This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.
The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an "in-incubator" video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.
Cell invasion and migration are involved in the dissemination of cancer cells, which is the main cause of resistance to treatment1 and can lead to locoregional or metastatic recurrence after cancer treatment2. The epithelial-mesenchymal transition (EMT) is the initial process of cell invasion-migration in which cancer cells switch from an epithelial to a mesenchymal phenotype. E-Cadherin is an extracellular marker of the epithelial phenotype3, and increased expression of N-cadherin and vimentin is characteristic of the mesenchymal phenotype4. Migration also depends on the intrinsic capacity of cancer cells to invade the extracellular matrix (ECM) through the action of matrix metalloproteases5.
This invasion–migration mechanism has been described for cancer at many locations, particularly in head and neck cancer6. Many researchers have focused on the migration and invasion processes to understand better how cancer cells disseminate in the hope that this knowledge will lead to new treatment strategies. It is crucial that these studies are performed using reliable and reproducible assays.
In vitro analysis of cell motility can be challenging. Developed many years ago, the Boyden chamber assay is considered to be the standard for invasion–migration analysis7. However, it is time consuming and is often inaccurate. A second test is the wound-healing assay8, which involves making a scratch on a cell monolayer culture and capturing images of cell invasion and migration at fixed time intervals. This technique has been criticized widely because of the large variations between the results of two successive tests. However, the application of modern technologies, especially in microscopy, has improved the reproducibility of the scratch wound assay. Video microscopes can be easily introduced in incubators and can generate real-time images of cell migration. These devices record microscopic data and provide automatic analysis of wound cell confluence over time. The aim of this paper is to describe the Boyden chamber assay and the optimized scratch wound assay, and to discuss the advantages and weaknesses of each approach.
NOTE: The Boyden chamber and scratch assays without inclusion of the ECM are referred to as the migration assay, and the same assays with the ECM is referred to as the invasion assay.
1. Boyden Chamber Assay
NOTE: This protocol is adapted for the SQ20B cell line, which is derived from a recurrent Head and Neck Squamous Cell Carcinoma (HNSCC) laryngeal cancer and was obtained from John Little (Boston, MA, USA).
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2. Scratch Wound Assay: Cell Migration
NOTE: Instructions must be adapted for each type of cell.
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3. Scratch Wound Assay: Cell Invasion
NOTE: Instructions must be adapted for each type of cell.
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We report here two different methods to analyze cell invasion and migration. Figure 1 shows the Boyden chamber experiment. The inserts are placed into a companion plate with chemoattractant medium, and the cells are seeded in CM. The membrane can be uncoated (migration assay) or coated (invasion assay). Cells are seeded into the upper chamber in s-CM. The lower chamber is filled with CM as a chemoattractant. The cells are fixed before the doubling time.
Figure 2 shows the Boyden membrane after cell fixation. Figure 2A shows a suboptimal result, with cell clusters on the upper side of the membrane. Figure 2B shows an optimal result with cells fixed and stained in blue on the membrane.
Figure 3 shows an optimal result of the scratch wound assay. The linear wound can be seen in Figure 3A. No cells are observed in the wound, and wound healing has occurred within 30 h. The time to heal the wound is dependent on the cell line and ranges from 24 to 50 h.
Figure 3B is a graphical representation of wound healing under four treatment conditions: control, ABT-199 (anti-Bcl-2), cetuximab (anti-EGFR), and ABT-199+cetuximab. The ABT-199+cetuximab combination significantly decreased cell migration. The curves are obtained using the video microscope software, which provides robust data analysis of cell migration with time. Error bars represent the standard deviation (SD) for each well.
Ninety percent confluence is the optimal cell density for the wound-healing assay. The optimal cell seeding depends on the cell line and ranges from 3 x 104 to 6 x 104 cells. Figure 4A shows the optimal cell density, and Figure 4C shows low cell density. Wells should be washed twice to avoid cell clusters, as shown in Figure 4B.
Figure 1: Schematic representation of the Boyden chamber experiment. Inserts are placed into a companion plate with chemoattractant medium, and the cells are seeded in CM. The membrane can be uncoated (A) or coated (B).
Figure 2: Suboptimal and optimal microscopic results obtained in the Boyden chamber experiment. (A) Suboptimal membrane with cell clusters on the upper side of the membrane and uninterpretable results. Scale bar = 300 µm. (B) Optimal membrane with countable cells in the lower part of the membrane stained in blue. Scale bar = 300 µm.
Figure 3: Optimal results obtained in the wound-healing experiment. (A) Representative image from the scratch wound assay shown wound healing observed at 0, 15, and 30 h. The criteria for a quality experiment are a linear wound, no cell fragments observed in the wound, and optimal cell confluence. Scale bar = 300 µm. (B) Graphical representation of wound healing showing quantification of parameters of wound cell confluence (percentage) according to time (h). Four treatment conditions are analyzed here, and the data are shown with the SD.
