migration Assay.

Proteases play crucial roles in all steps of tumor progression including cancer cell migration. In fact, uncontrolled proteolytic activity could lead to the degradation of different components of the extracellular matrix which facilitates dissemination of tumor cells. However, numerous studies have revealed that proteases may also exert tumor-protective actions which could impede progression of malignant cells. Consequently, it is crucial to distinguish those situations in which proteases promote tumor growth from those in which exhibit tumor-suppressive effects. In this regard, analysis of the influence of a particular protease on the capacity of a cell line to migrate can be employed as an approach to better understand its involvement in tumorigenesis. Different experimental designs have been developed to investigate cell migration. Herein, we describe a barrier assay to monitor cell migration, which overcomes some disadvantages of traditional methods such as the Boyden chamber or the wound healing assays. The version of the barrier assay explained in this chapter allows to examine cell migration through the analysis of the closure of a premade 500 µm wound. This method also facilitates comparison between two different situations in a given cell line (i.e., gene up- or downregulation) in the same assay and under the same conditions. Additionally, migration can be monitored and measured using a time lapse microscope which facilitates further analysis through different softwares.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing.

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate.

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor metastasis. In this assay, cells are grown to form a confluent monolayer and a mechanical wound is created by scratching with a device. Then the migration rate of the cells towards the denuded area can be monitored by imaging. Our 8-channel mechanical wounder is designed to tackle most of the problems associated with the cell migration assay. Firstly, our wounder can be easily sterilized by autoclaving or with common disinfectants. Secondly, the individual adjustable pins allow even contact with the cell culture plate so that sharp and reproducible wounds can be created. Thirdly, the guiding bars on both sides of the wounder ensure consistent wounding position in each well. The use of disposable plastic pipette tips for wounding can further provide better handling of the wounder as well as to minimize cross-contamination. In conclusion, our cell wounder can provide researchers with a user friendly and reproducible device for performing the cell migration assay using the standard 96-well culture plate.

A novel approach to quantify the wound closure dynamic.

The Wound Healing (WH) assay is widely used to investigate cell migration in vitro, in order to reach a better understanding of many physiological and pathological phenomena. Several experimental factors, such as uneven cell density among different samples, can affect the reproducibility and reliability of this assay, leading to a discrepancy in the wound closure kinetics among data sets corresponding to the same cell sample. We observed a linear relationship between the wound closure velocity and cell density, and suggested a novel methodological approach, based on transport phenomena concepts, to overcome this source of error on the analysis of the Wound Healing assay. In particular, we propose a simple scaling of the experimental data, based on the interpretation of the wound closure as a diffusion-reaction process. We applied our methodology to the MDA-MB-231 breast cancer cells, whose motility was perturbed by silencing or over-expressing genes involved in the control of cell migration. Our methodological approach leads to a significant improvement in the reproducibility and reliability in the in vitro WH assay.

Microdroplet chain array for cell migration assays.

Establishing cell migration assays in multiple different microenvironments is important in the study of tissue repair and regeneration, cancer progression, atherosclerosis, and arthritis. In this work, we developed a miniaturized and massive parallel microfluidic platform for multiple cell migration assays combining the traditional membrane-based cell migration technique and the droplet-based microfluidic technique. Nanoliter-scale droplets are flexibly assembled as building blocks based on a porous membrane to form microdroplet chains with diverse configurations for different assay modes. Multiple operations including in-droplet 2D/3D cell culture, cell co-culture and cell migration induced by a chemoattractant concentration gradient in droplet chains could be flexibly performed with reagent consumption in the nanoliter range for each assay and an assay scale-up to 81 assays in parallel in one microchip. We have applied the present platform to multiple modes of cell migration assays including the accurate cell migration assay, competitive cell migration assay, biomimetic chemotaxis assay, and multifactor cell migration assay based on the organ-on-a-chip concept, for demonstrating its versatility, applicability, and potential in cell migration-related research.

A parallel and quantitative cell migration assay using a novel multi-well-based device.

