Name: impedance-based real-time measurement of cancer cell migration and invasion
Uploaded: 2020-04-02T15:00:00
Description: Watch this Scientific Journal Video about Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion at JoVE.com

We thank Olivia Funk for her technical assistance with the real-time cell analysis system data. We also thank the Medical Writing Center at Children&#8217;s Mercy Kansas City for editing this manuscript. This work was supported by Tom Keaveny Endowed Fund for Pediatric Cancer Research (to TP) and by Children&#8217;s Mercy Hospital Midwest Cancer Alliance Partner Advisory Board funding (to TP).

NOTE: All cell culture materials need to be sterile and the entire experiment must be performed in a biosafety cabinet under sterile conditions.
1. Culture and Electroporation of the U-118MG Glioblastoma Cell Line
<ol>
	<li>Culture the U-118MG cell line in 5% fetal bovine serum (FBS) containing Dulbecco&#8217;s Modified Eagle Medium (DMEM) (culture medium) and Maintain at 37 &#176;C in a humid atmosphere containing 5% CO2 incubator (culture conditions).</li>
	<li>...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144 (5), 646-674 (2011).</li><li>Mudduluru, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Axl+receptor+tyrosine+kinase+expression+by+miR-34a+and+miR-199a/b+in+solid+cancer.">Regulation of Axl receptor tyrosine kinase expression by miR-34a and miR-199a/b in solid cancer.</a> Oncogene. 30 (25), 2888-2899 (2011).</li><li>Mudduluru, G., Vajkoczy, P., Allgayer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+zinc+finger+1+induces+migration,+invasion,+and+in+vivo+metastasis+through+Axl+gene+expression+in+solid+cancer.">Myeloid zinc finger 1 induces migration, invasion, and in vivo metastasis through Axl gene expression in solid cancer.</a> Molecular Cancer Research. 8 (2), 159-169 (2010).</li><li>Khalili, A. 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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crk+proteins+transduce+FGF+signaling+to+promote+lens+fiber+cell+elongation.">Crk proteins transduce FGF signaling to promote lens fiber cell elongation.</a> Elife. 7, (2018).</li><li>Fathers, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crk+adaptor+proteins+act+as+key+signaling+integrators+for+breast+tumorigenesis.">Crk adaptor proteins act as key signaling integrators for breast tumorigenesis.</a> Breast Cancer Research. 14 (3), 74 (2012).</li><li>Koptyra, M., Park, T. J., Curran, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crk+and+CrkL+are+required+for+cell+transformation+by+v-fos+and+v-ras.">Crk and CrkL are required for cell transformation by v-fos and v-ras.</a> Molecular Carcinogenesis. 55 (1), 97-104 (2016).</li><li>Lamorte, L., Royal, I., Naujokas, M., Park, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crk+adapter+proteins+promote+an+epithelial-mesenchymal-like+transition+and+are+required+for+HGF-mediated+cell+spreading+and+breakdown+of+epithelial+adherens+junctions.">Crk adapter proteins promote an epithelial-mesenchymal-like transition and are required for HGF-mediated cell spreading and breakdown of epithelial adherens junctions.</a> Molecular Biology of the Cell. 13 (5), 1449-1461 (2002).</li><li>Park, T. J., Curran, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+roles+of+Crk+and+CrkL+in+fibroblast+structure+and+motility.">Essential roles of Crk and CrkL in fibroblast structure and motility.</a> Oncogene. 33 (43), 5121-5132 (2014).</li><li>Rodrigues, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CrkI+and+CrkII+function+as+key+signaling+integrators+for+migration+and+invasion+of+cancer+cells.">CrkI and CrkII function as key signaling integrators for migration and invasion of cancer cells.</a> Molecular Cancer Research. 3 (4), 183-194 (2005).</li><li>Feller, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crk+family+adaptors-signalling+complex+formation+and+biological+roles.">Crk family adaptors-signalling complex formation and biological roles.</a> Oncogene. 20 (44), 6348-6371 (2001).</li><li>Park, T. J., Boyd, K., Curran, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiovascular+and+craniofacial+defects+in+Crk-null+mice.">Cardiovascular and craniofacial defects in Crk-null mice.</a> Molecular and Cellular Biology. 26 (16), 6272-6282 (2006).</li><li>Park, T. J., Curran, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crk+and+Crk-like+play+essential+overlapping+roles+downstream+of+disabled-1+in+the+Reelin+pathway.">Crk and Crk-like play essential overlapping roles downstream of disabled-1 in the Reelin pathway.</a> Journal of Neuroscience. 28 (50), 13551-13562 (2008).</li><li>Hallock, P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dok-7+regulates+neuromuscular+synapse+formation+by+recruiting+Crk+and+Crk-L.">Dok-7 regulates neuromuscular synapse formation by recruiting Crk and Crk-L.</a> Genes &amp; Development. 24 (21), 2451-2461 (2010).</li></ol>

