Name: Real-Time Quantitative Measurement of Tumor Cell Migration and Invasion Following Synthetic mRNA Transfection (Video) | JoVE Journal
Uploaded: 2023-06-23T13:10:00.000+00:00
Duration: 11 min
Description: Learn real-time tumor cell migration and invasion measurement using synthetic mRNA transfection with impedance-based analysis. This method identifies genes that stimulate metastasis for cancer therapy development.

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The authors thank the Medical Writing Center at Children&#39;s Mercy Kansas City for editing this manuscript. This work was supported by Natalie&#39;s A.R.T. Foundation (to T.P.) and by an MCA Partners Advisory Board grant from Children&#39;s Mercy Hospital (CMH) and the University of Kansas Cancer Center (KUCC) (to T.P.).

1. Synthesis of mRNA
NOTE: For the mRNA synthesis, all the reagents and equipment must be specially treated to inactivate the RNases before use. See the Table of Materials for details about all the materials, instruments, and reagents used in this protocol.
<ol>
	<li>Linearization of DNA 
	NOTE: Mouse cDNAs of CrkI and CrkL were cloned into the pFLAG-CMV-5a expression vector to add the FLAG epitope tag at the C-terminus and subcloned into the pcDNA3.1/myc-His vector to incorporate the T7 promoter, as previously described18,19.
	<ol>
		<li>Add 10 &#181;L of 10x reaction buffer, 1.5 &#181;L of PmeI (10,000 units/mL), and 10 &#181;g of a plasmid DNA to a microcentrifuge tube to linearize the plasmid DNA with the restriction enzyme. Add nuclease-free water to bring the reaction volume to 100 &#181;L.</li>
		<li>Mix by tapping, spin down, and incubate the reaction mixture at 37 &#176;C overnight.</li>
		<li>Spin down, and add 5 &#181;L of 10% sodium dodecyl sulfate (SDS) and 1 &#181;L of proteinase K (20 mg/mL, RNA grade) to the reaction mixture. Mix by tapping, spin down, and incubate at 50 &#176;C for 30 min.</li>
		<li>Under the fume hood, add 100 &#181;L of the bottom phase of phenol:chloroform:isoamyl alcohol to the plasmid reaction mixture for extraction. Vortex, and then centrifuge at 18,800 &#215; g for 5 min at room temperature. Move the upper phase to a new tube.</li>
		<li>Repeat step 1.1.4&#160;with chloroform:isoamyl alcohol at 24:1.</li>
		<li>In the new tube, bring the reaction volume to 300 &#181;L by adding 200 &#181;L of nuclease-free water, and then add 30 &#181;L of 3 M sodium acetate and 600 &#181;L of 100% ethanol.</li>
		<li>Mix by vortexing, and then place at &#8722;20 &#176;C for 30-60 min for the ethanol precipitation of the DNA.</li>
		<li>Centrifuge at 18,800 &#215; g for 20 min at 4 &#176;C, discard the supernatant, and rinse the pellet with 1 mL of 70% ethanol. Repeat the centrifugation for 10 min, and then remove the supernatant completely.</li>
		<li>Dry with the cap open for 2 min, add 30 &#181;L of nuclease-free water, and resuspend the DNA pellet.</li>
		<li>Determine the DNA concentration using a spectrophotometer.</li>
		<li>Verify the size and quantity of the linearized DNA using agarose gel electrophoresis.</li>
	</ol>
	</li>
	<li>RNA synthesis using T7 RNA polymerase
	<ol>
		<li>Add 2 &#181;L each of 10x transcription buffer, ATP, GTP, UTP, CTP, and T7 and 1 &#181;g of a linearized DNA to a microcentrifuge tube. Add nuclease-free water to bring the reaction volume to 20 &#181;L.</li>
		<li>Mix by tapping, spin down, and incubate the reaction mixture at 37 &#176;C for 2 h for RNA synthesis.</li>
		<li>Spin down, add 1 &#181;L of DNase (2 U/&#181;L), and incubate at 37 &#176;C for 15 min to remove the template DNA.</li>
	</ol>
	</li>
	<li>Lithium chloride precipitation of the RNA
	<ol>
		<li>Add 10 &#181;L of lithium chloride (7.5 M) to the reaction mixture. Mix by tapping, spin down, and incubate the reaction mixture at &#8722;20 &#176;C for 30 min.</li>
		<li>Centrifuge at 18,800 &#215; g for 20 min at 4 &#176;C, discard the supernatant, and rinse the pellet with 500 &#181;L of 70% ethanol. Repeat the centrifugation for 10 min, and then remove the supernatant completely.</li>
		<li>Dry with the cap open for 2 min, add 30 &#181;L of nuclease-free water, and resuspend the RNA pellet.</li>
	</ol>
	</li>
	<li>Capping
	<ol>
		<li>Heat the RNA sample at 65 &#176;C for 10 min, and then place it on ice for cooling.</li>
