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

Summary

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.

Abstract

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.

Introduction

Tumor cell motility plays a crucial role in metastasis^1,2. The spread of tumor cells to neighboring and remote healthy tissues makes cancer treatment difficult and contributes to recurrence^3,4. 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 motility^5,6.

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 diseases^15,16. 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.

Protocol

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.

1. 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 described^18,19.
   1. Add 10 µL of 10x reaction buffer, 1.5 µL of PmeI (10,000 units/mL), and 10 µg 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 µL.
   2. Mix by tapping, spin down, and incubate the reaction mixture at 37 °C overnight.
   3. Spin down, and add 5 µL of 10% sodium dodecyl sulfate (SDS) and 1 µL of proteinase K (20 mg/mL, RNA grade) to the reaction mixture. Mix by tapping, spin down, and incubate at 50 °C for 30 min.
   4. Under the fume hood, add 100 µL of the bottom phase of phenol:chloroform:isoamyl alcohol to the plasmid reaction mixture for extraction. Vortex, and then centrifuge at 18,800 × g for 5 min at room temperature. Move the upper phase to a new tube.
   5. Repeat step 1.1.4 with chloroform:isoamyl alcohol at 24:1.
   6. In the new tube, bring the reaction volume to 300 µL by adding 200 µL of nuclease-free water, and then add 30 µL of 3 M sodium acetate and 600 µL of 100% ethanol.
   7. Mix by vortexing, and then place at −20 °C for 30-60 min for the ethanol precipitation of the DNA.
   8. Centrifuge at 18,800 × g for 20 min at 4 °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.
   9. Dry with the cap open for 2 min, add 30 µL of nuclease-free water, and resuspend the DNA pellet.
   10. Determine the DNA concentration using a spectrophotometer.
   11. Verify the size and quantity of the linearized DNA using agarose gel electrophoresis.
2. RNA synthesis using T7 RNA polymerase
   1. Add 2 µL each of 10x transcription buffer, ATP, GTP, UTP, CTP, and T7 and 1 µg of a linearized DNA to a microcentrifuge tube. Add nuclease-free water to bring the reaction volume to 20 µL.
   2. Mix by tapping, spin down, and incubate the reaction mixture at 37 °C for 2 h for RNA synthesis.
   3. Spin down, add 1 µL of DNase (2 U/µL), and incubate at 37 °C for 15 min to remove the template DNA.
3. Lithium chloride precipitation of the RNA
   1. Add 10 µL of lithium chloride (7.5 M) to the reaction mixture. Mix by tapping, spin down, and incubate the reaction mixture at −20 °C for 30 min.
   2. Centrifuge at 18,800 × g for 20 min at 4 °C, discard the supernatant, and rinse the pellet with 500 µL of 70% ethanol. Repeat the centrifugation for 10 min, and then remove the supernatant completely.
   3. Dry with the cap open for 2 min, add 30 µL of nuclease-free water, and resuspend the RNA pellet.
4. Capping
   1. Heat the RNA sample at 65 °C for 10 min, and then place it on ice for cooling.
   2. Add 5 µL each of 10x capping buffer, 10 mM GTP, and 1 mM (10x) S-adenosylmethionine (SAM), 2 µL each of a capping enzyme mix and O-methyltransferase enzyme mix, and 1.25 µL of RNase inhibitor. Mix by tapping, spin down, and incubate the reaction mixture at 37 °C for 1 h.
5. Poly(A) tailing
   1. Spin down the sample, and then add 6 µL of nuclease-free water, 20 µL of 5x E-PAP buffer, 10 µL of 25 mM MnCl₂, 10 µL of 10 mM ATP, and 4 µL of E-PAP poly(A) tailing enzyme. Mix by tapping, spin down, and incubate the reaction mixture at 37 °C for 2 h.
6. Lithium chloride precipitation and quantification of the synthetic mRNA
   1. Add 50 µL of lithium chloride (7.5 M), and perform lithium chloride precipitation as described in steps 1.3.1-1.3.3.
   2. Measure the concentration of the mRNA using a spectrophotometer.
   3. Verify the size and quantity of mRNA using formaldehyde (1%-2%) agarose (1%) gel electrophoresis.

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.

1. Remove an aliquot of ECM gel stock from the freezer and keep it on ice.
2. Dilute the ECM gel (10 mg/mL) to a working concentration (100 µg/mL) by mixing 990 µL of Dulbecco's Modified Eagle Medium (DMEM) (without serum or antibiotics) with 10 µL of ECM gel in a microcentrifuge tube. Mix by gentle pipetting.
3. Add 60 µL 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 bubbles^20,21.
   NOTE: Optimize the concentration of the ECM gel for each cell line. For glioblastoma cell lines, 0.1 µg/µL to 1 µg/µL ECM gel was used for optimization.
4. 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.
   ​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's hands or the surfaces of the equipment, including the biosafety cabinet or the CO₂ incubator.

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.