Cell migration assays for different chemical environments are important for both scientists and clinicians searching for new therapeutics. In this study, we developed a multi-well-based microfluidic chip that has multiple units for different conditions. In each unit, cells can be patterned and then released to observe their migration. Automatic image analysis and model-based data processing were developed to describe the integrated cell migration assay precisely and quickly. As a demonstration, the migration behaviors of two types of cells in eight chemical conditions were studied. The results showed that supplementation with transforming growth factor-ß(TGF-ß) significantly promoted the migration of MCF-7 and MCF-10 A cells compared to several growth factors, such as Epidermal Growth Factor(EGF) and basic fibroblast growth factor(bFGF), as well as a control sample. Cells can migrate particularly fast with two or more mixed supplementary factors, such as TGF-ß + bFGF + EGF, which indicated a synergy effect. Thus, this chip could be used to quantitatively observe cancer cell migration and demonstrated great potential for use in quantitative migration studies and chemical screening.

[Downregulation of miR-18a or miR-328 inhibits the invasion and migration of lung adenocarcinoma A549 cells].

Objective To investigate the expressions of miR-18a and miR-328 in lung adenocarcinoma A549 cells, and explore the effect of miR-18a or miR-328 on invasion and migration of A549 cells. Methods The expressions of miR-18a and miR-328 in A549 cells were detected by real-time quantitative PCR. Then the specific miR-18a or miR-328 inhibitor sequences were transfected into A549 cells to downregulate the expression of miR-18a or miR-328. The invasion and migration abilities of A549 cells were evaluated by Transwell(TM) assay. Results The miR-18a and miR-328 were overexpressed in A549 cells. And with the corresponding inhibitors being transfected, the expressions of miR-18a and miR-328 in A549 cells were downregulated. In addition, TranswellTM assay showed that decreased expression of miR-18a or miR-328 significantly inhibited the invasion and migration of A549 cells. Conclusion Downregulation of miR-18a or miR-328 can inhibit the invasion and migration abilities of A549 cells effectively.

Time-lapse lens-free imaging of cell migration in diverse physical microenvironments.

Time-lapse imaging of biological samples is important for understanding complex (patho)physiological processes. A growing number of point-of-care biomedical assays rely on real-time imaging of flowing or migrating cells. However, the cost and complexity of integrating experimental models simulating physiologically relevant microenvironments with bulky imaging systems that offer sufficient spatiotemporal resolution limit the use of time-lapse assays in research and clinical settings. This paper introduces a compact and affordable lens-free imaging (LFI) device based on the principle of coherent in-line, digital holography for time-lapse cell migration assays. The LFI device combines single-cell resolution (1.2 µm) with a large field of view (6.4 × 4.6 mm(2)), thus rendering it ideal for high-throughput applications and removing the need for expensive and bulky programmable motorized stages. The set-up is so compact that it can be housed in a standard cell culture incubator, thereby avoiding custom-built stage top incubators. LFI is thoroughly benchmarked against conventional live-cell phase contrast microscopy for random cell motility on two-dimensional (2D) surfaces and confined migration on 1D-microprinted lines and in microchannels using breast adenocarcinoma cells. The quality of the results obtained by the two imaging systems is comparable, and they reveal that cells migrate more efficiently upon increasing confinement. Interestingly, assays of confined migration more readily distinguish the migratory potential of metastatic MDA-MB-231 cells from non-metastatic MCF7 cells relative to traditional 2D migration assays. Altogether, this single-cell migration study establishes LFI as an elegant and useful tool for live-cell imaging.

A ring barrier-based migration assay to assess cell migration in vitro.

Cell migration is a key feature of virtually every biological process, and it can be studied in a variety of ways. Here we outline a protocol for the in vitro study of cell migration using a ring barrier-based assay. A 'barrier' is inserted in the culture chamber, which prevents cells from entering a defined area. Cells of interest are seeded around this barrier, and after the formation of a peripheral monolayer the barrier is removed and migration into the cell-free area is monitored. This assay is highly reproducible and convenient to perform, and it allows the deduction of several parameters of migration, including total and effective migration, velocity and cell polarization. An advantage of this assay over the conventional scratch assay is that the cells move over an unaltered and virgin surface, and thus the effect of matrix components on cell migration can be studied. In addition, the cells are not harmed at the onset of the assay. Through computer automation, four individual barrier assays can be monitored at the same time. The procedure can be used in a 12-well standard plate allowing higher throughput, or it can be modified to perform invasion assays. The basic procedure takes 2-3 d to complete.

