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>Use 70&#8211; 80% confluent healthy cells for electroporation.</li>
	<li>For harvesting cells, wash the cells growing in culture dishes with 1x PBS and add 2 mL of 0.05% trypsin-EDTA. Place in the incubator for 30 s and remove the trypsin-EDTA. Collect cells in the culture medium into a 15 mL tube.</li>
	<li>Count the cells using a handheld automated cell counter, centrifuge cells at 100 x g for 5 min, and discard the supernatant.</li>
	<li>Suspend the cell pellet in the culture medium and take 600,000 cells for each condition into a microcentrifuge tube. Adjust the cell number depending on experimental designs and growth rates.&#160;</li>
	<li>Transfer the cell suspension to a microcentrifuge tube and add 800 &#181;L of Dulbecco&#8217;s phosphate-buffered saline (DPBS). Spin down using a minicentrifuge for 30 s and discard the supernatant.</li>
	<li>Add 60 &#181;L of resuspension buffer R to the cell pellet and add the siRNAs (i.e., non-targeting siRNA, Crk siRNA, CrkL siRNA, or both Crk and CrkL siRNAs) at a concentration of 6 &#181;M to the respective microcentrifuge tube. Mix them gently by tapping.</li>
	<li>Electroporate 10 &#181;L of cells with an electroporation system at 1,350 V for 10 ms with three pulses and transfer the electroporated cells into 5 mL of the culture medium. Repeat the electroporation for the rest of the cells prepared for each condition.</li>
	<li>Complete all respective electroporation. Transfer electroporated cells into two 35 mm dishes per condition and culture them under culture conditions for 3 days.</li>
	<li>On the third day, treat all the electroporated cells in 0.5% FBS containing DMEM (low serum medium) for 6 h prior to the actual cell impedance measurement.&#160; &#160;</li>
</ol>
2. Preparation of the Real-time Cell Analysis System, Cell Invasion and Migration (CIM) Plates, and Electroporated U-118MG Cells for Plating
<ol>
	<li>Place the real-time cell analysis system in a CO2 incubator under culture conditions 5&#8211;6 h prior to the start of experiment to stabilize the system to the culture conditions.</li>
	<li>For the invasion assay, plate 50 &#181;L of DMEM (plain medium) containing extracellular matrix (ECM) gel at 0.1 &#181;g/&#181;L in each well of the upper chamber of the CIM plate. To avoid having any air bubbles, use the reverse pipetting method. Immediately remove 30 &#181;L of ECM gel, leaving 20 &#181;L in the well.</li>
	<li>After the ECM gel coating, keep the plate in the incubator under culture conditions for 4 h with its lid on. During the ECM gel coating and drying, take preventive measures to avoid direct contact of the electrodes of the upper chamber of the plates with the hands, the surfaces of the biosafety cabinet, or the CO2 incubator.</li>
	<li>To set up the impedance measurement program, double-click the associated software icon (see Table of Materials) to open the system software application (control unit). Each cradle has an individual window with different tabs to set the experimental conditions, the impedance measuring time interval and duration, and data analysis.</li>
	<li>Under the Layout tab, set quadruplicate wells for each biological condition and set two-step cell impedance measurements under the Schedule tab. The first step is for a one-time baseline measurement (one sweep with a 1 min interval), and the second step is to measure the cell impedance in respective individual cradles for an actual experiment. For migration, use 145 sweeps with a 10 min interval, and for invasion, 577 sweeps with a 10 min interval, individually set) in respective individual cradles.</li>
	<li>One h prior to the start of the cell impedance measurement, add 160 &#181;L of DMEM with 10% FBS as a chemoattractant in the wells of the lower chamber of the plate. Assemble either the upper chamber containing the ECM gel-coated wells or uncoated wells with the lower chamber to measure invasion and migration, respectively.</li>
	<li>Fill the wells in the upper chamber with 50 &#181;L of low serum medium and place them in the cradle of the system. Check whether all the wells are recognized by the control unit by clicking the Message tab. If the message displays as OK, the plate in the cradle is ready for the experiment.&#160;</li>
	<li>Preincubate the completely packed plates in the incubator under culture conditions for 1 h prior to measurements in the real-time cell analysis system cradle, which is essential for acclimation of a packed plate to the cell culture conditions.</li>
	<li>For harvesting cells treated with low serum medium, trypsinize the cells as in step 1.3, collect them in low serum medium, and count the cells as in step 1.4.</li>
	<li>After counting, centrifuge cells at 100 x g for 5 min and discard the supernatant.</li>
	<li>Resuspend 800,000 cells in 800 &#181;L of low serum medium for the migration and invasion assays. Additionally, resuspend 300,000 cells in 2 mL of culture medium and seed in a 35 mm dish for Western blot analysis to confirm the regulated knockdown of Crk and CrkL.</li>
</ol>
3. Baseline Reading, Seeding of the Cells, and Cell Impedance Measurement and Visualization
<ol>
	<li>Measure the baseline reading by clicking the cradle Start button. The baseline should be read after preincubating the plates for 1 h in the cradle of the real-time cell analysis system under culture conditions in the CO2 incubator and before seeding the cells to the respective wells in the upper chamber. 
	NOTE: Once the cradle Start button is clicked, the control unit will ask whether to save the experimental file. After the file is saved, the cradle analyzer measures the baseline cell impedance as set in the program initially and enters a pause mode until the cradle Start button is clicked again to measure cell impedance in the second step.</li>
	<li>Take out both plates for migration and invasion from the cradles after the baseline measurement and keep them in the biosafety cabinet.</li>
	<li>Seed 100 &#181;L of cells (100,000 cells) in quadruplicates for each biological condition in the upper chamber of the CIM plate in the respective wells as programmed in the control unit of the cradle by reverse pipetting to avoid air bubbles.</li>
	<li>After seeding, keep the plate under a biosafety cabinet for 30 min at room temperature to allow cells to evenly settle down to the bottom. Transfer the plate back to the respective cradle and click the cradle start button to start measuring cell impedance as programmed in the second step of 2.5.</li>
	<li>After the last sweep, the experiment is finished, and the results are saved automatically.</li>
	<li>Under the Data Analysis tab, visualize the changes in cell impedance as cell index in a time-dependent manner during or after completion of the experiment. Each of the respective conditions of quadruplicates can be visualized either individually or as averages and/or standard deviations by clicking the Option boxes for average and standard deviation.</li>
	<li>To export cell index data to a spreadsheet file, open an empty spreadsheet file, place the cursor in the middle of the Data Analysis window, and right-click. In the dialog box that appears, choose the option Copy Data into List Format, and paste the data into the open spreadsheet.</li>
	<li>Adjust the time in the raw data to represent the actual start time of the cell impedance measurement. The second step start time is set as zero. 
	NOTE: The control unit has the option to obtain the cell index with or without normalization (i.e., normalized cell index) and visualize the results as a graphical presentation in a time-dependent manner. In this example, cell index data are exported without normalization for processing and graphical presentation.</li>
	<li>Release the experiment by clicking the Release button in each cradle.</li>
</ol>

