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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J., Curran, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crk+and+Crk-like+play+essential+overlapping+roles+downstream+of+disabled-1+in+the+Reelin+pathway.">Crk and Crk-like play essential overlapping roles downstream of disabled-1 in the Reelin pathway.</a> 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRK+proteins+selectively+regulate+T+cell+migration+into+inflamed+tissues.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+adaptor+protein+Crk+is+indispensable+for+malignant+feature+of+glioblastoma+cell+line+KMG4.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+regulation+of+Abl+kinase+by+Crk+Y251+and+Abi1+controls+invasive+phenotypes+in+glioblastoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crk+tyrosine+phosphorylation+regulates+PDGF-BB-inducible+Src+activation+and+breast+tumorigenicity+and+metastasis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engine+shutdown:+Migrastatic+strategies+and+prevention+of+metastases.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+anti-migratory+drugs+be+screened+in+vitro?+A+review+of+2D+and+3D+assays+for+the+quantitative+analysis+of+cell+migration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+exposure+to+triclosan+increases+migration+and+invasion+of+human+breast+epithelial+cells+in+vitro.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+flubendazole+on+adhesion+and+migration+in+SW480+and+SW620+colon+cancer+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PARK7/DJ-1+inhibition+decreases+invasion+and+proliferation+of+uveal+melanoma+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knock+down+of+TIMP-2+by+siRNA+and+CRISPR/Cas9+mediates+diverse+cellular+reprogramming+of+metastasis+and+chemosensitivity+in+ovarian+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGI174+protein+and+its+functional+domain+peptides+exert+antitumour+effects+on+renal+cell+carcinoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+aquaporin+3+expression+by+the+AhR+pathway+is+critical+to+cell+migration.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+different+cell-detaching+methods+on+the+viability+and+cell+surface+antigen+expression+of+synovial+mesenchymal+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difference+in+membrane+repair+capacity+between+cancer+cell+lines+and+a+normal+cell+line.">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 closure. The individual cell-tracking assay requires monitoring individual cells with time-lapse imaging, for which the cells can be tagged with fluorescent dyes. Both the wound-healing assay and the individual cell-tracking assay measure the spontaneous movement of cells.
With the help of time-lapse imaging and intensive post-requisition data processing, both assays can provide quantitative comparisons among samples28. However, these assays are not suitable for studying cell invasion through an ECM protein layer. In contrast, the transwell migration and invasion assays measure directed cell migration through a transwell insert membrane with or without an ECM protein layer. However, continual monitoring is not feasible with these assays because the migrated cells need to be collected at a given time point, and the transwell cannot be used again. All these assays require significant time and effort for data processing or for hands-on experiments to collect and count the cells, resulting in potential operator-related variations. The biggest challenge for these assays is making sophisticated quantitative comparisons among multiple, combined treatments at various time points.
The use of the real-time cell analysis system presented in this work allows for quantitative, continuous, and comprehensive monitoring to measure cell migration and invasion, and this system has many advantages over other simple cell motility assays, which provide results at limited, fixed time points. As with other assays, the experimental conditions of the real-time cell analyses need to be optimized for each cell line, as the migration and invasion of different cell lines can be differentially affected by the cell number. Furthermore, the rate of cell invasion decreases as the concentration of the ECM gel increases (Figure 2B,C). Therefore, it is recommended to test different ECM gel concentrations and compare the effects of gene expression changes on cell invasion under these different ECM gel concentrations.
With the real-time cell analysis system, optimization is easy and straightforward, as the assay system produces data in real time with no hands-on time. The assay system identifies how soon the cells migrate or invade and when they reach the maximal cell migration or invasion level. Obtaining this information on cell motility enables detailed comparative analyses among various treatment groups, which can be done simply by using the program&#39;s built-in features. Furthermore, the sensitivity of the real-time assay system allows for the identification and quantification of subtle changes in cell migration and invasion by concentration-dependent gene expression, as demonstrated in Figure 1 and Figure 2.