Facile preparation of a photoactivatable surface on a 96-well plate: a versatile and multiplex cell migration assay platform.

Cell migration is an essential cellular activity in various physiological and pathological processes, such as wound healing and cancer metastasis. Therefore, in vitro cell migration assays are important not only for fundamental biological studies but also for evaluating potential drugs that control cell migration activity in medical applications. In this regard, robust control over cell migrating microenvironments is critical for reliable and quantitative analysis as cell migration is highly dependent upon the microenvironments. Here, we developed a facile method for making a commercial glass-bottom 96-well plate photoactivatable for cell adhesion, aiming to develop a versatile and multiplex cell migration assay platform. Cationic poly-d-lysine was adsorbed to the anionic glass surface via electrostatic interactions and, subsequently, functionalized with poly(ethylene glycol) (PEG) bearing a photocleavable reactive group. The initial PEGylated surface is non-cell-adhesive. However, upon near-ultraviolet (UV) irradiation, the photorelease of PEG switches the surface from non-biofouling to cell-adhesive. With this platform, we assayed cell migration in the following procedure: (1) create cell-attaching regions of precise geometries by controlled photoirradiation, (2) seed cells to allow them to attach selectively to the irradiated regions, (3) expose UV light to the remaining PEGylated regions to extend the cell-adhesive area, (4) analyse cell migration using microscopy. Surface modification of the glass surface was characterized by ζ-potential and contact angle measurements. The PEGylated surface showed cell-resistivity and became cell-adhesive upon releasing PEG by near-UV irradiation. The method was applied for parallelly evaluating the effect of model drugs on the migration of epithelial MDCK cells in the multiplexed platform. The dose-response relationship for cytochalasin D treatment on cell migration behavior was successfully evaluated with high reproducibility. Interestingly, the impact of blebbistatin on cell migration was dependent upon the widths of the migrating regions, resulting in both cases of migration acceleration and deceleration. These results clearly demonstrate that the cellular response to certain drugs is highly affected by their migrating geometries. Therefore, the obtained novel photoactivatable 96-well plate serves as a useful high-throughput platform for the identification of drug candidates that have an effect on cell migration behavior.

Tapered microtract array platform for antimigratory drug screening of human glioblastoma multiforme.

Understanding the effects of topographic characteristics on tumor cell migration is important for the development of new anti-migratory therapies. However, simplified in vitro culture systems often lead to inaccurate results regarding the efficacy of drugs. Histopathologically, glioblastoma multiform (GBM) cells migrate along the orientation of thin, elongated anatomical structures, such as white-matter tracts. Here, a tapered microtract array platform which mimics the anatomical features of brain tissue is introduced. This platform enables optimization of design for platform fabrication depending on topographic effects. By monitoring the migration of GBM cells on a simple tapered microtract, a saltatory migration resembling the migratory phenotype of human GBM cells in vivo is observed. The platform effectively induces the native characteristics and behavior of cells by topographic cues, allowing to observe the critical point for crawling to saltatory transition. Furthermore, this platform can be applied to efficiently screen anti-cancer drug by inhibiting associated signaling pathways on GBM cells. In conclusion, the microtract array platform reported here may provide a better understanding of the effects of topographic characteristics on cell migration, and may also be useful to determine the efficacy of antimigratory drugs for glioblastoma cells with cellular and molecular research and high-throughput screening.