		<li>Add 5 &#181;L each of 10x capping buffer, 10 mM GTP, and 1 mM (10x) S-adenosylmethionine (SAM), 2 &#181;L each of a capping enzyme mix and O-methyltransferase enzyme mix, and 1.25 &#181;L of RNase inhibitor. Mix by tapping, spin down, and incubate the reaction mixture at 37 &#176;C for 1 h.</li>
	</ol>
	</li>
	<li>Poly(A) tailing
	<ol>
		<li>Spin down the sample, and then add 6 &#181;L of nuclease-free water, 20 &#181;L of 5x E-PAP buffer, 10 &#181;L of 25 mM MnCl2, 10 &#181;L of 10 mM ATP, and 4 &#181;L of E-PAP poly(A) tailing enzyme. Mix by tapping, spin down, and incubate the reaction mixture at 37 &#176;C for 2 h.</li>
	</ol>
	</li>
	<li>Lithium chloride precipitation and quantification of the synthetic mRNA
	<ol>
		<li>Add 50 &#181;L of lithium chloride (7.5 M), and perform lithium chloride precipitation as described in steps 1.3.1-1.3.3.</li>
		<li>Measure the concentration of the mRNA using a spectrophotometer.</li>
		<li>Verify the size and quantity of mRNA using formaldehyde (1%-2%) agarose (1%) gel electrophoresis.</li>
	</ol>
	</li>
</ol>
2. Extracellular matrix (ECM) gel coating of the cell invasion and migration (CIM) plates
NOTE: A cell invasion and migration (CIM) plate is a commercially manufactured 16-well plate for impedance-based real-time cell analysis. For the cell invasion assay, coat CIM plates with ECM gel, as previously described but with some modifications11.
<ol>
	<li>Remove an aliquot of ECM gel stock from the freezer and keep it on ice.</li>
	<li>Dilute the ECM gel (10 mg/mL) to a working concentration (100 &#181;g/mL) by mixing 990 &#181;L of Dulbecco&#39;s Modified Eagle Medium (DMEM) (without serum or antibiotics) with 10 &#181;L of ECM gel in a microcentrifuge tube. Mix by gentle pipetting.</li>
	<li>Add 60 &#181;L of diluted ECM gel to each of the 16 wells of the upper chamber of the CIM plate. Apply the reverse pipetting method while avoiding air bubbles20,21. 
	NOTE: Optimize the concentration of the ECM gel for each cell line. For glioblastoma cell lines, 0.1 &#181;g/&#181;L to 1 &#181;g/&#181;L ECM gel was used for optimization.</li>
	<li>Place the upper chamber of the CIM plate with the plate cover off on a protective plastic sheet inside a CO2 incubator for approximately 4 h to form a gel layer. 
	&#8203;CAUTION: During the coating of the CIM plate with ECM gel, the electrodes of the upper chamber of the CIM plate should not have direct contact with the experimenter&#39;s hands or the surfaces of the equipment, including the biosafety cabinet or the CO2 incubator.</li>
</ol>
3. Preparation of the tumor cells
NOTE: All the cell culture materials must be kept sterile. Harvest and electroporate the tumor cells under a biological safety cabinet with appropriate personal protective equipment (PPE), as previously described but with some modifications11.
<ol>
	<li>Culture of the tumor cells
	<ol>
		<li>Culture U-118MG cells in 10 mL of DMEM containing 5% fetal bovine serum (FBS) and 1% antibiotics per 100 mm x 20 mm polystyrene tissue culture dish at 37 &#176;C in a 5% CO2 incubator (culture condition).</li>
	</ol>
	</li>
	<li>Serum depletion of the tumor cells 
	NOTE: The exposure of the cells to the chemoattractants present in FBS must be minimized before the cell migration and invasion assays by using a high concentration of FBS.
	<ol>
		<li>Remove the old medium, and add 10 mL of prewarmed DMEM containing 0.5% FBS and 1% antibiotics per dish (low-serum medium).</li>
		<li>Repeat step 3.2.1.</li>
		<li>Incubate the cells at 37 &#176;C in a 5% CO2 incubator for 4 h or longer.</li>
	</ol>
	</li>
	<li>Harvesting of the tumor cells
	<ol>
		<li>Remove the old medium, add 8 mL of prewarmed Dulbecco&#39;s phosphate-buffered saline (DPBS) per dish, remove the DPBS, add prewarmed 0.05% trypsin-EDTA (2 mL/dish) to cover the surface, and incubate in the CO2 incubator for 30 s.</li>
		<li>Aspirate the trypsin-EDTA carefully, add low-serum medium (7 mL/dish), and then collect cells into a 50 mL or 15 mL centrifuge tube.</li>
		<li>Aliquot a small volume of cells, and use a handheld automated cell counter to count the cells.</li>
		<li>Calculate the total number of cells and the required volume for 10,000 cells/&#181;L.</li>