Previously, a detailed procedure was provided to measure tumor cell migration and invasion following gene knockdown using the impedance-based real-time cell analysis system. Since gene knockdown takes 3-4 days after the cells are electroporated with siRNAs, the cells were re-plated after electroporation. The electroporated cells were cultured for 3 days before they were reharvested for the real-time cell analyses, making the entire experiment a two-step process: electroporation on day 1 and real-time cell analysis on day 4, as shown in Figure 3. In contrast, gene expression upon the electroporation of cells with synthetic mRNA is rapid and efficient, as the time-lapse analysis of fibroblasts electroporated with synthetic mRNA for GFP showed a strong GFP signal 5 h after transfection; the fluorescence intensity reached a maximum around 24 h, after which there was a gradual decline in the fluorescence signal18.
In addition, in this work, the U-118MG cells showed robust expression of the exogenous protein when electroporated with synthetic mRNA for CrkI (Figure 1A) and for CrkL (Figure 2A), consistent with previous observations8. Therefore, it is appropriate to carry out the real-time cell analyses right after electroporation. Some of the steps for the real-time cell analysis should be performed before harvesting the cells for electroporation. The entire experiment is a one-step process involving electroporation and real-time cell analysis on day 1. The impedance-based real-time cell analysis system has been used extensively to study tumor cell migration and invasion in various solid tumor cells, including breast cancer29, colorectal cancer30, melanoma31, ovarian cancer32, head and neck squamous cancer33, renal cell carcinoma34, pancreatic carcinoma35, hepatocellular carcinoma36, and non-small-cell lung cancer cells10. Therefore, the combined use of gene overexpression using mRNA or gene knockdown using siRNAs makes the real-time measurement of cell migration and invasion more useful and applicable.
The limitation of this protocol is that this method requires dissociating and harvesting cells right before the measurement of the cell migration and invasion. The enzymatic and mechanical treatments during dissociation, harvest, and resuspension may affect the analysis37. In addition, there can be a delay in cell migration while the cells recover from the enzymatic and mechanical treatments. This method may not be appropriate if the cells are easily damaged by trypsinization or other mechanical treatments during single-cell dissociation and collection or require a long recovery time after those manipulations. This limitation also applies to the transwell migration assay, which is another method for measuring directed cell migration. In addition, the electroporation following cell preparation may make cells more vulnerable to damage38. Therefore, it is important to optimize the conditions for electroporation for each cell line and also to electroporate the control cells for more accurate comparisons.
The manufacturer&#39;s homepage for the electroporation system provides the recommended parameters for electroporation (see the footnote in the Table of Materials). Using consistent and minimally damaging experimental conditions during cell dissociation and resuspension is critical for obtaining reproducible results. Furthermore, correlating the amount of mRNA, the protein level, and the cell motility is crucial for distinguishing between the specific and nonspecific effects of mRNA transfection. In this work, it was observed that if the mRNA concentration exceeded a certain level, it nonspecifically inhibited cellular functions, including cell migration and invasion (data not shown). Therefore, it is important to titrate the concentration of mRNA. In addition, it is critical to perform the real-time cell analysis when the protein expression is at its peak level, since protein expression is transient with mRNA transfection. As with other cell motility assays, the results of this real-time cell analysis can be confounded by the proliferation of cells during migration. Therefore, it is recommended to additionally carry out a cell proliferation assay to understand the influence of cell proliferation on the cell migration and invasion results.
The protein levels of Crk and CrkL are known to be elevated in some types of human cancers. As the expression of Crk and CrkL correlates with various tumor cell functions and their overexpression contributes to poor prognosis, Crk and CrkL have been proposed as therapeutic targets for cancer treatment13. Previously, gene knockdown was induced in glioblastoma cells to demonstrate that glioblastoma cell migration and invasion are robust markers of Crk and CrkL activity. The current study provides a systematic gene expression approach to induce the overexpression of Crk and CrkL using synthetic mRNA. A close correlation was obtained between the protein levels of Crk and CrkL and glioblastoma cell migration and invasion using the real-time cell analysis system. The results further support the hypothesis that Crk and CrkL play essential roles in glioblastoma cell migration and invasion. Together with the previous methods paper11, this study provides a proof-of-concept approach for investigating a potential correlation between changes in gene expression and tumor cell migration and invasion.