Figure 4: Suboptimal results obtained in the wound-healing experiment. (A) Ideal cell density. (B) Problems washing the cell pellet. (C) Low cell density. Scale bars = 300 µm
Advantages | Disadvantages | |
Boyden Chamber Experiment | - 3D-Cell Chemotaxis | - Time-Consuming |
- Both invasion and migration with coated and non coated layer matrix | - Low reproducibility | |
- Expensive inserts | ||
- No Time-Lapse exploration | ||
- Need of adherent cells | ||
Wound Healing Assay | - Automatic Highly reproducible wound | - 2D-Cell motility |
- Both invasion and migration with coated and non coated layer matrix | - Need of adherent cells | |
- Cell proliferation included in the analysis | ||
- Time-Lapse microscopic visual analysis | ||
- Data exportation of wound healing metrics with precise analysis | ||
- Post-treatment robust software for cell migration and invasion curves |
Table 1: Advantages and disadvantages of the Boyden chamber experiment and video microscope wound-healing assay. The advantages and disadvantages of the Boyden chamber experiment and scratch wound assay.
We report here two different modalities to study the cell invasion and migration process. The analysis of this process is important to understanding the factors involved in metastatic recurrence, which might be explained by increased motility of a subpopulation of cancer cells called cancer stem cells10,11.
The Boyden chamber experiment is one the most frequently used techniques to explore cell invasion and migration. One advantage of this approach is its reproducibility, although this technique is highly dependent on the experimenter's expertise. Cell counting is manual and can vary between experimenters. Many critical steps must be standardized and followed with care, such as ensuring the use of the appropriate chemoattractant and starvation medium specific to each cell line. Removing cells from the upper membrane with a cotton swab is also critical to ensuring optimal results and avoiding the suboptimal results shown in Figure 2A. If this assay is used to evaluate cell motility, invasion, and migration capacities through a three-dimensional (3D) porous membrane, it is not possible to track cells through a time-lapse experiment. Moreover, commercial inserts with a porous membrane are often expensive.
The video microscope-based scratch wound assay presented here is a robust technique to evaluate cell migration and invasion. The assay is performed with a mechanical wound maker and provides a reproducible comparison between treatment conditions. The video microscopic analysis can be used to generate precise quantitative data for wound healing and cell confluence through a time-lapse experiment. Time-to-time analysis of different treatment conditions can be corroborated by microscopic observations. This analysis integrates cell proliferation during the assay and takes into account the cell-doubling time. Video or raw data content can be exported to obtain a highly visual representation of the results, as shown in the cell migration curves in Figure 3B.
Many critical steps are required to obtain interpretable results. The optimal conditions for cell seeding are dependent on the cell line and must be tested with different dilutions to obtain 90% cell confluence in the wells. To obtain a linear wound, cell plating must not exceed 16 h. The washing step must be done twice and rapidly to avoid introducing cell debris into the wound that could alter the results. For the invasion scratch wound, proper care of the ECM is also a critical step. The cooling rates must be constant, and the ECM should be manipulated with chilled pipette tips and maintained at 4 °C.
These techniques have many advantages, which are summarized in Table 1. Adherent cells are needed for this technique. Scratching causes mechanical injury to the cells, which causes release of cellular contents into the surrounding medium and may influence the migration process. The scratch wound assay provides an estimate of 2D cell motility but not of cell chemotaxis, which cannot be studied using this technique. We have recently developed a video microscopic chemotaxis assay using a commercial plating system. This assay needs cells with a high migration ability, such as cancer stem cells, as described previously12. This new technique may become the gold standard for invasion-migration assays in the future.
The authors have nothing to disclose.
These techniques were developed with the support of LabEx PRIMES (ANR-11-LABX-0063), the Contrat Plan-Etat-Region (CPER) within the scientific framework of ETOILE (CPER 2009-2013), and Lyric Grant INCa-DGOS-4664.
Fetal Calf Serum Gold | GE Healthcare | A15-151 | |
Hydrocortisone water soluble | Sigma-Aldrich | H0396-100MG | |
Penicillin/Streptomycin 100 X | Dominique Dutscher | L0022-100 | |
DMEM | Gibco | 61965-026 | |
F12 Nut Mix (1X) + GlutaMAX-I | Gibco | 31765-027 | |
EGF | Promega | G5021 | The solution must be prepared just before use because it is very unstable |
Z1 coulter particle | Beckman Coulter | 6605698 | |
Optical microscope | Olympus | CKX31 | |
SQ20B cell line | Gift from the John Little’s Laboratory | – | |
Wound Maker | Essen Bioscience | 4494 | Store in safe and dry place |
96-well ImageLock Plate | Essen Bioscience | 4379 | |
CoolBox 96F System with CoolSink 96F | Essen Bioscience | 1500-0078-A00 | |
CoolBox with M30 System | Essen Bioscience | 1500-0078-A00 | |
Boyden Insert | Dominique Dutcher | 353097 | |
Boyden Coated Insert | Dominique Dutcher | 354483 | Store at -20 °C |
Companion 24-well Plate | Dominique Dutcher | 353504 | |
BD Matrigel standard | BD BioScience | BD 354234 | Store at -20°C. |
RAL 555 Staining Kit | RAL Diagnostics | 361550 | Store in safe and dry place |
Microcentrifuge tubes | Eppendorf | 33511 |