		<li>Spin down the cells by centrifuging them at 100 &#215; g for 5 min at room temperature, aspirate the supernatant, add the calculated volume of DMEM containing 0.5% FBS (without antibiotics), and gently resuspend the cell pellet.</li>
		<li>Transfer 110 &#181;L of the cell suspension, containing 1.1 million cells, to a microcentrifuge tube for each treatment.</li>
		<li>Repeat step 3.3.6 to transfer the cells to four microcentrifuge tubes for four different treatments. 
		&#8203;NOTE: A CIM plate has a total of 16 wells. Design four different treatments for comparison so that four wells of the CIM plate can be assigned for each treatment. Use the electroporated cells for the following experiments: western blot analyses (0.3 million cells per 35 mm tissue culture dish), four wells of real-time cell migration assays (0.1 million cells/well), and four wells of real-time cell invasion assays (0.1 million cells/well) for each treatment. Adjust the number of cells that need to be electroporated if the experimental design changes.</li>
	</ol>
	</li>
</ol>
4. Electroporation of the tumor cells
<ol>
	<li>To remove the medium, add 1 mL of DPBS to the cell suspension in each tube, spin down the cell suspension three times for 10 s each time while rotating the tube 180&#176; every 10 s using a mini centrifuge, and remove the supernatant using a micropipette. 
	NOTE: It is important to form a compact but easily resuspendable cell pellet.</li>
	<li>Add 110 &#956;L of resuspension buffer R to the cell pellet to obtain 0.1 million cells in 10 &#956;L.</li>
	<li>Add synthetic mRNA to the cell pellet to obtain a concentration of 0.2-20 ng/&#181;L depending on the desired expression level. Mix and resuspend the cell pellet gently by tapping or gentle pipetting. 
	NOTE: Use different concentrations of synthetic mRNA, examine the protein expression using western blot analysis, and determine the concentration of synthetic mRNA that leads to the desired expression level in order to investigate the specific correlation between the protein expression and biological effects.</li>
	<li>Electroporate 10 &#181;L of the cell suspension with an electroporation system at 1,350 V for 10 ms with three pulses each time.</li>
	<li>Transfer the electroporated cells into a new microcentrifuge tube with 1.1 mL of DMEM containing 0.5% FBS.</li>
	<li>Repeat the electroporation until the rest of the cell suspension is electroporated. Combine the electroporated cells in the microcentrifuge tube to obtain 1.1 million cells in 1.1 mL. 
	NOTE: Both 100 &#181;L and 10 &#181;L electroporation tips may be used to electroporate a large volume of cells, but the electroporation tips for 10 &#181;L and 100 &#181;L are included in two separate kits and need to be purchased separately. Resuspension buffer R is included in both kits.</li>
	<li>Upon completing all the respective electroporations, gently resuspend the pooled cells.</li>
	<li>Plate 0.3 million cells in a 35 mm x 10 mm polystyrene tissue culture dish with 2 mL of DMEM containing 5% FBS, and culture the cells for 1 day under the culture condition for total cell lysate preparation and western blot analyses.</li>
	<li>Keep the rest of the electroporated cells at room temperature until the real-time cell analysis system is ready.</li>
</ol>
5. Setting up the real-time cell analyzer, the program, and the CIM plates
NOTE: Prepare the real-time cell analyzer and two CIM plates, as previously described11.
<ol>
	<li>Equilibration of the real-time cell analyzer
	<ol>
		<li>Move the real-time cell analyzer into a CO2 incubator several hours before use to equilibrate the system under the culture condition.</li>
	</ol>
	</li>
	<li>Setting up the analysis program
	<ol>
		<li>Open the analysis program by double-clicking on the real-time cell analysis software icon&#160;on the desktop.</li>
		<li>Once the Default Experiment Pattern Setup option is open, select the option for running three experiments separately. 
		NOTE: Each cradle has a separate window. There are different tabs to set up the measurement interval and duration, data analysis, and other experimental conditions.</li>
		<li>Open each cradle by clicking on the Number tab.</li>