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

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 variant of Crk, on glioblastoma cell migration were studied in this work. U-118MG cells were electroporated with different concentrations of synthetic CrkI mRNA and analyzed for protein levels and cell migration. The electroporation of U-118MG glioblastoma cells with varying concentrations of synthetic CrkI mRNA resulted in a concentration-dependent increase in FLAG-tagged CrkI protein 1 day after transfection (Figure 1A). While 0.2 ng/&#181;L and 2 ng/&#181;L mRNA led to an undetectable or modest expression of the exogenous CrkI protein, 20 ng/&#181;L mRNA resulted in a much higher expression level than the endogenous CrkI protein.
The results from the cell migration assay using the real-time cell analysis system indicated that the electroporation with 0.2 ng/&#181;L or 2 ng/&#181;L CrkI mRNA did not greatly affect the cell migration. However, electroporation with 20 ng/&#181;L CrkI mRNA led to a clear stimulation of cell migration, with more cells migrating between 2 h and 13 h (Figure 1B). The comparison between the CrkI protein level and the cell migration revealed that glioblastoma cell migration was stimulated by the increase in the CrkI protein level. It appears that the CrkI protein level should be higher than a certain threshold to cause a substantial stimulation of cell migration. If the cell migration had been measured in different ways to count or observe the migrated cells at specific time points, much more effort might have been required to identify this kind of change in cell migration.
To study how CrkL overexpression influences glioblastoma cell invasion through ECM gel layers with different concentrations of ECM proteins, U-118MG cells were electroporated with synthetic CrkL mRNA and analyzed in terms of the protein levels and cell invasion through an ECM gel layer. The electroporation of U-118MG glioblastoma cells with synthetic CrkL mRNA led to a robust expression of FLAG-tagged CrkL protein 1 day after transfection (Figure 2A). As the concentration of ECM proteins increased, the invasion of the control cells slowed down (Figure 2B). CrkL-overexpressing cells also showed an ECM gel concentration-dependent decrease in cell invasion (Figure 2C). The comparison between the control and CrkL-overexpressing cells at different ECM gel concentrations indicated that CrkL overexpression generally stimulated cell invasion through the ECM gel layer (Figure 2D-G). However, the difference between the two cell populations became obvious at different time points depending on the ECM gel concentration.
For the 0.1 &#181;g/&#181;L ECM gel, the CrkL overexpression-mediated stimulation of cell invasion was evident between 8 h and 20 h (Figure 2D), but the difference in cell invasion was negligible after 32 h. For the 0.2 &#181;g/&#181;L ECM gel, the difference in cell invasion with and without CrkL overexpression was minimal at all times (Figure 2E). For the 0.5 &#181;g/&#181;L ECM gel, the difference in cell invasion was evident between 24 h and 36 h (Figure 2F). For the 1 &#181;g/&#181;L ECM gel, the CrkL overexpression effect on cell invasion became slightly apparent at 48 h (Figure 2G). The results suggest that the window to detect the difference between the control and CrkL-overexpressing cells shifts to later times as the concentration of the ECM gel increases. The results also suggest that the two cell populations were differentially affected by the increase in the ECM gel concentration at different time points. For example, at 12 h, the CrkL-overexpressing cells showed substantially higher invasion only at 0.1 &#181;g/&#181;L ECM gel (Figure 2H). However, at 24 h, the CrkL-overexpressing cells showed a little higher or similar invasion at the tested ECM gel concentrations (Figure 2I). Therefore, it is important to investigate both the time-dependent and ECM gel concentration-dependent differences in cell invasion to obtain a comprehensive view of the differences between the two cell populations with and without CrkL overexpression. These results demonstrate that combining transient overexpression using synthetic mRNA with impedance-based real-time cell analyses provides a powerful tool for analyzing the potential correlation between gene overexpression and tumor cell migration and invasion. Examining the effects of concentration variations in the synthetic mRNA and ECM gel would provide more accurate and detailed information.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64274/64274fig01.jpg" /> 
Figure 1: The effects of CrkI overexpression on glioblastoma cell migration. U-118MG cells were electroporated with the indicated concentrations (ng/&#181;L) of FLAG-tagged CrkI mRNA. (A) For the western blot analyses, the electroporated cells were cultured for 1 day before the total cell lysate preparation. The protein levels upon transfection with synthetic CrkI mRNA were compared. Anti-Crk and anti-CrkL antibodies were used to detect both the endogenous and the FLAG-tagged proteins and to compare the ratio between the endogenous proteins and FLAG-tagged proteins. Vinculin and &#945;-tubulin were used as loading controls. (B) For the cell migration analyses, the electroporated cells were plated onto a CIM plate without further culture. Cell index values were obtained from four wells for each sample, and their mean &#177; SD values are shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64274/64274fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64274/64274fig02.jpg" /> 
Figure 2: Effects of CrkL overexpression on glioblastoma cell invasion. U-118MG cells were electroporated with nuclease-free H2O or 20 ng/&#181;L FLAG-tagged CrkL mRNA. (A) For the western blot analyses, the electroporated cells were cultured for 1 day before the total cell lysate preparation. The protein levels upon transfection with synthetic CrkL mRNA were compared. Anti-Crk and anti-CrkL antibodies were used to detect both the endogenous and the FLAG-tagged proteins and to compare the ratio between the endogenous proteins and FLAG-tagged proteins. Vinculin and &#945;-tubulin were used as loading controls. (B-G) For the cell invasion analysis, the electroporated cells were plated onto a CIM plate with an ECM gel coating without further culture. The cell index values were obtained from four wells for each sample, and their mean &#177; SD values are shown. (B) The cell invasion data from the control cells with different ECM gel concentrations were compared. (C) The cell invasion data from the CrkL-overexpressing cells with various ECM gel concentrations were compared. (D-G) The cell invasion data between the control and CrkL-overexpressing cells were compared for the indicated ECM gel concentration. (H) A comparison of the cell invasion at 12 h between the control and CrkL-overexpressing cells. (I) A comparison of the cell invasion at 24 h between the control and CrkL-overexpressing cells. Abbreviations: ECM = extracellular matrix; CIM = cell invasion and migration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64274/64274fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64274/64274fig03.jpg" /> 
Figure 3: Schematic diagrams of the experimental procedures following gene knockdown or gene overexpression. (A) The experimental procedure of the real-time cell analysis following the siRNA transfection. Since 3-4 days are required to induce complete gene knockdown after siRNA transfection under the experimental conditions, the cells were replated and cultured for 3 days after the electroporation before they were ready for real-time cell analyses. (B) The experimental procedure for the real-time cell analysis following the synthetic mRNA transfection. Since the protein expression from synthetic mRNA transfection is rapid, the electroporated cells were used for real-time cell analyses on the same day. Note the difference between the two experimental procedures; whereas real-time cell analysis was performed 3 days after the electroporation for gene knockdown using siRNAs, real-time cell analysis was performed right after the electroporation for gene overexpression with synthetic mRNA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64274/64274fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Real-Time Quantitative Measurement of Tumor Cell Migration and Invasion Following Synthetic mRNA Transfection at JoVE.com