The real-time measurement of cell migration and invasion using the real-time cell analysis system is a simple, quick, and continuous monitoring process with multiple, significant advantages over the traditional methods that provide data at a single time point. As with the traditional methods, experimental conditions must be optimized for each cell line for the real-time cell analysis system, because each cell line may be different in terms of its adhesion to the substrate, growth, cell-to-cell contacts, and migratory and...

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining cancer genotypes and phenotypes is critical to understanding and designing effective therapeutic strategies. Decades of cancer research have led to the development and adaptation of different methods to determine cancer genotypes and phenotypes. One of the latest technical developments is real-time measurement of cell migration and invasion based on cell impedance. Cell adhesion to substrates and cell-cell contacts play an important role in cell-to-cell communication and regulation, development, and maintenance of tissues. Abnormalities in cell adhesion lead to the loss of cell-cell contact, degradation of extracellular matrix (ECM), and gain of migratory and invading capabilities by cells, all of which contribute to metastasis of cancer cells to different organs1,2. Various methods are available to determine cell migration (wound healing and Boyden chamber assays) and invasion (Matrigel-Boyden chamber assay)3,4,5. These conventional methods are semiquantitative because cells need to be labeled with a fluorescent dye or other dyes either before or after the experiment to measure cell phenotypes. In addition, mechanical disruptions are needed in some cases for creating a wound for measuring the migration of cells to the wound site. Moreover, these existing methods are time-consuming, labor-intensive, and measure the results at only one time point. In addition, these methods are prone to making inaccurate measurements due to inconsistent handling during the experimental procedure6.
Unlike conventional methods, the real-time cell analysis system measures cell impedance in real-time without requiring pre- or poststaining and mechanical damage of cells. More importantly, the duration of an experiment can be extended so that biological effects can be determined in a time-dependent manner. Executing the experiment is time-efficient and not labor-intensive. Analyzing data is relatively simple and accurate. Compared to other methods, this method is one of the best real-time measurements to measure cell migration and invasion6,7,8,9.
Giaever and Keese were the first to describe the impedance-based measurement of a cell population on the surface of electrodes10. The real-time cell analysis system works on the same principle. The area of each microplate well is approximately 80% covered with an array of gold microelectrodes. When the electrode surface area is occupied by cells due to adherence or spreading of the cells, the electrical impedance changes. This impedance is displayed as the cell index, which is directly proportional to the cells covering the electrode surface area after they penetrate the microporous membrane (the median pore size of this membrane is 8 &#956;m)11.
Crk and CrkL are adaptor proteins containing SH2 and SH3 domains and play important roles in various cellular functions, such as cytoskeleton regulation, cell transformation, proliferation, adhesion, epithelial-mesenchymal transition, migration, invasion, and metastasis by mediating protein-protein interactions in many signaling pathways1,12,13,14,15,16,17,18. Therefore, it is important to determine the Crk/CrkL-dependent migratory and invasive capabilities of cancer cells. Real-time cell analysis was performed to determine the migratory and invasive abilities of glioblastoma cells upon gene knockdown of Crk and CrkL.
This method paper describes detailed measurements of Crk- and CrkL-mediated migration and invasion of human glioblastoma cells.&#160;