		<li>Click on the Experiment Notes tab, select the folder that the data will be saved to, and enter the name of the experiment.</li>
		<li>Click on the Layout tab, set up quadruplicate wells for each treatment condition by selecting four wells at a time and entering the sample information, and then click on Apply.</li>
		<li>Click on the Schedule tab | Add a step to set up the two-step mode of the cell impedance measurements. Then, click on Apply to set up the first step.</li>
		<li>Click on Add a step again, enter 10 min for the interval and 48 h for the duration for migration and invasion, and click on Apply to set up the second step. 
		NOTE: The first step takes a one-time baseline measurement (one sweep with a 1 min interval). The second step measures the cell impedance for the experiment (for example, 289 sweeps with a 10 min interval for 48 h) at the cradle. Adjust the interval and duration depending on the experimental design.</li>
		<li>Move to the next cradle, and set it up by repeating steps 5.2.3-5.2.7.</li>
	</ol>
	</li>
	<li>Preparation of the CIM plates
	<ol>
		<li>One hour before the cell impedance measurement starts, fill the wells of the lower chamber of the CIM plate with 160 &#181;L of DMEM containing 10% serum or other chemoattractants.</li>
		<li>Assemble the upper chamber that contains the ECM gel-coated wells (for invasion) or uncoated wells (for migration) with the lower chamber.</li>
		<li>Add 50 &#181;L of low-serum medium to the wells of the upper chamber of the CIM plate for the cell migration assay, and place the CIM plate in the cradle of the system.</li>
		<li>Click on the Message tab, and ensure that the Connections ok message is displayed. The CIM plate is now ready for the experiment.</li>
		<li>Preincubate the assembled CIM plate in the CO2 incubator for 30-60 min before the real-time cell analysis to acclimate the CIM plate to the culture condition.</li>
	</ol>
	</li>
</ol>
6. Real-time cell analysis and data export
NOTE: Perform a baseline reading, cell seeding, cell impedance measurement, and data export as previously described11.
<ol>
	<li>Baseline reading 
	NOTE: The baseline should be read after the CIM plate is acclimated and before the cells are added to the wells of the upper chamber.
	<ol>
		<li>Click on the Start button for each cradle. When the Save As window appears, save the experimental file to perform the baseline reading.</li>
	</ol>
	</li>
	<li>Cell seeding
	<ol>
		<li>Remove the CIM plate from the cradle, and place it in the biosafety cabinet on the plate holder.</li>
		<li>Add 100 &#181;L (containing 100,000 cells) of electroporated cells to the wells of the upper chamber of the CIM plate according to the program in the control unit. Apply the reverse pipetting method while avoiding air bubbles.</li>
		<li>Leave the CIM plate under the biosafety cabinet for 30 min at room temperature to make sure the cells spread evenly on the well bottom.</li>
	</ol>
	</li>
	<li>Cell impedance measurement
	<ol>
		<li>Move the fully assembled CIM plate back to the respective cradle. Click on the cradle Start button to begin the cell impedance measurement for the second step.</li>
		<li>Click on the Plot tab, then click the Add All button, and select the Average and STD DEV boxes to visualize the data in real time. 
		&#8203;NOTE: The default plotting option is Time for the x-axis and Cell Index for the y-axis. The Plot Selection section allows the user to select alternative options for the y-axis: Normalized Cell Index or Delta Cell Index. Once the final sweep is made, the experiment is completed, and the results are saved automatically.</li>
	</ol>
	</li>
	<li>Export of the data for analysis
	<ol>
		<li>Click on the Plot tab and select the Average and STD DEV boxes to copy the data for each well individually.</li>
		<li>Right-click on the plot area, and select Copy Data in List Format.</li>
		<li>Open an empty spreadsheet file, paste the data, and then save the file.</li>
		<li>Go back to the analysis program, click on the Plate tab for each cradle, and select Release to close the experiment for the cradle.</li>
		<li>Go back to the spreadsheet file, and adjust the time of the raw data so that the start time at the second step becomes the actual start time for the measurement.</li>
	</ol>
	</li>
</ol>

