Cell Invasion Assays for Cancer Research

Cell invasion across the basement membrane is an important step in cancer metastasis.  Metastasis occurs when cancer cells pass through the basement membrane of the organ where they originated, and subsequently spread into different organs of the body, where they form secondary tumors [1].  

Cancer Cell Invasion. Image from Ref. [2].

Cell invasion assays are important tools for cancer research.  Scientists studying cancer perform cell invasion assays to measure the cell movement through an extracellular matrix. By varying the conditions of the cell culture (e.g. adding new drugs), scientists can identify conditions that prevent or accelerate cell invasion.  Through these studies, new treatments for cancer can be identified for potential therapeutic use.

SIMPLE & RELIABLE CELL ASSAYS FOR BREAKTHROUGH SCIENCE

To accelerate scientific breakthroughs in cancer research, Platypus Technologies has introduced a simple, reproducible assay platform to study cell invasion.  The OrisTM platform comprises 96-well plates with removable “stoppers” that create a central cell-free detection zone.  For a cell invasion assay, this detection zone can be filled with an extracellular matrix of choice (e.g. collagen, basement membrane extract (BME), hyaluronic acid, hydrogels, etc.).  The cells invade into the matrix in the detection zone and the extent and rate of cell invasion is easily quantified, in real-time, using a plate reader or from microscope images.  Compared to other approaches, the OrisTM platform offers unique advantages for studying cell invasion:

  • Very simple set-up and straightforward data interpretation
  • Accurately identify specific drugs or conditions that inhibit or stimulate cell invasion
  • Reproducible detection zone enables high accuracy of results
  • Supports real-time and end-point measurements of cell invasion
  • Easy analysis:
    • Use the OrisTM Detection Mask to quantify invasion using plate readers
    • Doing image analysis? No stains or mask required
  • Results are physiologically relevant: cells invade directly into the extracellular matrix; no artificial membranes are needed.

EXAMPLE OF CELL INVASION ASSAY

Below, an experiment illustrates the use of the OrisTM platform for studying cell invasion through basement membrane extract (BME).  The main materials required to perform this assay include: (1) OrisTM cell migration assay coated with BME, (2) BME solution, (3) cells and (4) cell culture media.  HT1080 fibrosarcoma cells were live-stained and cultured on the OrisTM plate coated with BME.  Following a period of incubation to allow for cell attachment, the stoppers that create the central cell-free detection zone were removed, and the wells of the plate were filled with chilled BME solution.  Subsequently, the cells embedded in the BME were incubated at 37 °C.  Images of the cell culture were collected at various intervals and analyzed with ImageJ. 

[SEE DETAILED PROTOCOL FOR ORISTM CELL MIGRATION ASSAY]

The figure below captures representative images of the cell culture before removal of the stoppers, and 40 hours of incubation of the cells in BME. 

(a) Image of cell culture at the start of the experiment. The cells (bright dots) collect around the cell-free detection zone (dark circle), which is filled with basement membrane extract (BME). (b) Image of cell culture after 40 hours of incubation. Note that the cells have invaded into the BME in the detection zone.

As shown in these images, the OrisTM platform allowed for the cells to attach around the circular cell-free detection zone.  This detection zone, filled with BME, makes it easy to characterize cell invasion: cells that move into the circular detection zone can be readily quantified. 

In this case, the extent of cell invasion was characterized by using ImageJ to measure the area of the cell-free zone: the smaller the area, the larger the extent of cell invasion into the BME.  As shown in the figure below, within 24 hours of incubation, a significant number of cells invaded into the BME.  Longer incubation times result in significantly higher rates of cell invasion.    

Cell invasion of HT1080 into basement membrane extract (BME) as a function of time

CONCLUSION

This experiment demonstrates that HT1080 exhibits significant cell invasion into the basement membrane.  Follow-up experiments could explore parameters that influence cell invasion such as (i) stiffness of BME, (ii) drugs and concentration of drugs present in the culture media, or (iii) presence of secondary cell culture (co-culture) present in the BME.  In conclusion, the OrisTM platform provides a simple and reliable platform for carrying cell invasion assays. 