1. Culture of the tumor cells
   1. 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 °C in a 5% CO₂ incubator (culture condition).
2. 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.
   1. Remove the old medium, and add 10 mL of prewarmed DMEM containing 0.5% FBS and 1% antibiotics per dish (low-serum medium).
   2. Repeat step 3.2.1.
   3. Incubate the cells at 37 °C in a 5% CO₂ incubator for 4 h or longer.
3. Harvesting of the tumor cells
   1. Remove the old medium, add 8 mL of prewarmed Dulbecco'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.
   2. 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.
   3. Aliquot a small volume of cells, and use a handheld automated cell counter to count the cells.
   4. Calculate the total number of cells and the required volume for 10,000 cells/µL.
   5. Spin down the cells by centrifuging them at 100 × 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.
   6. Transfer 110 µL of the cell suspension, containing 1.1 million cells, to a microcentrifuge tube for each treatment.
   7. Repeat step 3.3.6 to transfer the cells to four microcentrifuge tubes for four different treatments.
      ​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.

4. Electroporation of the tumor cells

1. 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° 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.
2. Add 110 μL of resuspension buffer R to the cell pellet to obtain 0.1 million cells in 10 μL.
3. Add synthetic mRNA to the cell pellet to obtain a concentration of 0.2-20 ng/µL 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.
4. Electroporate 10 µL of the cell suspension with an electroporation system at 1,350 V for 10 ms with three pulses each time.
5. Transfer the electroporated cells into a new microcentrifuge tube with 1.1 mL of DMEM containing 0.5% FBS.
6. 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 µL and 10 µL electroporation tips may be used to electroporate a large volume of cells, but the electroporation tips for 10 µL and 100 µL are included in two separate kits and need to be purchased separately. Resuspension buffer R is included in both kits.
7. Upon completing all the respective electroporations, gently resuspend the pooled cells.
8. 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.
9. Keep the rest of the electroporated cells at room temperature until the real-time cell analysis system is ready.

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.

1. Equilibration of the real-time cell analyzer
   1. Move the real-time cell analyzer into a CO₂ incubator several hours before use to equilibrate the system under the culture condition.
2. Setting up the analysis program
   1. Open the analysis program by double-clicking on the real-time cell analysis software icon on the desktop.
   2. 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.
   3. Open each cradle by clicking on the Number tab.
   4. Click on the Experiment Notes tab, select the folder that the data will be saved to, and enter the name of the experiment.
   5. 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.
   6. 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.
   7. 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.
   8. Move to the next cradle, and set it up by repeating steps 5.2.3-5.2.7.
3. Preparation of the CIM plates
   1. One hour before the cell impedance measurement starts, fill the wells of the lower chamber of the CIM plate with 160 µL of DMEM containing 10% serum or other chemoattractants.
   2. Assemble the upper chamber that contains the ECM gel-coated wells (for invasion) or uncoated wells (for migration) with the lower chamber.
   3. Add 50 µL 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.
   4. Click on the Message tab, and ensure that the Connections ok message is displayed. The CIM plate is now ready for the experiment.
   5. 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.

6. Real-time cell analysis and data export

NOTE: Perform a baseline reading, cell seeding, cell impedance measurement, and data export as previously described¹¹.

1. 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.
   1. Click on the Start button for each cradle. When the Save As window appears, save the experimental file to perform the baseline reading.
2. Cell seeding
   1. Remove the CIM plate from the cradle, and place it in the biosafety cabinet on the plate holder.
   2. Add 100 µL (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.
   3. 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.
3. Cell impedance measurement
   1. 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.
   2. 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.
      ​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.
4. Export of the data for analysis
   1. Click on the Plot tab and select the Average and STD DEV boxes to copy the data for each well individually.
   2. Right-click on the plot area, and select Copy Data in List Format.
   3. Open an empty spreadsheet file, paste the data, and then save the file.
   4. Go back to the analysis program, click on the Plate tab for each cradle, and select Release to close the experiment for the cradle.
   5. 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.

Representative Results

Crk and CrkL proteins play important roles in the motility of many cell types, including neurons²², T cells²³, fibroblasts^18,19, and a variety of tumor cells¹³. Since Crk and CrkL proteins have been reported to be elevated in glioblastoma^24,25,26, 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/µL and 2 ng/µL mRNA led to an undetectable or modest expression of the exogenous CrkI protein, 20 ng/µL 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/µL or 2 ng/µL CrkI mRNA did not greatly affect the cell migration. However, electroporation with 20 ng/µL 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 µg/µL 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 µg/µL ECM gel, the difference in cell invasion with and without CrkL overexpression was minimal at all times (Figure 2E). For the 0.5 µg/µL ECM gel, the difference in cell invasion was evident between 24 h and 36 h (Figure 2F). For the 1 µg/µL 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 µg/µL 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.

Figure 1: The effects of CrkI overexpression on glioblastoma cell migration. U-118MG cells were electroporated with the indicated concentrations (ng/µL) 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 α-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 ± SD values are shown. Please click here to view a larger version of this figure.

Figure 2: Effects of CrkL overexpression on glioblastoma cell invasion. U-118MG cells were electroporated with nuclease-free H₂O or 20 ng/µL 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 α-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 ± 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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Discussion

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 interventions^2,27. 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'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'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.

