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.
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 cancer genotypes and phenotypes is critical to understanding and designing effective therapeutic strategies. Decades of cancer research have led to the development and adaptation of different methods to determine cancer genotypes and phenotypes. One of the latest technical developments is real-time measurement of cell migration and invasion based on cell impedance. Cell adhesion to substrates and cell-cell contacts play an important role in cell-to-cell communication and regulation, development, and maintenance of tissues. Abnormalities in cell adhesion lead to the loss of cell-cell contact, degradation of extracellular matrix (ECM), and gain of migratory and invading capabilities by cells, all of which contribute to metastasis of cancer cells to different organs1,2. Various methods are available to determine cell migration (wound healing and Boyden chamber assays) and invasion (Matrigel-Boyden chamber assay)3,4,5. These conventional methods are semiquantitative because cells need to be labeled with a fluorescent dye or other dyes either before or after the experiment to measure cell phenotypes. In addition, mechanical disruptions are needed in some cases for creating a wound for measuring the migration of cells to the wound site. Moreover, these existing methods are time-consuming, labor-intensive, and measure the results at only one time point. In addition, these methods are prone to making inaccurate measurements due to inconsistent handling during the experimental procedure6.
Unlike conventional methods, the real-time cell analysis system measures cell impedance in real-time without requiring pre- or poststaining and mechanical damage of cells. More importantly, the duration of an experiment can be extended so that biological effects can be determined in a time-dependent manner. Executing the experiment is time-efficient and not labor-intensive. Analyzing data is relatively simple and accurate. Compared to other methods, this method is one of the best real-time measurements to measure cell migration and invasion6,7,8,9.
Giaever and Keese were the first to describe the impedance-based measurement of a cell population on the surface of electrodes10. The real-time cell analysis system works on the same principle. The area of each microplate well is approximately 80% covered with an array of gold microelectrodes. When the electrode surface area is occupied by cells due to adherence or spreading of the cells, the electrical impedance changes. This impedance is displayed as the cell index, which is directly proportional to the cells covering the electrode surface area after they penetrate the microporous membrane (the median pore size of this membrane is 8 μm)11.
Crk and CrkL are adaptor proteins containing SH2 and SH3 domains and play important roles in various cellular functions, such as cytoskeleton regulation, cell transformation, proliferation, adhesion, epithelial-mesenchymal transition, migration, invasion, and metastasis by mediating protein-protein interactions in many signaling pathways1,12,13,14,15,16,17,18. Therefore, it is important to determine the Crk/CrkL-dependent migratory and invasive capabilities of cancer cells. Real-time cell analysis was performed to determine the migratory and invasive abilities of glioblastoma cells upon gene knockdown of Crk and CrkL.
This method paper describes detailed measurements of Crk- and CrkL-mediated migration and invasion of human glioblastoma cells.
NOTE: All cell culture materials need to be sterile and the entire experiment must be performed in a biosafety cabinet under sterile conditions.
1. Culture and Electroporation of the U-118MG Glioblastoma Cell Line
- Culture the U-118MG cell line in 5% fetal bovine serum (FBS) containing Dulbecco’s Modified Eagle Medium (DMEM) (culture medium) and Maintain at 37 °C in a humid atmosphere containing 5% CO2 incubator (culture conditions).
- Use 70– 80% confluent healthy cells for electroporation.
- For harvesting cells, wash the cells growing in culture dishes with 1x PBS and add 2 mL of 0.05% trypsin-EDTA. Place in the incubator for 30 s and remove the trypsin-EDTA. Collect cells in the culture medium into a 15 mL tube.
- Count the cells using a handheld automated cell counter, centrifuge cells at 100 x g for 5 min, and discard the supernatant.
- Suspend the cell pellet in the culture medium and take 600,000 cells for each condition into a microcentrifuge tube. Adjust the cell number depending on experimental designs and growth rates.
- Transfer the cell suspension to a microcentrifuge tube and add 800 µL of Dulbecco’s phosphate-buffered saline (DPBS). Spin down using a minicentrifuge for 30 s and discard the supernatant.