REFERENCES:

[1] T. A. Martin et al. “Cancer Invasion and Metastasis : Molecular and Cellular Perspective” In: Madame Curie Bioscience Database [Internet]. Austin (TX):  Landes Bioscience; 2000-2013. LINK

[2] M. Malboubi et al. “An open access microfluidic device for the study of the physical limits of cancer cell deformation during migration in confined environments” Microelectron. Eng. vol. 144, 16 Aug. 2015 pp. 42-45. LINK

Comparison of the Oris Cell Migration Assay to the Scratch Assay

Introduction

Cell migration is integral to many physiological processes, including embryonic development, tissue regeneration, and wound healing.  In addition, cell migration is involved in tumor metastasis and atherosclerosis.[1]  One assay commonly used to study cell migration in vitro is the scratch assay.  The scratch assay is performed by creating a cell-free gap, or “scratch”, on a confluent cell monolayer upon which cells at the edge of the opening move inward to close the scratch.  Cell migration can be assessed by comparing images captured at the onset of the scratch creation and at user-defined intervals during scratch closure.  The scratch assay is straightforward to perform and is inexpensive.  However, methods for creating the scratch vary from lab to lab and results can be highly variable.  Furthermore, the process of scratch formation has been shown to damage the underlying extracellular matrix (ECM).[2] 

The Oris™ Cell Migration Assay (CMA) was designed to address the limitations of the scratch assay.  The Oris™ CMA (Figure 1) uses a 96-well plate populated with silicone stoppers that exclude cells from the central Detection Zone of the well.  After cells are seeded and allowed to adhere, the silicone stoppers are removed to reveal an unseeded region in the center of each well, into which cells are permitted to migrate.  Assay reproducibility is greater in the Oris™ CMA compared to the scratch assay as a result of uniformly sized Detection Zones, and the underlying ECM is not damaged by the silicone stopper. 

This application note offers a direct comparison of the Oris™ CMA and the scratch assay in assessing cell migration.  Furthermore, ECM integrity is assessed in both assay formats.

Figure 1. Schematic of Oris Cell Migration Assay

Materials and Methods

All steps for the Oris™ CMA and the scratch assay were performed in parallel on the same day.

Oris™ Cell Migration Assay:  Each well of a 96-well Oris™ TC plate was coated with 9µg/mL collagen I (Trevigen) and incubated overnight at 37oC/5%CO2.  Following incubation, wells were rinsed and Oris™ Cell Seeding Stoppers removed stoppers were inserted.  MDA-MB-231 human breast epithelial cells (25,000 cells/100µL) were seeded into all test wells of the Oris™ assay plate.  Once the confluent monolayer was formed, cells were serum-starved for 24 hours, stoppers were removed, and media was replaced with serum-containing media.  Phase images were captured, using a Zeiss Axiovert microscope with an attached CCD camera, immediately following stopper removal to document the pre-migration area of the cell-free Detection Zone.  The Oris™ assay plate was then placed at 37oC/5% CO2 to permit cell migration. 

Scratch Assay:  Each well of a Costar® 6-well plate was coated with 9µg/mL collagen I (Trevigen) and incubated overnight at 37oC/5% CO2.  Following incubation, wells were rinsed, and MDA-MB-231 cells (500,000 cells/2mL) were seeded into each well of the Costar® plate.  Once the confluent monolayer was formed, cells were serum-starved for 24 hours, then cell monolayers were scratched using a 1000µL pipette tip, and serum-containing media was added to each well.  A sharpie marker was used to create a reference point near the scratch in each well, and phase images were captured of each scratch to document the pre-migration area of the cell-free Detection Zone.  In parallel with the Oris™ assay plate, the scratch assay plate was placed at 37oC/5% CO2 to permit cell migration. 

After 24 hours, test wells of both the Oris™ assay plate and scratch assay plate were fixed with 0.25% glutaraldehyde.  Phase images were captured in both assays to document the migration time points.  Using the reference point, the same region of the scratch from the pre-migration image was imaged.  In both assay formats, cell migration was assessed by measuring the area of the Detection Zone at the pre-migration and corresponding migration time-points using ImageJ v1.42l analysis software (http://rsb.info.nih.gov/ij).[3]  Cell migration is presented as percent closure, calculated using the following equation:

((Pre-migration)area – (Migration)area ) x 100  /     (Pre-migration)area  

ECM Integrity Assay:  To test the integrity of the ECM in each assay, 100µg/mL of Collagen Type I – FITC conjugate (Sigma-Aldrich) was coated onto wells of an Oris™  assay plate (in the absence of stoppers) and a Costar® plate, and incubated overnight at 37oC/5% CO2.  Following incubation, stoppers were inserted into the Oris™ assay plate and sterile PBS was added to the wells of both assay plates.  At 1 hour, 24 hours, and 48 hours post-insertion, stoppers were removed and fluorescence images were captured using a Zeiss Axiovert inverted microscope.  At each aforementioned time point, scratches were made in the Costar® plate and images were captured using the same settings as those used for the Oris™ assay plate. 