Ação da proteína amiloide sérica A em melanomas / Activity of serum amyloid A protein in melanomas

Concentrações séricas basais da proteína amiloide sérica A (SAA) estão significativamente aumentadas em pacientes com câncer e alguns autores sugerem uma relação causal. Trabalho anterior do grupo mostrou que a SAA induz a proliferação de duas linhagens de glioblastoma humano e afeta os processos de invasividade in vitro, sustentando um papel pró-tumoral para esta proteína. Com base nesse trabalho, investigamos a abrangência dos efeitos de SAA para outro tipo de célula tumoral e para isso escolhemos um painel de linhagens de melanoma humano e uma linhagem primária obtida a partir de aspirado de linfonodo de paciente com melanoma, por nós isolada. Observamos que apesar da célula precursora de melanomas, isto é, melanócito, não produzir SAA, todas as linhagens de melanoma produziram a proteína e expressaram alguns dos seus receptores. Além disso, quando estas células foram estimuladas com SAA houve uma inibição da proliferação em tempos curtos de exposição (48 horas) e efeitos citotóxicos após um tempo maior (7 dias). A SAA também afetou processos de invasividade e a produção das citocinas IL-6, IL-8 e TNF-α. Aos avaliarmos o efeito da SAA na interação das células de melanoma com células do sistema imune, vimos que a SAA ativou uma resposta imune anti-tumoral aumentando a expressão de moléculas co-estumolatórias, como CD69 e HLA-DR, e sua função citotóxica. Ainda, vimos que a produção de TNF-α, IFN-γ, IL-10, IL-1ß e IL-8 estimuladas por SAA podem contribuir com os efeitos desta. De forma geral estes resultados nos levam a crer que a SAA tem atividade anti-tumoral em melanomas. Finalizando, com base na importância do desenvolvimento da resistência às terapias atuais para o melanoma, observamos que em células resistentes ao PLX4032, um inibidor de BRAF, os efeitos imunomodulatórios induzidos pela SAA estão abolidos, possivelmente identificando um novo componente da resistência

Basal serum concentrations of the protein serum amyloid A are significantly increased in cancer patients and some authors suggest a causal relationship. Previous work of our research group showed that SAA induces proliferation of two cell lines of human glioblastoma and affects invasiveness processes in vitro, supporting a pro-tumor role for this protein. Based on this work, we investigated the extent of SAA effects to another type of tumor cell and we chose a panel of human melanoma cell lines and primary line obtained from a patient with melanoma by lymph node aspirate. Melanoma cells were isolated by us. We observed that while the precursor cells of melanoma, melanocytes, do not produce SAA, all melanoma cell lines expressed the protein and produced some of their receptors. Moreover, when these cells were stimulated with SAA there was an inhibition of proliferation in short exposure times (48 hours) and cytotoxic effects after a longer period (7 days). SAA also affected invasive procedures and the production of the cytokines IL-6, IL-8 and TNF-α. To evaluate the SAA effect in the interaction of melanoma cells with immune system cells, we found that SAA activated an anti-tumor immune response by increasing the expression of co-estimulatory molecules such as CD69 and HLA-DR, and their cytotoxic function. Furthermore, we found that the production of TNF-α, IFN-γ, IL-10, IL-1ß and IL-8 stimulated by SAA can contribute to this effect. In general these results lead us to believe that the SAA has anti-tumor activity in melanomas. Finally, based on the importance of the resistance development to current therapies for melanoma we observed that in cells resistant to PLX4032, a BRAF inhibitor, the immunomodulatory effects induced by SAA are abolished, possibly identifying a new resistance component

Utilizing custom-designed galvanotaxis chambers to study directional migration of prostate cells.

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and epithelial wound healing. Applying a direct current electric field to cultured cells in vitro induces directional cell migration, or galvanotaxis. The 2-dimensional galvanotaxis method we demonstrate here is modified with custom-made poly(vinyl chloride) (PVC) chambers, glass surface, platinum electrodes and the use of a motorized stage on which the cells are imaged. The PVC chambers and platinum electrodes exhibit low cytotoxicity and are affordable and re-useable. The glass surface and the motorized microscope stage improve quality of images and allow possible modifications to the glass surface and treatments to the cells. We filmed the galvanotaxis of two non-tumorigenic, SV40-immortalized prostate cell lines, pRNS-1-1 and PNT2. These two cell lines show similar migration speeds and both migrate toward the cathode, but they do show a different degree of directionality in galvanotaxis. The results obtained via this protocol suggest that the pRNS-1-1 and the PNT2 cell lines may have different intrinsic features that govern their directional migratory responses.