- Add 60 µL of resuspension buffer R to the cell pellet and add the siRNAs (i.e., non-targeting siRNA, Crk siRNA, CrkL siRNA, or both Crk and CrkL siRNAs) at a concentration of 6 µM to the respective microcentrifuge tube. Mix them gently by tapping.
- Electroporate 10 µL of cells with an electroporation system at 1,350 V for 10 ms with three pulses and transfer the electroporated cells into 5 mL of the culture medium. Repeat the electroporation for the rest of the cells prepared for each condition.
- Complete all respective electroporation. Transfer electroporated cells into two 35 mm dishes per condition and culture them under culture conditions for 3 days.
- On the third day, treat all the electroporated cells in 0.5% FBS containing DMEM (low serum medium) for 6 h prior to the actual cell impedance measurement.
2. Preparation of the Real-time Cell Analysis System, Cell Invasion and Migration (CIM) Plates, and Electroporated U-118MG Cells for Plating
- Place the real-time cell analysis system in a CO2 incubator under culture conditions 5–6 h prior to the start of experiment to stabilize the system to the culture conditions.
- For the invasion assay, plate 50 µL of DMEM (plain medium) containing extracellular matrix (ECM) gel at 0.1 µg/µL in each well of the upper chamber of the CIM plate. To avoid having any air bubbles, use the reverse pipetting method. Immediately remove 30 µL of ECM gel, leaving 20 µL in the well.
- After the ECM gel coating, keep the plate in the incubator under culture conditions for 4 h with its lid on. During the ECM gel coating and drying, take preventive measures to avoid direct contact of the electrodes of the upper chamber of the plates with the hands, the surfaces of the biosafety cabinet, or the CO2 incubator.
- To set up the impedance measurement program, double-click the associated software icon (see Table of Materials) to open the system software application (control unit). Each cradle has an individual window with different tabs to set the experimental conditions, the impedance measuring time interval and duration, and data analysis.
- Under the Layout tab, set quadruplicate wells for each biological condition and set two-step cell impedance measurements under the Schedule tab. The first step is for a one-time baseline measurement (one sweep with a 1 min interval), and the second step is to measure the cell impedance in respective individual cradles for an actual experiment. For migration, use 145 sweeps with a 10 min interval, and for invasion, 577 sweeps with a 10 min interval, individually set) in respective individual cradles.
- One h prior to the start of the cell impedance measurement, add 160 µL of DMEM with 10% FBS as a chemoattractant in the wells of the lower chamber of the plate. Assemble either the upper chamber containing the ECM gel-coated wells or uncoated wells with the lower chamber to measure invasion and migration, respectively.
- Fill the wells in the upper chamber with 50 µL of low serum medium and place them in the cradle of the system. Check whether all the wells are recognized by the control unit by clicking the Message tab. If the message displays as OK, the plate in the cradle is ready for the experiment.
- Preincubate the completely packed plates in the incubator under culture conditions for 1 h prior to measurements in the real-time cell analysis system cradle, which is essential for acclimation of a packed plate to the cell culture conditions.
- For harvesting cells treated with low serum medium, trypsinize the cells as in step 1.3, collect them in low serum medium, and count the cells as in step 1.4.
- After counting, centrifuge cells at 100 x g for 5 min and discard the supernatant.
- Resuspend 800,000 cells in 800 µL of low serum medium for the migration and invasion assays. Additionally, resuspend 300,000 cells in 2 mL of culture medium and seed in a 35 mm dish for Western blot analysis to confirm the regulated knockdown of Crk and CrkL.
3. Baseline Reading, Seeding of the Cells, and Cell Impedance Measurement and Visualization
- Measure the baseline reading by clicking the cradle Start button. The baseline should be read after preincubating the plates for 1 h in the cradle of the real-time cell analysis system under culture conditions in the CO2 incubator and before seeding the cells to the respective wells in the upper chamber.
NOTE: Once the cradle Start button is clicked, the control unit will ask whether to save the experimental file. After the file is saved, the cradle analyzer measures the baseline cell impedance as set in the program initially and enters a pause mode until the cradle Start button is clicked again to measure cell impedance in the second step.