Results

MDA-MB-231 cell migration was compared on collagen I coated surfaces using two different migration assays; the Oris™ CMA and the scratch assay.  Four separate experiments were performed in parallel on different days to compare the performance of each assay.  For each independent experiment, the average area closure achieved using the Oris™ CMA (Figure 2A and B) ranged from 87%-89% with a coefficient of variance between 3.7-6.5% (Figure 2E).  Conversely, the average area closure obtained using the scratch assay (Figure 2C and D) ranged from 69%-77% with a coefficient of variance between 11.3-25.6% (Figure 2E).  These results demonstrate that the Oris™ CMA yields more consistent results between experiments with greater reproducibility compared to results obtained using the scratch assay. 

Figure 2.  Comparison of cell migration using the Oris™ CMA and the scratch assay.  Representative phase images of pre-migration (A and C) and migration after 20 hours (B and D) in the Oris™ CMA (A and B) and the scratch assay (C and D).  Scale bar = 500µm.  2E.  Graph comparing cell migration using the Oris™ CMA and scratch assay (performed in parallel over four separate experiments, Exp 1-4).  Data are presented as average percent closure ± SD (n≥12).

As indicated in Figure 2E, cell migration in the scratch assay was consistently less than that observed in the Oris™ assay.  One reason for the diminished amount of cell migration in the scratch assay may be due to the damage caused to the ECM during scratch formation.  Kam et al. (2008) demonstrated that the ECM can be damaged during scratch formation.2  To assess whether this might be true in this study, the integrity of the collagen coating following stopper removal and scratch formation was assessed.  The Oris™ Cell Seeding Stopper did not adversely affect the collagen coating, as indicated by uniform fluorescence across the image field (Figure 3A).  Conversely, using assay plates coated with FITC-labeled collagen I, the process of scratch formation damaged the collagen coating, consistent with published findings (Figure 3B).2  These results suggest that damage to the underlying collagen coating may contribute to the reduced cell migration observed using the scratch assay.  

Figure 3. Comparison of the effects of the Oris™ CMA and the Scratch Assay on ECM integrity.Representative images of FITC-labeled collagen I (Collagen I-FITC) after stopper removal from Oris™ CMA plates (A) and following scratch formation in scratch assay plates (B).  Arrows indicate the edge of the stopper or scratched region.  Scale bar = 100µm.

Conclusions

This application note compares the performance of the Oris™ Cell Migration Assay to the commonly used scratch assay.  Results of this study demonstrate that the Oris™ CMA permits more consistent results between experiments with greater reproducibility compared to the scratch assay.  Also, in contrast to mechanical scratch formation in the scratch assay, the use of a silicone stopper in the Oris™ CMA does not damage the underlying ECM.  Collectively, these results illustrate the benefits of the Oris™ CMA over the scratch assay with regards to reproducibility and integrity of the ECM.



[1] 1.  Horwitz R and Webb D. (2003). “Cell migration”.  Current Biology.  13:R756-759. LINK

[2] 2.  Kam Y, Guess C, Estrada L, Weidow B, Quaranta V. (2008). “A novel circular invasion assay mimics in vivo invasive behavior of cancer cell lines and distinguishes single-cell motility in vitro”.  BMC Cancer. 8:198. LINK

[3] Hulkower and Gehler.  “How to Measure Area Closure Using the OrisTM Pro Cell Migration Assay.”  https://www.platypustech.comPlatypus Technologies, LLC.  Web.  May 14, 2010. LINK

Counting Cells in Migration Assays with ImageJ

This application note describes a method to measure cell migration, using ImageJ, by counting the number of cells that have migrated into the Detection Zone in an Oris™ Cell Migration Assay. ImageJ is a freeware image analysis program developed at the National Institutes of Health (https://imagej.nih.gov/ij/).

Cell migration is critical for many physiological events, including embryonic development, wound healing, and the inflammatory response. Furthermore, aberrant motile behavior of cells contributes to pathological processes including tumor metastasis and arthritis (1).

The Oris™ Cell Migration Assay (Figure 1) uses a 96-well plate populated with silicone stoppers that exclude cells from the central Detection Zone of the well. After cells are seeded and allowed to adhere, the silicone stoppers are removed to reveal a 2mm diameter unseeded region in the center of each well, into which cells are permitted to migrate.

Figure 1. Schematic of Oris™ Cell Migration Assays

METHODS

MDA-MB-231 breast epithelial cells and HT-1080 fibrosarcoma cells were cultured on an Oris™ Cell Migration Assay – TriCoated plate having Tissue Culture Treated, Collagen I coated, or Fibronectin coated wells. After 16 hours, cells were fixed with 0.25% glutaraldehyde and cell nuclei were stained with 1:2000 DAPI (Pierce). Images were acquired using a 5X objective on a Zeiss Axiovert 200 inverted microscope equipped with a CCD camera.

