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
2. Preparation of the Real-time Cell Analysis System, Cell Invasion and Migration (CIM) Plates, and Electroporated U-118MG Cells for Plating
3. Baseline Reading, Seeding of the Cells, and Cell Impedance Measurement and Visualization
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 |
Crk siRNA | Dharmacon | J-010503-10 | |
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 |
Trypsin-EDTA | Gibco | 25300-054 | |
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 |