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 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...

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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 ...

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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 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, ...

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 ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...