<table border="0" cellpadding="0" cellspacing="0" style="width:1140px;" width="1140">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:323px;">Biosafety cabinet</td>
			<td style="width:339px;">ThermoFisher Scientific</td>
			<td style="width:235px;">1300 Series Class II, Type A2</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CIM plates</td>
			<td>Cell Analysis Division of Agilent Technologies, Inc</td>
			<td>5665825001</td>
			<td>Cell invasion and migration plates</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Crk siRNA</td>
			<td>Dharmacon</td>
			<td>J-010503-10</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CrkL siRNA</td>
			<td>Ambion</td>
			<td>ID: 3522 and ID: 3524</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#8217;s modified eagle&#8217;s medium (DMEM)</td>
			<td>ATCC</td>
			<td>302002</td>
			<td>Culture medium used for cell culture</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td>Gibco</td>
			<td>21-031-CV</td>
			<td>DPBS used to wash the cells</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal bovine serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heracell VIOS 160i CO2 incubator</td>
			<td>ThermoFisher Scientific</td>
			<td>51030285</td>
			<td>Co2 incubator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Matrigel</td>
			<td>BD Bioscience</td>
			<td>354234</td>
			<td>Extracellular matrix gel</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neon electroporation system</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK5000</td>
			<td>Electroporation system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Neon transfection system 10 &#181;L kit</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK1025</td>
			<td>Electroporation kit</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Non-targeting siRNA</td>
			<td>Dharmacon</td>
			<td>D-001810-01</td>
			<td>siRNA for non targated control</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Odyssey CLx (Imaging system)</td>
			<td>LI-COR Biosciences</td>
			<td></td>
			<td>Western blot imaging system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RTCA software</td>
			<td>Cell Analysis Division of Agilent Technologies, Inc</td>
			<td></td>
			<td>Instrument used for experiment</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Scepter</td>
			<td>Millipore</td>
			<td>C85360</td>
			<td>Handheld automated cell counter</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">U-118MG</td>
			<td>ATCC</td>
			<td>ATCC HTB15</td>
			<td>Cell lines used for experiments</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">xCELLigence RTCA DP</td>
			<td>Cell Analysis Division of Agilent Technologies, Inc</td>
			<td>380601050</td>
			<td>Instrument used for experiment</td>
		</tr>
	</tbody>
</table>

impedance-based real-time measurement of cancer cell migration and invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

It has been suggested that Crk and CrkL are important for cell migration and invasion in different cancer cell lines13,17. Although Crk and CrkL proteins are structurally and functionally similar to each other and play essential overlapping functions16,19,20,21, many gene knockdown studies for Crk and CrkL have not clearly addressed whe...

Watch this Scientific Journal Video about Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion at JoVE.com

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line.&#160;