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<li>Pushparaj, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Revisiting+the+micropipetting+techniques+in+biomedical+sciences%3A+A+fundamental+prerequisite+in+good+laboratory+practice%5BTitle%5D)+AND+%22Bioinformation%22%5BJournal%5D)">Revisiting the micropipetting techniques in biomedical sciences: A fundamental prerequisite in good laboratory practice.</a> Bioinformation. 16 (1), 8-12 (2020).</li>
<li>Park, T. J., Curran, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crk+and+Crk-like+play+essential+overlapping+roles+downstream+of+disabled-1+in+the+Reelin+pathway%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">Crk and Crk-like play essential overlapping roles downstream of disabled-1 in the Reelin pathway.</a> The Journal of Neuroscience. 28 (50), 13551-13562 (2008).</li>
<li>Huang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CRK+proteins+selectively+regulate+T+cell+migration+into+inflamed+tissues%5BTitle%5D)+AND+%22The+Journal+of+Clinical+Investigation%22%5BJournal%5D)">CRK proteins selectively regulate T cell migration into inflamed tissues.</a> The Journal of Clinical Investigation. 125 (3), 1019-1032 (2015).</li>
<li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Signaling+adaptor+protein+Crk+is+indispensable+for+malignant+feature+of+glioblastoma+cell+line+KMG4%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">Signaling adaptor protein Crk is indispensable for malignant feature of glioblastoma cell line KMG4.</a> Biochemical and Biophysical Research Communications. 362 (4), 976-981 (2007).</li>
<li>Kumar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reciprocal+regulation+of+Abl+kinase+by+Crk+Y251+and+Abi1+controls+invasive+phenotypes+in+glioblastoma%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">Reciprocal regulation of Abl kinase by Crk Y251 and Abi1 controls invasive phenotypes in glioblastoma.</a> Oncotarget. 6 (35), 37792-37807 (2015).</li>
<li>Kumar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Crk+tyrosine+phosphorylation+regulates+PDGF-BB-inducible+Src+activation+and+breast+tumorigenicity+and+metastasis%5BTitle%5D)+AND+%22Molecular+Cancer+Research%22%5BJournal%5D)">Crk tyrosine phosphorylation regulates PDGF-BB-inducible Src activation and breast tumorigenicity and metastasis.</a> Molecular Cancer Research. 16 (1), 173-183 (2018).</li>
<li>Raudenska, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engine+shutdown%3A+Migrastatic+strategies+and+prevention+of+metastases%5BTitle%5D)+AND+%22Trends+in+Cancer%22%5BJournal%5D)">Engine shutdown: Migrastatic strategies and prevention of metastases.</a> Trends in Cancer. 9 (4), 293-308 (2023).</li>
<li>Decaestecker, C., Debeir, O., Van Ham, P., Kiss, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Can+anti-migratory+drugs+be+screened+in+vitro%3F+A+review+of+2D+and+3D+assays+for+the+quantitative+analysis+of+cell+migration%5BTitle%5D)+AND+%22Medicinal+Research+Reviews%22%5BJournal%5D)">Can anti-migratory drugs be screened in vitro? A review of 2D and 3D assays for the quantitative analysis of cell migration.</a> Medicinal Research Reviews. 27 (2), 149-176 (2007).</li>
<li>Farasani, A., Darbre, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+exposure+to+triclosan+increases+migration+and+invasion+of+human+breast+epithelial+cells+in+vitro%5BTitle%5D)+AND+%22Journal+of+Applied+Toxicology%22%5BJournal%5D)">Long-term exposure to triclosan increases migration and invasion of human breast epithelial cells in vitro.</a> Journal of Applied Toxicology. 41 (7), 1115-1126 (2021).</li>
<li>Hanusova, V., Skalova, L., Kralova, V., Matouskova, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+flubendazole+on+adhesion+and+migration+in+SW480+and+SW620+colon+cancer+cells%5BTitle%5D)+AND+%22Anti-Cancer+Agents+in+Medicinal+Chemistry%22%5BJournal%5D)">The effect of flubendazole on adhesion and migration in SW480 and SW620 colon cancer cells.</a> Anti-Cancer Agents in Medicinal Chemistry. 18 (6), 837-846 (2018).</li>
<li>Lago-Baameiro, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PARK7%2FDJ-1+inhibition+decreases+invasion+and+proliferation+of+uveal+melanoma+cells%5BTitle%5D)+AND+%22Tumori%22%5BJournal%5D)">PARK7/DJ-1 inhibition decreases invasion and proliferation of uveal melanoma cells.</a> Tumori. 109 (1), 47-53 (2023).</li>
<li>Escalona, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Knock+down+of+TIMP-2+by+siRNA+and+CRISPR%2FCas9+mediates+diverse+cellular+reprogramming+of+metastasis+and+chemosensitivity+in+ovarian+cancer%5BTitle%5D)+AND+%22Cancer+Cell+International%22%5BJournal%5D)">Knock down of TIMP-2 by siRNA and CRISPR/Cas9 mediates diverse cellular reprogramming of metastasis and chemosensitivity in ovarian cancer.</a> Cancer Cell International. 22 (1), 422(2022).</li>
<li>Mosca, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+S-adenosyl-L-methionine+on+the+invasion+and+migration+of+head+and+neck+squamous+cancer+cells+and+analysis+of+the+underlying+mechanisms%5BTitle%5D)+AND+%22International+Journal+of+Oncology%22%5BJournal%5D)">Effects of S-adenosyl-L-methionine on the invasion and migration of head and neck squamous cancer cells and analysis of the underlying mechanisms.</a> International Journal of Oncology. 56 (5), 1212-1224 (2020).</li>
<li>Zhao, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((VEGI174+protein+and+its+functional+domain+peptides+exert+antitumour+effects+on+renal+cell+carcinoma%5BTitle%5D)+AND+%22International+Journal+of+Oncology%22%5BJournal%5D)">VEGI174 protein and its functional domain peptides exert antitumour effects on renal cell carcinoma.</a> International Journal of Oncology. 54 (1), 390-398 (2019).</li>
<li>Witte, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TGF-beta1-induced+cell+migration+in+pancreatic+carcinoma+cells+is+RAC1+and+NOX4-dependent+and+requires+RAC1+and+NOX4-dependent+activation+of+p38+MAPK%5BTitle%5D)+AND+%22Oncology+Reports%22%5BJournal%5D)">TGF-beta1-induced cell migration in pancreatic carcinoma cells is RAC1 and NOX4-dependent and requires RAC1 and NOX4-dependent activation of p38 MAPK.</a> Oncology Reports. 38 (6), 3693-3701 (2017).</li>
<li>Bui, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+aquaporin+3+expression+by+the+AhR+pathway+is+critical+to+cell+migration%5BTitle%5D)+AND+%22Toxicological+Sciences%22%5BJournal%5D)">Regulation of aquaporin 3 expression by the AhR pathway is critical to cell migration.</a> Toxicological Sciences. 149 (1), 158-166 (2016).</li>
<li>Tsuji, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+different+cell-detaching+methods+on+the+viability+and+cell+surface+antigen+expression+of+synovial+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Cell+Transplantation%22%5BJournal%5D)">Effects of different cell-detaching methods on the viability and cell surface antigen expression of synovial mesenchymal stem cells.</a> Cell Transplantation. 26 (6), 1089-1102 (2017).</li>
<li>Frandsen, S. K., McNeil, A. K., Novak, I., McNeil, P. L., Gehl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Difference+in+membrane+repair+capacity+between+cancer+cell+lines+and+a+normal+cell+line%5BTitle%5D)+AND+%22The+Journal+of+Membrane+Biology%22%5BJournal%5D)">Difference in membrane repair capacity between cancer cell lines and a normal cell line.</a> The Journal of Membrane Biology. 249 (4), 569-576 (2016).</li>
</ol>