Materials

Name	Company	Catalog Number	Comments
AlphaImager HP	ProteinSimple	92-13823-00	Agarose gel imaging system
α-Tubulin antibody	Sigma	T9026	Used to detect α-tubulin protein (dilution 1:3,000)
CIM-plate 16	Agilent Technologies, Inc	5665825001	Cell invasion and migration plates
Crk antibody	BD Biosciences	610035	Used to detect CrkI and CrkII proteins (dilution 1:1,500)
CrkL antibody	Santa Cruz	sc-319	Used to detect CrkL protein (dilution 1:1,500)
Dulbecco’s Modified Eagle’s Medium (DMEM)	ATCC	302002	Cell culture medium
Dulbecco's phosphate-buffered saline (DPBS)	Corning	21-031-CV	Buffer used to wash cells
Fetal bovine serum (FBS)	Hyclone	SH30910.03	Culture medium supplement
Heracell VIOS 160i CO2 incubator	Thermo Scientific	51030285	CO2 incubator
IRDye 800CW goat anti-mouse IgG secondary antibody	Li-Cor	926-32210	Secondary antibody for Western blot analysis (dilution 1:10,000)
IRDye 800CW goat anti-rabbit IgG secondary antibody	Li-Cor	926-32211	Secondary antibody for Western blot analysis  (dilution 1:10,000)
Lithium chloride	Invitrogen	AM9480	Used for RNA precipitation
Matrigel matrix	Corning	354234	Extracellular matrix (ECM) gel
MEGAscript T7 transcription kit	Invitrogen	AM1334	Used for RNA synthesis
Millennium RNA markers	Invitrogen	AM7150	Used for formaldehyde agarose gel electrophoresis
Mini centrifuge	ISC BioExpress	C1301P-ISC	Used to spin down cells
Mouse brain QUICK-Clone cDNA	TaKaRa	637301	Source of genes (inserts) for cloning
NanoQuant	Tecan	M200PRO	Nucleic acid quantification system
Neon electroporation system	ThermoFisher Scientific	MPK5000	Electroporation system1
Neon transfection system 10 µL kit	ThermoFisher Scientific	MPK1025	Electroporation kit
Neon transfection system 100 µL kit	ThermoFisher Scientific	MPK10096	Electroporation kit
NorthernMax denaturing gel buffer	Invitrogen	AM8676	Used for formaldehyde agarose gel electrophoresis
NorthernMax formaldehyde load dye	Invitrogen	AM8552	Used for formaldehyde agarose gel electrophoresis
NorthernMax running buffer	Invitrogen	AM8671	Used for formaldehyde agarose gel electrophoresis
Nuclease-free water	Teknova	W3331	Used for various reactions during mRNA synthesis
Odyssey CLx Imager	Li-Cor		Imager for Western blot analysis
pcDNA3.1/myc-His	Invitrogen	V80020	The vector into which inserts (mouse CrkI and CrkL cDNAs) were cloned
pFLAG-CMV-5a	Millipore Sigma	E7523	Source of the FLAG epitope tag
Phenol:chloroform:isoamyl alcohol	Sigma	P2069	Used for DNA extraction
PmeI	New England BioLabs	R0560L	Used to linearize the plasmids for mRNA synthesis
Poly(A) tailing kit	Invitrogen	AM1350	Used for poly(A) tail reaction
Polystyrene tissue culture dish (100 x 20 mm style)	Corning	353003	Used for culturing cells before transfection
Polystyrene tissue culture dish (35 x 10 mm style)	Corning	353001	Used for culturing transfected cells
Proteinase K	Invitrogen	25530049	Used to remove protein in the reaction mixture
Purifier Axiom Class II, Type C1	Labconco Corporation	304410001	Biosafety cabinet for sterile handling of cells
Resuspension Buffer R	ThermoFisher Scientific		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.
RNaseZap	Invitrogen	AM9780	RNA decontamination solution
Scepter	Millipore	C85360	Handheld automated cell counter
ScriptCap 2'-O-methyltransferase kit	Cellscript	C-SCMT0625	Used for capping reaction
ScriptCap m7G capping system	Cellscript	C-SCCE0625	Used for capping reaction
Sodium dodecyl sulfate solution	Invitrogen	15553-035	Detergent used for the proteinase K reaction
Sorvall Legend XT centrifuge	Thermo Scientific	75004532	Benchtop centrifuge to spin down cells
Trypsin-EDTA	Gibco	25300-054	Used for dissociation of cells
U-118MG	ATCC	HTB15	An adherent cell line derived from a human glioblastoma patient
Vinculin antibody	Sigma	V9131	Used to detect vinculin protein (dilution 1:100,000)
xCELLigence RTCA DP	Agilent Technologies, Inc	380601050	Instrument used for real-time cell analysis
1Electroporation parameters and other related information for various cell lines are available on the manufacturer's homepage (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/neon-transfection-system/neon-transfection-system-cell-line-data.html?).