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

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

Nanyang Technological University

Research

JoVE Journal

Cancer Research

936 Views.  Children’s Mercy Kansas City. Dit protocol toont aan dat het combineren van sensatieboodschapper-RNA-transactie met impedantiegebaseerde real-time metingen van celmigratie en -invasie de identificatie van genen vergemakkelijkt die de migratie en invasie van tumorcellen stimuleren. Het op impedantie gebaseerde real-time celanalysesysteem maakt kwantitatieve, continue en uitgebreide monitoring van tumorcelmigratie bij veranderingen in genexpressie mogelijk. Genen die tumoren tot overexpressie brengen en de migratie van tumo...

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

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. ...

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

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

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.

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Bioengineering

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

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.

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Medicine

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 ...

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 ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Immunology and Infection

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

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.

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 ...

Quantifying the Brain Metastatic Tumor Micro-Environment using an Organ-On-A Chip 3D Model, Machine Learning, and Confocal Tomography

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, depending on the cancer type. To reduce the brain metastatic tumor burden, gaps in basic and translational knowledge need to be addressed. Major challenges include a paucity of reproducible preclinical models and associated tools. Three-dimensional models of brain metastasis can yield the relevant molecular and phenotypic data used to address these needs when combined with dedicated analysis tools. Moreover, compared to murine models, organ-on-a-chip models of patient tumor cells traversing the blood brain barrier into the brain microenvironment generate results rapidly and are more interpretable with quantitative methods, thus amenable to high throughput testing. Here we describe and demonstrate the use of a novel 3D microfluidic blood brain niche (&#181;mBBN) platform where multiple elements of the niche can be cultured for an extended period (several days), fluorescently imaged by confocal microscopy, and the images reconstructed using an innovative confocal tomography technique; all aimed to understand the development of micro-metastasis and changes to the tumor micro-environment (TME) in a repeatable and quantitative manner. We demonstrate how to fabricate, seed, image, and analyze the cancer cells and TME cellular and humoral components, using this platform. Moreover, we show how artificial intelligence (AI) is used to identify the intrinsic phenotypic differences of cancer cells that are capable of transit through a model &#181;mBBN and to assign them an objective index of brain metastatic potential. The data sets generated by this method can be used to answer basic and translational questions about metastasis, the efficacy of therapeutic strategies, and the role of the TME in both.

Here, we present a protocol for preparing and culturing a blood brain barrier metastatic tumor micro-environment and then quantifying its state using confocal imaging and artificial intelligence (machine learning).

Brain metastases are the most lethal cancer lesions; 10-30% of all cancers metastasize to the brain, with a median survival of only ~5-20 months, ...

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

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

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.

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

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

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