- Take out both plates for migration and invasion from the cradles after the baseline measurement and keep them in the biosafety cabinet.
- Seed 100 µL of cells (100,000 cells) in quadruplicates for each biological condition in the upper chamber of the CIM plate in the respective wells as programmed in the control unit of the cradle by reverse pipetting to avoid air bubbles.
- After seeding, keep the plate under a biosafety cabinet for 30 min at room temperature to allow cells to evenly settle down to the bottom. Transfer the plate back to the respective cradle and click the cradle start button to start measuring cell impedance as programmed in the second step of 2.5.
- After the last sweep, the experiment is finished, and the results are saved automatically.
- Under the Data Analysis tab, visualize the changes in cell impedance as cell index in a time-dependent manner during or after completion of the experiment. Each of the respective conditions of quadruplicates can be visualized either individually or as averages and/or standard deviations by clicking the Option boxes for average and standard deviation.
- To export cell index data to a spreadsheet file, open an empty spreadsheet file, place the cursor in the middle of the Data Analysis window, and right-click. In the dialog box that appears, choose the option Copy Data into List Format, and paste the data into the open spreadsheet.
- Adjust the time in the raw data to represent the actual start time of the cell impedance measurement. The second step start time is set as zero.
NOTE: The control unit has the option to obtain the cell index with or without normalization (i.e., normalized cell index) and visualize the results as a graphical presentation in a time-dependent manner. In this example, cell index data are exported without normalization for processing and graphical presentation.
- Release the experiment by clicking the Release button in each cradle.
It has been suggested that Crk and CrkL are important for cell migration and invasion in different cancer cell lines13,17. Although Crk and CrkL proteins are structurally and functionally similar to each other and play essential overlapping functions16,19,20,21, many gene knockdown studies for Crk and CrkL have not clearly addressed whether the knockdown is specific to either Crk, CrkL, or both. Therefore, it is unclear which of the two proteins contributes to cell migration and invasion. As a proof-of-principle study, we used siRNAs specific to Crk or CrkL and studied their effects on migration and invasion of the U-118MG GBM cell line. The knockdown of Crk decreased CrkII and CrkI protein levels by 85% and 86%, respectively, without reducing the CrkL protein level. The knockdown of CrkL reduced the CrkL protein level by 85% (Figure 1). CrkL knockdown slightly reduced the CrkII and CrkI levels, too. Combined use of siRNAs for Crk and CrkL reduced CrkII, CrkI, and CrkL levels by more than 80% (Figure 1B). On the other hand, knockdown of Crk and CrkL did not affect the vinculin and α-tubulin levels (Figure 1).
The U-118MG cells migrated to high serum (10% FBS), reaching the maximal level of migration at 13 h, which served as the experiment internal control (Figure 2A). With Crk knockdown, cell migration was delayed, and the cells continued to migrate until 23 h. CrkL knockdown substantially inhibited cell migration. U-118MG cells lost their migratory ability upon knockdown of both Crk and CrkL (Figure 2A), suggesting that Crk and CrkL play essential overlapping roles in cancer cell migration. However, this conclusion is not clearly evident if cell migration is examined at a fixed time point. When cell migrations at 6 or 13 h were compared, inhibitions by Crk and CrkL knockdowns were obvious (Figure 2B,C). In contrast, Crk knockdown did not have an inhibitory effect on cell migration at 18 h (Figure 2D), leading to conflicting results depending on the time point selected for comparison. The inhibitory effects of CrkL knockdown and Crk/CrkL double knockdown were clearly visible at all three time points. These results clearly demonstrate that cell migration must be assessed over the entire period of cell migration to accurately analyze effects by genetic manipulations or drugs.