The authors have no conflicts of interest to disclose.

Migration and invasion are important features of tumor cells. Measuring the motility of tumor cells and understanding the underlying mechanism that controls tumor cell motility provide critical insights into therapeutic interventions2,27. Several methods have been developed to study cell migration7. The wound-healing assay using scratches or culture inserts is a simple and frequently used method that provides contrasting images of gap clos...

Tumor cell motility plays a crucial role in metastasis1,2. The spread of tumor cells to neighboring and remote healthy tissues makes cancer treatment difficult and contributes to recurrence3,4. Therefore, it is essential to understand the mechanisms of tumor cell motility and develop relevant therapeutic strategies. Since many tumor cells have altered gene expression profiles, it is crucial to understand which changes in the gene expression profile lead to altered tumor cell motility5,6.
Several assays have been developed to measure cell migration in vitro. Some assays only provide limited information due to only allowing measurements at specific time points, whereas others offer comprehensive information on tumor cell motility in real time7. Although many of these cell motility assays can provide quantitative results at a given time or the endpoint, they fail to provide sufficiently detailed information on dynamic changes in the rate of cell migration over the experimental period. In addition, it may be difficult to examine potential changes in the cell migration rate depending on experimental design, cell types, and cell numbers. Furthermore, the effects of uncomplicated treatments can be investigated by the simple quantification of traditional motility assays, but more sophisticated quantification may be required to study the complex effects of various combined treatments8.
An instrument to monitor the electrical current of a microtiter plate well bottom covered with microelectrodes has been developed9. The adhesion of cells to the surface of the well impedes the electron flow, and the impedance correlates with the quantitative and qualitative binding of the cells. The presence of the microelectrodes on the well bottom allows for the measurement of cell adhesion, spreading, and proliferation. The presence of the microelectrodes underneath a microporous membrane of the upper chamber allows for the measurement of cell migration and invasion into the lower chamber, with the upper chamber coated with extracellular matrix (ECM) proteins to allow for invasion10.
Previously, it was demonstrated that impedance-based real-time measurements of tumor cell migration and invasion provide real-time data during the whole experiment, as well as instant comparisons and quantifications under various experimental conditions11. In that method paper, gene knockdown was induced to test the role of proteins of interest in tumor cell migration and invasion. Since a full-blown gene knockdown effect under the tested experimental conditions took 3-4 days after electroporation with small interfering RNAs (siRNAs)8, the cells were replated after the electroporation and reharvested 3 days later for the impedance-based real-time measurement of tumor cell migration and invasion.
CT10 regulator of kinase (Crk) and Crk-like (CrkL) are adaptor proteins that mediate protein-protein interactions downstream of various growth factor receptor kinase pathways and nonreceptor tyrosine kinase pathways12. Elevated levels of Crk and CrkL proteins contribute to poor prognosis in several human cancers, including glioblastoma13. However, it is unclear how elevated Crk and CrkL proteins lead to a poor prognosis. Therefore, it is important to define the effect of Crk and CrkL overexpression on tumor cell functions. Previously, a gene knockdown study was performed to demonstrate that endogenous levels of Crk and CrkL proteins are required for glioblastoma cell migration and invasion8. Here, a modified assay system has been developed to address the effect of Crk and CrkL overexpression on tumor cell migration and invasion.
Recently, the in vitro synthesis of mRNA and its therapeutic applications have drawn renewed attention due to the development of the mRNA vaccines against SARS-CoV-2 (reviewed by Verbeke et al.14). In addition, remarkable advances have been made in using synthetic mRNA in cancer and other diseases15,16. The electroporation of cells is an effective method to deliver synthetic mRNA and induce transient genetic modification (reviewed by Campillo-Davo et al.17), and the use of synthetic mRNA enables rapid and efficient gene expression in immortalized fibroblasts18. This method paper combines gene overexpression using synthetic mRNA with real-time cell analyses to study tumor cell migration and invasion. However, the experimental scheme used for siRNAs does not work with synthetic mRNA transfection, as the level of exogenous proteins increases rapidly and decreases gradually upon synthetic mRNA transfection18. Therefore, the method has been modified to carry out the real-time analysis of cell migration and invasion right after the transfection without additionally culturing the cells.
This method paper demonstrates that combining impedance-based real-time measurements with the transfection of tumor cells with synthetic mRNAs provides a rapid and comprehensive analysis of the effects of gene upregulation on tumor cell migration and invasion. This method paper describes detailed procedures for measuring how the migration and invasion of glioblastoma cells are affected by the overexpression of Crk and CrkL. By examining the concentration-dependent effects of synthetic mRNA on tumor cell migration, the paper clearly describes how an increase in protein levels stimulates tumor cell migration. In addition, an approach of varying the concentration of the ECM gel is presented to assess the effects of changes in gene expression on tumor cell invasion.