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The authors have nothing to disclose.

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><tbody><tr><td>Biosafety cabinet</td><td>ThermoFisher Scientific</td><td>1300 Series Class II, Type A2</td><td></td></tr><tr><td>CIM plates</td><td>Cell Analysis Division of Agilent Technologies, Inc</td><td>5665825001</td><td>Cell invasion and migration plates</td></tr><tr><td>Crk siRNA</td><td>Dharmacon</td><td>J-010503-10</td><td></td></tr><tr><td>CrkL siRNA</td><td>Ambion</td><td>ID: 3522 and ID: 3524</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified eagle&amp;rsquo;s medium (DMEM)</td><td>ATCC</td><td>302002</td><td>Culture medium used for cell culture</td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS)</td><td>Gibco</td><td>21-031-CV</td><td>DPBS used to wash the cells</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Hyclone</td><td>SH30910.03</td><td></td></tr><tr><td>Heracell VIOS 160i CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>ThermoFisher Scientific</td><td>51030285</td><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td></tr><tr><td>Matrigel</td><td>BD Bioscience</td><td>354234</td><td>Extracellular matrix gel</td></tr><tr><td>Neon electroporation system</td><td>ThermoFisher Scientific</td><td>MPK5000</td><td>Electroporation system</td></tr><tr><td>Neon transfection system 10 &amp;micro;L kit</td><td>ThermoFisher Scientific</td><td>MPK1025</td><td>Electroporation kit</td></tr><tr><td>Non-targeting siRNA</td><td>Dharmacon</td><td>D-001810-01</td><td>siRNA for non targated control</td></tr><tr><td>Odyssey CLx (Imaging system)</td><td>LI-COR Biosciences</td><td></td><td>Western blot imaging system</td></tr><tr><td>RTCA software</td><td>Cell Analysis Division of Agilent Technologies, Inc</td><td></td><td>Instrument used for experiment</td></tr><tr><td>Scepter</td><td>Millipore</td><td>C85360</td><td>Handheld automated cell counter</td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>U-118MG</td><td>ATCC</td><td>ATCC HTB15</td><td>Cell lines used for experiments</td></tr><tr><td>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 ...

impedance-based-real-time-measurement-cancer-cell-migration

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

6.3K Views.  Children's Mercy Research Institute, Children's Mercy Kansas City. 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. 