The U-118MG cells invaded high serum, reaching the maximum level of invasion at 52 h, which served as the experiment internal control (Figure 3A). With Crk knockdown, cell invasion was delayed, but it reached a similar maximum level at 60 h. With CrkL knockdown, U-118MG cells showed delayed and reduced invasion compared with the control cells. Combined knockdown of Crk and CrkL further inhibited cell invasion (Figure 3A). Comparison of cell invasion at 36 h, when the control cells were actively undergoing invasion, clearly demonstrated inhibition by individual knockdown of Crk and CrkL and a synergistic inhibition by Crk/CrkL double knockdown (Figure 3B). However, a comparison of cell invasion at 52 or 60 h exhibited a slight or no inhibitory effect by Crk knockdown (Figure 3C,D). These results clearly support the suggestion that cell invasion should be analyzed over the entire period of the experiment.
These results demonstrate that both Crk and CrkL mediate cell migration and invasion, and that the real-time cell analysis system has a clear advantage over the traditional methods in understanding the different kinetics of cell migration and invasion and the specific effects on cell phenotypes in a time-dependent manner.
Figure 1: siRNA-mediated knockdown of CrkI, CrkII, and CrkL in U-118MG cells. (A) Total cell lysates were prepared 4 days after U-118MG cells were electroporated with non-targeting control siRNA (NT), Crk siRNA, CrkL siRNA, or both Crk and CrkL siRNAs, and protein levels were determined by Western blot analyses as described previously1. (B) The signal intensities of respective bands were quantified using the imaging system and calculated as percentages of NT. Their mean ± SD values are shown in the graph. Vinculin and a-tubulin served as internal controls. Statistical analyses of data were performed using unpaired two-tailed Student’s t-test for comparison between the two experimental groups. *p < 0.05 and **p < 0.01, compared to NT. Please click here to view a larger version of this figure.
Figure 2: Effects of Crk/CrkL knockdown on U-118MG cell migration: (A) Three days after U-118MG cells were electroporated with non-targeting control siRNA (NT), Crk siRNA, CrkL siRNA, or both Crk and CrkL siRNAs, cells were harvested and cell migration was examined using the real-time analysis system. Migration of U-118MG cells was inhibited with a single knockdown of Crk or CrkL in a time-dependent manner. The knockdown of both Crk and CrkL completely blocked cell migration. Cell index values at 6 (B), 13 (C), and 18 h (D) are presented to compare cell migration at different time points (arrows). At 13 h the control cells (NT) reached the maximal migration. At 18 h both control and Crk knockdown cells showed similar levels of cell migration. Statistical analyses of data were performed using unpaired two-tailed Student’s t-test for comparison between the two experimental groups. **p < 0.01, compared to NT. Please click here to view a larger version of this figure.
Figure 3: Effects of Crk/CrkL knockdown on U-118MG cell invasion: (A) Three days after U-118MG cells were electroporated with non-targeting control siRNA (NT), Crk siRNA, CrkL siRNA, or both Crk and CrkL siRNAs, cells were harvested and cell invasion was examined for 4 days using the real-time analysis system. The invasion of U-118MG cells was inhibited with a single knockdown of Crk or CrkL in a time-dependent manner. The knockdown of both Crk and CrkL in the U-118MG cell line reduced its invasive capacity up to 48 h compared to NT. Cell index values at 36 (B), 52 (C), and 60 h (D) are presented to compare cell invasion at different time points (arrows). At 52 h, the control cells (NT) reached the initial peak of invasion. At 60 h, Crk knockdown cells reached the initial peak of invasion. Statistical analyses of data were performed using unpaired two-tailed Student’s t-test for comparison between the two experimental groups. *p < 0.05 and **p < 0.01, compared to NT. Please click here to view a larger version of this figure.