<table><tbody><tr><td>AlphaImager HP</td><td>ProteinSimple</td><td>92-13823-00</td><td>Agarose gel imaging system</td></tr><tr><td>&amp;alpha;-Tubulin antibody</td><td>Sigma</td><td>T9026</td><td>Used to detect &amp;alpha;-tubulin protein (dilution 1:3,000)</td></tr><tr><td>CIM-plate 16</td><td>Agilent Technologies, Inc</td><td>5665825001</td><td>Cell invasion and migration plates</td></tr><tr><td>Crk antibody</td><td>BD Biosciences</td><td>610035</td><td>Used to detect CrkI and CrkII proteins (dilution 1:1,500)</td></tr><tr><td>CrkL antibody</td><td>Santa Cruz</td><td>sc-319</td><td>Used to detect CrkL protein (dilution 1:1,500)</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>ATCC</td><td>302002</td><td>Cell culture medium</td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS)</td><td>Corning</td><td>21-031-CV</td><td>Buffer used to wash cells</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Hyclone</td><td>SH30910.03</td><td>Culture medium supplement</td></tr><tr><td>Heracell VIOS 160i CO2 incubator</td><td>Thermo Scientific</td><td>51030285</td><td>CO2 incubator</td></tr><tr><td>IRDye 800CW goat anti-mouse IgG secondary antibody</td><td>Li-Cor</td><td>926-32210</td><td>Secondary antibody for Western blot analysis (dilution 1:10,000)</td></tr><tr><td>IRDye 800CW goat anti-rabbit IgG secondary antibody</td><td>Li-Cor</td><td>926-32211</td><td>Secondary antibody for Western blot analysis&amp;nbsp; (dilution 1:10,000)</td></tr><tr><td>Lithium chloride&amp;nbsp;</td><td>Invitrogen</td><td>AM9480</td><td>Used for RNA precipitation</td></tr><tr><td>Matrigel matrix</td><td>Corning</td><td>354234</td><td>Extracellular matrix (ECM) gel</td></tr><tr><td>MEGAscript T7 transcription kit</td><td>Invitrogen</td><td>AM1334</td><td>Used for RNA synthesis</td></tr><tr><td>Millennium RNA markers</td><td>Invitrogen</td><td>AM7150</td><td>Used for formaldehyde agarose gel electrophoresis</td></tr><tr><td>Mini centrifuge</td><td>ISC BioExpress</td><td>C1301P-ISC</td><td>Used to spin down cells</td></tr><tr><td>Mouse brain QUICK-Clone cDNA</td><td>TaKaRa</td><td>637301</td><td>Source of genes (inserts) for cloning</td></tr><tr><td>NanoQuant</td><td>Tecan</td><td>M200PRO</td><td>Nucleic acid quantification system</td></tr><tr><td>Neon electroporation system&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>MPK5000</td><td>Electroporation system&lt;sup&gt;1&lt;/sup&gt;</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>Neon transfection system 100 &amp;micro;L kit</td><td>ThermoFisher Scientific</td><td>MPK10096</td><td>Electroporation kit</td></tr><tr><td>NorthernMax denaturing gel buffer</td><td>Invitrogen</td><td>AM8676</td><td>Used for formaldehyde agarose gel electrophoresis</td></tr><tr><td>NorthernMax formaldehyde load dye</td><td>Invitrogen</td><td>AM8552</td><td>Used for formaldehyde agarose gel electrophoresis</td></tr><tr><td>NorthernMax running buffer</td><td>Invitrogen</td><td>AM8671</td><td>Used for formaldehyde agarose gel electrophoresis</td></tr><tr><td>Nuclease-free water</td><td>Teknova</td><td>W3331</td><td>Used for various reactions during mRNA synthesis</td></tr><tr><td>Odyssey CLx Imager</td><td>Li-Cor</td><td></td><td>Imager for Western blot analysis</td></tr><tr><td>pcDNA3.1/myc-His</td><td>Invitrogen</td><td>V80020</td><td>The vector into which inserts (mouse CrkI and CrkL cDNAs) were cloned</td></tr><tr><td>pFLAG-CMV-5a</td><td>Millipore Sigma</td><td>E7523</td><td>Source of the FLAG epitope tag</td></tr><tr><td>Phenol:chloroform:isoamyl alcohol&amp;nbsp;</td><td>Sigma</td><td>P2069</td><td>Used for DNA extraction</td></tr><tr><td>PmeI</td><td>New England BioLabs</td><td>R0560L</td><td>Used to linearize the plasmids for mRNA synthesis</td></tr><tr><td>Poly(A) tailing kit</td><td>Invitrogen</td><td>AM1350</td><td>Used for poly(A) tail reaction</td></tr><tr><td>Polystyrene tissue culture dish (100 x 20 mm style)</td><td>Corning</td><td>353003</td><td>Used for culturing cells before transfection</td></tr><tr><td>Polystyrene tissue culture dish (35 x 10 mm style)</td><td>Corning</td><td>353001</td><td>Used for culturing transfected cells</td></tr><tr><td>Proteinase K</td><td>Invitrogen</td><td>25530049</td><td>Used to remove protein in the reaction mixture</td></tr><tr><td>Purifier Axiom Class II, Type C1</td><td>Labconco Corporation</td><td>304410001</td><td>Biosafety cabinet for sterile handling of cells</td></tr><tr><td>Resuspension Buffer R</td><td>ThermoFisher Scientific</td><td></td><td>A buffer included in the electroporation kits, MPK1025 and MPK10096. The buffer is used to resupend cells before electroporation, and its composition is proprietary information.