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

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

Medicine

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. ...

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 ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

Genetics

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Bioengineering

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of cancer cell populations typically generate a single endpoint measurement that does not distinguish between cancer cell subpopulations with different invasive potential. Also, the tumor microenvironment consists of different resident stromal and immune cells that alter and participate in the invasive behavior of cancer cells. Invasion into tissues also plays a role in immune cell subpopulations fending off microorganisms or eliminating diseased cells from the parenchyma and endothelial cells during tissue remodeling and angiogenesis. Real-Time Cellular Analysis (RTCA) that utilizes impedance biosensors to monitor cell invasion was a major step forward beyond endpoint measurement of invasion: this provides continuous measurements over time and thus can reveal differences in invasion rates that are lost in the endpoint assay. Using current RTCA technology, we expanded dual-chamber arrays by adding a further chamber that can contain stromal and/or immune cells and allows measuring the rate of invasion under the influence of secreted factors from co-cultured stromal or immune cells over time. Beyond this, the unique design allows for detaching chambers at any time and isolating of the most invasive cancer cell, or other cell subpopulations that are present in heterogeneous mixes of tumor isolates tested. These most invasive cancer cells and other cell subpopulations drive malignant progression to metastatic disease, and their molecular characteristics are important for in-depth mechanistic studies, the development of diagnostic probes for their detection, and the assessment of vulnerabilities. Thus, the inclusion of small- or large-molecule drugs can be used to test the potential of therapies that target cancer and/or stromal cell subpopulations with the goal of inhibiting (e.g., cancer cells) or enhancing (e.g., immune cells) invasive behavior.

We describe an approach to detect and capture invasive cell subpopulations in real-time. The experimental design uses Real-Time Cellular Analysis by monitoring changes in the electric impedance of cells. Invasive cancer, immune, endothelial or stromal cells in complex tissues can be captured, and the impact of co-cultures can be assessed.

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of ...

Real-Time Quantitative Measurement of Tumor Cell Migration and Invasion Following Synthetic mRNA Transfection

Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell migration and invasion is essential for understanding the mechanisms of tumor cell infiltration into neighboring healthy tissues and metastasis. Previously, it was demonstrated that gene knockdown followed by the impedance-based real-time measurement of tumor cell migration and invasion enables the identification of the genes required for tumor cell migration and invasion. Recently, the mRNA vaccines against SARS-CoV-2 have increased interest in using synthetic mRNA for therapeutic purposes. Here, the method using synthetic mRNA was revised to study the effect of gene overexpression on tumor cell migration and invasion. This study demonstrates that elevated gene expression with synthetic mRNA transfection followed by impedance-based real-time measurement may help identify the genes that stimulate tumor cell migration and invasion. This method paper provides important details on the procedures for examining the effect of altered gene expression on tumor cell migration and invasion.

Many upregulated genes stimulate tumor cell migration and invasion, leading to poor prognosis. Determining which genes regulate tumor cell migration and invasion is critical. This protocol presents a method for investigating the effects of the increased expression of a gene on the migration and invasion of tumor cells in real time.

Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Biology

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Three-Chambered Array-Based Impedance Assay: A Real-Time Analysis Technique to Assess Invasive Potential of Cancer Cells by Measuring Electrical Impedance

Source: Sharif, G. M. et al., <a href="https://jove.unicartagenaproxy.elogim.com/v/63512">Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures.</a> J. Vis. Exp. (2022).
This video describes an invasion assay using a three-chambered array based on electrical impedance. This technique measures the invasive potential of cancer cells under the influence of soluble factors secreted by the resident stromal cells in tumor microenvironments.

Source: Sharif, G. M. et al., Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures. J. Vis. Exp. (2022).
This video describes ...

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

Summary

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Chapters in this video

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

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

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

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

Real-Time Quantitative Measurement of Tumor Cell Migration and Invasion Following Synthetic mRNA Transfection

Measurement of Cellular Chemotaxis with ECIS/Taxis

In vitro Cell Migration and Invasion Assays

Three-Chambered Array-Based Impedance Assay: A Real-Time Analysis Technique to Assess Invasive Potential of Cancer Cells by Measuring Electrical Impedance