The real-time measurement of cell migration and invasion using the real-time cell analysis system is a simple, quick, and continuous monitoring process with multiple, significant advantages over the traditional methods that provide data at a single time point. As with the traditional methods, experimental conditions must be optimized for each cell line for the real-time cell analysis system, because each cell line may be different in terms of its adhesion to the substrate, growth, cell-to-cell contacts, and migratory and invasive abilities. Due to these differences, each cell line may show different cellular kinetics and cell impedances. Impedance is greatly influenced by the number of cells seeded in a well, the time for cell adhesion, the lag time before cells start to migrate or invade, and the concentration of ECM gel on CIM plates. First, real-time cell analysis makes the optimization easier because it provides results in real time over a specific time period, enabling researchers to identify the time point when the control cells show active cell migration and invasion and when the control cells reach the maximal levels of migration and invasion. Second, ectopic gene overexpression or gene knockdown studies may need additional optimizations, because the cells need to adopt the phenotypes from the modified genotypic changes. In addition, effective drug concentrations and the efficacy of drugs can be determined in combination with normal or modified genetic conditions using the real-time cell analysis system.
Traditional methods such as wound healing, soft agar, Boyden chamber migration, or invasion assays have been used to determine that knockdown of either Crk or CrkL leads to reduced migration and invasion in different cancer cell lines13,17. In this study, we induced single or double knockdown of Crk and CrkL in the U-118MG cell line and investigated cell migration and invasion. Real-time measurement of cell impedances over the entire experiment provided in-depth information about the kinetics of cell migration and invasion, allowing us to identify two different modes of inhibition. Whereas Crk knockdown delayed migration and invasion, CrkL knockdown inhibited migration and invasion over the entire time period. Furthermore, the double knockdown of both Crk and CrkL completely blocked cell migration and substantially inhibited cell invasion.
This study provides a proof-of-concept that combining the systematic knockdown approach to induce single and double knockdown of Crk and CrkL with real-time analyses of cell migration and invasion over the entire period of the experiments is necessary for comprehensive analyses of Crk- and CrkL-mediated functions in cancer cells. The data presented in this study suggest that this method can also be used to test candidate drugs for their inhibitory effects on Crk and CrkL. Overall, the real-time cell analysis system is useful in setting up experiments for cell migration or cell invasion and makes real-time, in-depth, and comprehensive analyses possible.
The authors have nothing to disclose.
We thank Olivia Funk for her technical assistance with the real-time cell analysis system data. We also thank the Medical Writing Center at Children’s Mercy Kansas City for editing this manuscript. This work was supported by Tom Keaveny Endowed Fund for Pediatric Cancer Research (to TP) and by Children’s Mercy Hospital Midwest Cancer Alliance Partner Advisory Board funding (to TP).
|Biosafety cabinet||ThermoFisher Scientific||1300 Series Class II, Type A2|
|CIM plates||Cell Analysis Division of Agilent Technologies, Inc||5665825001||Cell invasion and migration plates|
|CrkL siRNA||Ambion||ID: 3522 and ID: 3524|
|Dulbecco’s modified eagle’s medium (DMEM)||ATCC||302002||Culture medium used for cell culture|
|Dulbecco's phosphate-buffered saline (DPBS)||Gibco||21-031-CV||DPBS used to wash the cells|
|Fetal bovine serum (FBS)||Hyclone||SH30910.03|
|Heracell VIOS 160i CO2 incubator||ThermoFisher Scientific||51030285||CO2 incubator|
|Matrigel||BD Bioscience||354234||Extracellular matrix gel|
|Neon electroporation system||ThermoFisher Scientific||MPK5000||Electroporation system|
|Neon transfection system 10 µL kit||ThermoFisher Scientific||MPK1025||Electroporation kit|
|Non-targeting siRNA||Dharmacon||D-001810-01||siRNA for non targated control|
|Odyssey CLx (Imaging system)||LI-COR Biosciences||Western blot imaging system|
|RTCA software||Cell Analysis Division of Agilent Technologies, Inc||Instrument used for experiment|
|Scepter||Millipore||C85360||Handheld automated cell counter|
|U-118MG||ATCC||ATCC HTB15||Cell lines used for experiments|
|xCELLigence RTCA DP||Cell Analysis Division of Agilent Technologies, Inc||380601050||Instrument used for experiment|
- Park, T., Koptyra, M., Curran, T. Fibroblast Growth Requires CT10 Regulator of Kinase (Crk) and Crk-like (CrkL). Journal of Biological Chemistry. 291, (51), 26273-26290 (2016).