Cell migration into the Detection Zone was measured by counting cell number using ImageJ analysis software (version 1.42l). First, the threshold was set for each grayscale image (Image–> Adjust–> Threshold). By selecting “Apply” in the threshold window, the thresholded image was converted to a binary image. Slightly overlapping nuclei were separated by performing a Watershed segmentation process (Process–> Binary–> Watershed).

Using the binary image, a circular region-of-interest (ROI) measuring 2mm in diameter (the same diameter as the tip of the stopper) was made using the menu command Edit–> Selection–> Specify. In the Specify window, “width” and “height” were set at 2mm, and the “oval” box was checked. The ROI was centered over the Detection Zone within each well. The number of nuclei contained in the ROI was quantified using the menu command Analyze–> Analyze particles. Values to define the minimum and maximum particle size were 100 and 1000 pixels2, respectively. “Show Masks” was selected to display a drawing of the detected objects. “Summary” and “Exclude on Edges” were checked for analysis.

The cell counts from the Summary Window (i.e., counts) were exported into Windows Excel for statistical analysis. The number of nuclei for each condition was averaged from 8 wells. Additional information regarding the use of ImageJ for particle analysis can be found at https://imagej.nih.gov/ij/docs/menus/analyze.html#ap).

RESULTS

In this application note, MDA-MB-231 and HT-1080 cell migration on three surfaces (Tissue Culture Treated, Collagen I, or Fibronectin; Oris™ Cell Migation Assay – TriCoated), was assessed by counting the number of cells in the Detection Zone using ImageJ. MDA-MB-231 cells exhibited varying degrees of migration into the Detection Zone dependent upon the surface coating of the well (Figure 2). Phase images of cells acquired immediately after stopper removal (migration control) and 16 hours after stopper removal demonstrate differences in cell migration depending upon whether cells were seeded on a Tissue Culture Treated surface, a Collagen I coated surface, or a Fibronectin coated surface (Figure 2A-D).

Using ImageJ, DAPI-labeled cells were counted by creating a 2mm circular region-of-interest (ROI) similar in size to the initial Detection Zone (Figure 2E-H). Performing the particle analysis function in ImageJ yielded drawings of detected objects that were counted within the circular ROI (Figure 2I-L). Differences in MDA-MB-231 cell migration into the Detection Zone (ROI) were highlighted by overlaying the particle analysis drawing on the original fluorescent image (Figure 2M-P).

Figure 2. ImageJ Analysis of Cell Migration by Counting Cell Number in the Detection Zone. (A-D) Phase images of migration control (A) and MDA-MB-231 cell migration on Tissue Culture Treated (B), Collage I (C), and Fibronectin (D) 16 hours after removal of stoppers from Oris™ Cell Migration Assay. (E-H), Fluorescent images of DAPI-labeled cells with a 2-mm circular ROI (red circle) defining the region for particle analysis. (I-L) Particle analysis drawings of objects in the ROI. (M-P) Merged images of DAPI and particle analysis drawings. Scale bar = 500 micrometers.

Figure 3 shows the average number of MDA-MB-231 (3A) and HT-1080 (3B) cells that migrated into the Detection Zone when seeded on Tissue Culture treated, Collagen I coated, and Fibronectin coated wells. Both MDA-MB-231 and HT-1080 cells exhibited the most robust migration on Collagen I. Furthermore, this method of analysis yielded statistical differences in the migration of model cell lines on all three plate coatings (i.e., MDA-MB-231 migration on Collagen I versus Fibronectin).

Figure 3. Quantitation of Cell Number using ImageJ. (A) MDA-MB-231 and (B) HT-1080 cell migration on three surfaces (Tissue Culture Treated, Collagen I, or Fibronectin). Data are represented as average cell number +\- SD from 8 wells for each condition. p<0.001; statistical difference between control vs. 16 hour migration on all surfaces; p<0.005; statistical difference in cell migration between surfaces (two-sample t-test).

CONCLUSIONS

This application note demonstrates a method to measure cell migration in the Oris™ Cell Migration Assay by the use of ImageJ analysis software for counting cells. This study, using ImageJ to quantify cell number in the Detection Zone, demonstrates that both MDA-MB-231 and HT-1080 cells exhibited statistically significant differences in migration when seeded on Tissue Culture Treated, Collagen I coated, and Fibronectin coated wells. Using the detailed analysis method described here, ImageJ can provide an accurate measure of cell migration when using the Oris™ Cell Migration Assay.