</td></tr><tr><td>RNaseZap</td><td>Invitrogen</td><td>AM9780</td><td>RNA decontamination solution</td></tr><tr><td>Scepter</td><td>Millipore</td><td>C85360</td><td>Handheld automated cell counter&amp;nbsp;</td></tr><tr><td>ScriptCap 2&#039;-O-methyltransferase kit</td><td>Cellscript</td><td>C-SCMT0625</td><td>Used for capping reaction</td></tr><tr><td>ScriptCap m&lt;sup&gt;7&lt;/sup&gt;G capping system</td><td>Cellscript</td><td>C-SCCE0625</td><td>Used for capping reaction</td></tr><tr><td>Sodium dodecyl sulfate solution</td><td>Invitrogen</td><td>15553-035</td><td>Detergent used for the proteinase K reaction</td></tr><tr><td>Sorvall Legend XT centrifuge</td><td>Thermo Scientific</td><td>75004532</td><td>Benchtop centrifuge to spin down cells</td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td>Used for dissociation of cells</td></tr><tr><td>U-118MG&amp;nbsp;</td><td>ATCC</td><td>HTB15</td><td>An adherent cell line derived from a human glioblastoma patient</td></tr><tr><td>Vinculin antibody</td><td>Sigma</td><td>V9131</td><td>Used to detect vinculin protein (dilution 1:100,000)</td></tr><tr><td>xCELLigence RTCA DP</td><td>Agilent Technologies, Inc</td><td>380601050</td><td>Instrument used for real-time cell analysis</td></tr><tr><td>&lt;sup&gt;1&lt;/sup&gt;Electroporation parameters and other related information for various cell lines are available on the manufacturer&#039;s homepage (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/neon-transfection-system/neon-transfection-system-cell-line-data.html?).</td><td></td><td></td><td></td></tr></tbody></table>

real-time quantitative measurement of tumor cell migration and invasion following synthetic mrna transfection

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.

Crk and CrkL proteins play important roles in the motility of many cell types, including neurons22, T cells23, fibroblasts18,19, and a variety of tumor cells13. Since Crk and CrkL proteins have been reported to be elevated in glioblastoma24,25,26, the effects of the overexpression of CrkI, a splice v...

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 ...

real-time-quantitative-measurement-tumor-cell-migration-invasion

Research

JoVE Journal

Cancer Research

An assay to quantitatively measure Transforming Growth Factor (TGF)-β-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

Spheroid Assay to Measure TGF-&#946;-induced Invasion

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modeling and Imaging 3-Dimensional Collective Cell Invasion

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

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.

In vitro Cell Migration and Invasion Assays

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.

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

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.

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

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.

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

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.

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

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

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...

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

Learn real-time tumor cell migration and invasion measurement using synthetic mRNA transfection with impedance-based analysis. This method identifies genes that stimulate metastasis for cancer therapy development.

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

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