- Hanahan, D., Weinberg, R. A. Hallmarks of cancer: the next generation. Cell. 144, (5), 646-674 (2011).
- Mudduluru, G., et al. Regulation of Axl receptor tyrosine kinase expression by miR-34a and miR-199a/b in solid cancer. Oncogene. 30, (25), 2888-2899 (2011).
- Mudduluru, G., Vajkoczy, P., Allgayer, H. Myeloid zinc finger 1 induces migration, invasion, and in vivo metastasis through Axl gene expression in solid cancer. Molecular Cancer Research. 8, (2), 159-169 (2010).
- Khalili, A. A., Ahmad, M. R. A Review of Cell Adhesion Studies for Biomedical and Biological Applications. International Journal of Molecular Sciences. 16, (8), 18149-18184 (2015).
- Katt, M. E., Placone, A. L., Wong, A. D., Xu, Z. S., Searson, P. C. In Vitro Tumor Models: Advantages, Disadvantages, Variables, and Selecting the Right Platform. Frontiers in Bioengineering and Biotechnology. 4, 12 (2016).
- Hamidi, H., Lilja, J., Ivaska, J. Using xCELLigence RTCA Instrument to Measure Cell Adhesion. Bio Protocols. 7, (24), 2646 (2017).
- Scrace, S., O'Neill, E., Hammond, E. M., Pires, I. M. Use of the xCELLigence system for real-time analysis of changes in cellular motility and adhesion in physiological conditions. Methods in Molecular Biology. 1046, 295-306 (2013).
- Kumar, S., et al. Crk Tyrosine Phosphorylation Regulates PDGF-BB-inducible Src Activation and Breast Tumorigenicity and Metastasis. Molecular Cancer Research. 16, (1), 173-183 (2018).
- Giaever, I., Keese, C. R. Monitoring fibroblast behavior in tissue culture with an applied electric field. Proceeding of the National Academy of Science U. S. A. 81, (12), 3761-3764 (1984).
- Tiruppathi, C., Malik, A. B., Del Vecchio, P. J., Keese, C. R., Giaever, I. Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function. Proceedings of the National Academy of Sci U.S.A. 89, (17), 7919-7923 (1992).
- Collins, T. N., et al. Crk proteins transduce FGF signaling to promote lens fiber cell elongation. Elife. 7, (2018).
- Fathers, K. E., et al. Crk adaptor proteins act as key signaling integrators for breast tumorigenesis. Breast Cancer Research. 14, (3), 74 (2012).
- Koptyra, M., Park, T. J., Curran, T. Crk and CrkL are required for cell transformation by v-fos and v-ras. Molecular Carcinogenesis. 55, (1), 97-104 (2016).
- Lamorte, L., Royal, I., Naujokas, M., Park, M. Crk adapter proteins promote an epithelial-mesenchymal-like transition and are required for HGF-mediated cell spreading and breakdown of epithelial adherens junctions. Molecular Biology of the Cell. 13, (5), 1449-1461 (2002).
- Park, T. J., Curran, T. Essential roles of Crk and CrkL in fibroblast structure and motility. Oncogene. 33, (43), 5121-5132 (2014).
- Rodrigues, S. P., et al. CrkI and CrkII function as key signaling integrators for migration and invasion of cancer cells. Molecular Cancer Research. 3, (4), 183-194 (2005).
- Feller, S. M. Crk family adaptors-signalling complex formation and biological roles. Oncogene. 20, (44), 6348-6371 (2001).
- Park, T. J., Boyd, K., Curran, T. Cardiovascular and craniofacial defects in Crk-null mice. Molecular and Cellular Biology. 26, (16), 6272-6282 (2006).
- Park, T. J., Curran, T. Crk and Crk-like play essential overlapping roles downstream of disabled-1 in the Reelin pathway. Journal of Neuroscience. 28, (50), 13551-13562 (2008).
- Hallock, P. T., et al. Dok-7 regulates neuromuscular synapse formation by recruiting Crk and Crk-L. Genes & Development. 24, (21), 2451-2461 (2010).