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Using this protocol, cell migration and invasion can be unveiled under real-time conditions enabling cell kinetics under loss or gain of gene function and drugs to be determined. Unlike other methods, no staining, mechanical cell damage, or fluorescent markers needed. Most importantly, the real-time cell kinetics can be determined in an effective manner.When the U-118MG cell line culture reaches 70 to 80%confluency, wash the cells with PBS before treating the cells with two milliliters of 0.5%trypsin-EDTA. After 30 seconds at 37 degrees Celsius, stop the reaction with 10 milliliters of fresh culture medium and transfer the detached cells to a 15 milliliter conical tube. After counting, collect the cells by centrifugation and resuspend the pellet in a six times 10 to the fifth cells per condition concentration in the appropriate volume of fresh medium.Transfer six times 10 to the fifth cells into one microcentrifuge tube per condition and add 800 microliters of Dulbecco's PBS to each tube. After centrifugation, resuspend each pellet in 60 microliters of resuspension buffer R and six micromolar of the siRNA of interest. Mix each tube with gentle tapping before electroporating 10 microliter aliquots of each cell suspension with three 10-millisecond 1, 350 volt pulses.After each treatment, pool the electroporated cells from each condition in individual five milliliter aliquots of fresh culture medium. Then seed the cells into two 35 millimeter culture dishes per condition and place the cells in the cell culture incubator for three days. Five to six hours before the real-time cell analysis, place the real-time cell analysis system into the cell culture incubator.To set up an invasion assay, use reverse pipetting to place 50 microliters of DMEM supplemented with 0.1 microgram per milliliter of extracellular matrix gel into each well of the upper chamber of a cell invasion plate. Immediately after plating, remove 30 microliters of the extracellular matrix solution from each well and place the plate into the cell culture incubator for four hours. To set up an impedance measurement program, six hours before the measurement, replace the medium in all of the electroporated cell cultures with low serum medium.Under the Layout tab in the associated impedance measurement software, select quadruplicate wells for each biological condition. Under the Schedule tab, set a one-time baseline measurement sweep step with a one-minute interval and set a second step to measure the cell impedance in respective individual cradles for the actual experiment. One hour before the start of the cell impedance measurement, add 160 microliters of medium supplemented with 10%fetal bovine serum as the chemoattractant into the wells of the lower chamber of the cell invasion and migration plate.To measure invasion, assemble the upper chamber containing the extracellular matrix gel coated wells. To measure migration, use uncoated wells in the upper chamber. For either type of experiment, fill the wells in the upper chamber with 50 microliters of low serum medium and place the chambers into the cradle of the system.Click the Message tab in the software to determine whether all of the wells are recognized by the control unit. If the message displays as expected, the plate in the cradle is ready for the experiment. Then place the completely packed plates into the cell culture incubator in the real-time cell analysis system cradle for one hour to acclimate the plate to the cell culture conditions.While the plates are equilibrating, harvest the glioblastoma cells as demonstrated and resuspend the cells from each condition at an eight times 10 to the fifth cells per 800 microliters of low serum medium concentration. Additionally, set aside a three times 10 to the fifth cells in two milliliters of culture medium for seeding in a 35 millimeter dish for downstream Western blot analysis. To measure the baseline pre-migration reading, at the end of the acclimatization, click the cradle Start button.After the baseline measurement has been acquired, transfer the migration and the invasion plates from their respective cradles into a biosafety cabinet. Reverse pipette 100 microliters of cells into quadruplicate upper chamber wells for each biological condition in the appropriate well of the cell invasion and migration plates as programmed in the control unit of the cradle. After seeding, keep the plates in the biosafety cabinet for 30 minutes at room temperature to allow the cells to settle evenly on the plate bottom before transferring the plates to their respective cradles.Click the cradle Start button to begin measuring the cell impedance. To visualize the changes in cell impedance as the cell index in a time-dependent manner during or after completion of the experiment, open the Data Analysis tab. To visualize the data for each of the respective conditions either individually or as averages and/or standard deviations, click the option boxes for average and standard deviation.To export the cell index data to a spreadsheet file, place the cursor in the middle of the data analysis window and right-click. In the dialogue box that appears, select the copy data into list format option and paste the data into an open spreadsheet. To release the experiment, click the Release button in each cradle.Crk adapter protein knockdown decreases Crk1 and Crk2 protein levels by 85%and 86%respectively without inducing Crk-like protein levels. The knockdown of Crk-like reduces Crk-like protein expression by 85%with slight reductions in Crk1 and Crk2 protein levels. Combining both siRNAs reduces Crk1, Crk2, and Crk-like expression by over 80%while neither Vinculin nor alpha-Tubulin expression is affected by any combination of Crk or Crk-like knockdown.Human brain glioblastoma cells migrate along a gradient in response to high serum concentrations reaching the maximum level of migration at 13 hours. In Crk knockdown cells, although the migration is initially delayed, the cells continued to migrate until 23 hours. Crk-like knockdown substantially reduces cell migration with the glioblastoma cell line completely losing their migratory ability upon knockdown of both Crk and Crk-like suggesting that Crk and Crk-like play essential overlapping roles in cancer cell migration.However, when cell invasion is monitored over several days, the Crk knockdown cells reach a similar maximum level of cell invasion compared to control glioblastoma cells at 60 hours with Crk-like cells partially recovering their invasive ability by 90 hours and double Crk-Crk cells demonstrating a reduced ability to invade after 40 hours. The results clearly support the suggestion that cell invasion should be analyzed over the entire period of the experiment. Seeding the right number of cells and avoiding the air bubbles while recording the extracellular matrix for invasion assay and seeding cells are key points for success of this experiment.Following similar procedure but using e-plates, cell adhesion and cell proliferation kinetics can be determined.