Cells on the Move: Do surface coatings influence cell migration?

Experiments show that surface coatings play an important role in cell movement

When performing cell migration experiments, a perennial question is: what surface coatings should be used to culture a particular cell type? Scientists working in Cancer Research, Wound Healing, or Drug Discovery utilize cell cultures to make important experiments and advance our understanding of biological mechanisms.  In particular, assays for cell migration enable characterization of conditions and substances that influence movement of cells.  For example, scientists using the OrisTM Cell Migration Assays successfully identified proteins, mRNA and antioxidants that inhibit migration of tumor cells. 

Each cell type requires specific conditions for optimal growth, so it is important to choose the right surface to culture each cell type.  For example, fibroblasts produce and inhabit the connective tissue of the body; thus we may hypothesize that surfaces and scaffolds that are rich in collagen and other fibers (components of connective tissue) are better suited for culturing fibroblasts.  This means that collagen-coated surfaces are a good choice when culturing fibroblasts for cell migration assays.  But does the choice of surface coating influence cell migration?

In new experiments, scientists at Platypus Technologies demonstrate that the choice of surface coating for cell culture may influence the results for cell migration experiments.  

Oris™ Cell Migration Assays use a 96-well plate with “stopper” barriers that create a central cell-free Detection Zone for cell migration experiments.  Removing the stoppers allows the cells to migrate into the Detection Zone at the center of each well:

Principle of OrisTM Cell Migration Assay: OrisTM stoppers are used to create a central cell free Detection Zone (red dotted circle).  Following 20 hours of incubation, the cells migrate into the detection zone.

In this experiment, the scientists used a cell line called HT1080, which are fibroblasts isolated from a malignant human tumor.  The cells were incubated on the OrisTM Cell Migration Assays that had wells coated with five different bioactive surfaces: Tissue Culture, Collagen I, Fibronectin, Poly-L-Lysine, and Basement Membrane Extract (BME).  Following cell attachment, the OrisTM stoppers were removed to permit cell migration into the detection zone.  The cells were then imaged following 24 hours of incubation without the OrisTM stoppers.  Representative images from this experiment are shown below:

Representative images of cells (HT1080) cultured in surfaces treated with various bioactive coatings.  OrisTM stoppers are used to create a central cell-free Detection Zone.  Following 24 hours of incubation, the cells migrate into the detection zone. HT1080 cells cultured in Collagen I surfaces migrate faster than when cultured in other surfaces.

Through visual inspection of these images, we observe that the cells cultured on Collagen I migrate deeper into the detection zone, compared to cells cultured on other bioactive surfaces.  There is no noticeable difference in the migration of the cells cultured on tissue culture, fibronectin, poly-l-lysine or basement membrane extract. 

Twelve (12) different wells from each plate were imaged prior and after cell migration, and the area of the cell-free zone was measured using ImageJ.  The difference in the area of the cell-free zone pre- and post- migration was used to calculate the percentage area closure of the assay.  These measurements are presented in the graph below, which show unequivocally that HT1080 cells exhibit higher migration rates in Collagen I than in any other surface coating. 

Quantitative Migration of Cells incubated on different bioactive surfaces.  These data indicate that the HT1080 cells incubated on Collagen I (Col I) exhibit higher rates of cell migration. 

In conclusion, the choice of surface coating has a strong effect on the cell migration of HT1080 cells.  Cells cultured in collagen I exhibit the fastest rate of cell migration, compared to cells cultured in other surfaces.  These results demonstrate that the choice of surface coatings plays an important role on the measured cell migration.

Learn more about OrisTM Cell Migration Assays: https://www.platypustech.com/cell-based-assays/oris-cell-migration

Learn more about 96-well plates coated with bioactive surfaces for cell culture: https://www.platypustech.com/cell-culture-solutions/microplates

External Links:

https://www.nature.com/articles/s41598-019-52480-3

https://www.thoughtco.com/how-and-why-cells-move-373377

New Surfaces for Cell Migration Assays

Platypus Technologies introduces new surfaces for Oris™ Cell Migration Assays: Poly-L-lysine and basement membrane extract.

Polylysine surfaces contain positively-charged amine groups that enhance attachment of negatively-charged proteins and cells. Polylysine surfaces are popular for culturing cell lines derived from nerve tissue (e.g. neurons, glial cells, fibroblasts, epithelial cells).

The basement membrane extract (BME) contains multiple extracellular matrix proteins, including laminin and collagen. Surfaces coated with BME support cell culture assays of epithelial cells, endothelial cells, muscle cells and stem cells.

Learn more about Cell Migration Assays: https://www.platypustech.com/cell-based-assays/oris-cell-migration