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Research

JoVE Journal

Cancer Research

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

Using the Dot Assay to Analyze Migration of Cell Sheets

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move within tissues in a tightly orchestrated manner. During tumor development, this equilibrium is disturbed, and tumor cells leave the epithelium of origin to invade the local microenvironment, to travel to distant sites, and to ultimately form metastatic tumors at distant sites. The dot assay is a simple, two-dimensional unconstrained migration assay, to assess the net movement of cell sheets into a cell-free area, and to analyze parameters of cell migration using time-lapse imaging. Here, the dot assay is demonstrated using a human invasive, lung colony forming breast cancer cell line, MCF10CA1a, to analyze the cells' migratory response to epidermal growth factor (EGF), which is known to increase malignant potential of breast cancer cells and to alter the migratory phenotype of cells.

Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

Serum and Plasma Copy Number Detection Using Real-time PCR

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance. Starting from the hypothesis that androgen receptor (AR) gene copy number (CN) gain is a frequent event in metastatic castration resistance prostate cancer (mCRPC), we propose to analyze this event in cfDNA as a potential predictive biomarker.
We evaluated AR CN in cfDNA using 2 different real-time PCR assays and 2 reference genes (RNaseP and AGO1). DNA amount of 60 ng was used for each assay combination. AR CN gain was confirmed using Digital PCR as a more accurate method. CN variation analysis has already been demonstrated to be informative for the prediction of treatment resistance in the setting of mCRPC, but it could be useful also for other purposes in different patient settings. CN analysis on cfDNA has several advantages: it is non-invasive, rapid and easy to perform, and it starts from a small volume of serum or plasma material.

This manuscript describes the copy number variation analysis performed in serum or plasma DNA using real-time PCR approach. This method is suitable for the prediction of drug resistance in castration resistant prostate cancer patients, but it could be informative also for other diseases.

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better understand the molecular mechanisms underlying glioma cell migration, an ability to study the interaction between hGCs and axons within the tumor microenvironment is essential. In order to model this cellular interaction, we developed a mixed culture system consisting of hGCs and dorsal root ganglia (DRG) axon-oligodendrocyte co-cultures. DRG cultures were selected because they can be isolated efficiently and can form the long, extensive projections which are ideal for migration studies of this nature. Purified rat oligodendrocytes were then added on purified rat DRG axons and induced to myelinate. After confirming the formation of compact myelin, hGCs were finally added to the co-culture and their interactions with DRG axons and oligodendrocytes was monitored in real-time using time-lapse microscopy. Under these conditions, hGCs form tumor-like aggregate structures that express GFAP and Ki67, migrate along both myelinated and non-myelinated axonal tracks and interact with these axons through the formation of pseudopodia. Our ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of hGC migration and could potentially be used for in vitro drug efficacy testing.

Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...