Analysis of Random Migration of Cancer Cells in 3D

Materials and Reagents

1. µ-slide 8 well with a glass bottom (Ibidi, catalog number: 80827)
2. Cell line: MDA-MB-231 (ATCC, catalog number: HTB-26)
3. L-15 medium (Thermo Fisher Scientific, catalog number: 11415049)
4. Fetal bovine serum (FBS) (Thermo Fisher Scientific, catalog number: A31605)
5. Penicillin-Streptomycin (100x) (Thermo Fisher Scientific, catalog number: 15140122)
6. Trypsin (0.25%) (Thermo Fisher Scientific, catalog number: 15050065)
7. DPBS (Thermo Fisher Scientific, catalog number: 14190250)
8. 10x MEM (Thermo Fisher Scientific, catalog number: 11430030)
9. HEPES (Thermo Fisher Scientific, catalog number: 15630080)
10. Collagen I (Rat-tail, 4.41 mg/ml) (BD Biosciences, catalog number: 354236)
11. Fibronectin (5 mg) (FN) (Sigma, catalog number: F1141)
12. MDA-MB-231 culture medium (see Recipes)
13. Collagen-Fibronectin gel mix (see Recipes)
    

Equipment

1. Phase contrast microscope with 20x air objective, temperature module, CO₂ module and auto focus module (Zeiss Axio Observer microscope)
2. 37 °C cell culture incubator
3. Laminar airflow hood
   

Software

1. Fiji: ImageJ with MTrackJ plugin
   Note: Fiji: ImageJ, software could be downloaded from https://imagej.net/Fiji/Downloads and for MTrackJ plugin information go to https://imagescience.org/meijering/software/mtrackj/.
2. Microsoft (MS) Excel 2007 or later
   

Procedure

1. Prepare the appropriate volume of collagen-fibronectin mix as described in Recipe 2.
2. Add 100 µl of gel mix per well of µ-Slide 8 well and let it polymerize at 37 °C for 1 h (Figure 1A).
   Note: After gelification, collagen-fibronectin gel becomes slightly milky white and collagen fibers become uniformly visible.
3. During this gelification period, trypsinize the cells and count them. Set the cell concentration to 10⁴ cells per ml.
4. After gel polymerizes, put 200 µl of medium containing cells (i.e., 2,000 cells) on top of the gel.
5. Put µ-slide 8 well in the incubator at 37 °C and allow the cells to adhere on the gel surface for about 2 h (Figure 1A).
6. Once cells have adhered, remove the medium gently. This can be done by slowly pipetting out the medium from the corners of the µ-slide well.
   Note: This step is delicate. While removing the medium, extra care must be taken not to disturb the gel and cells. If the gel gets disturbed/damaged, then they are not good for imaging.
7. After removing the medium, keep the µ-slide aside in the laminar airflow hood and quickly, re-prepare the collagen-fibronectin mix.
8. Put 100 µl of gel mix on the top of the cells adhering to the previously casted gel (Figure 1A).
9. Incubate for 1 h at 37 °C in the incubator for the gel to polymerize.
   Note: After gelification of the second layer of gel, one can observe cells that are sandwiched between two gels in a single z plane.
10. Meanwhile, switch on the microscope and the microscope stage heater. Adjust the temperature to 37 °C.
11. After gelification of the second layer gel, add 150 µl of complete medium on top of the second gel to ensure the gels do not dry out (Figure 1A).
12. Carefully place the µ-slide into a microscope insert designed to hold the slides and allowing for temperature, humidity, and CO₂ control.
13. Place the insert onto the microscope stage. Set the focus on the cells that are at the center of well. Use a 20x objective. Use an objective with phase contrast (Figure 1B).
    Note: The center of the µ-slide wells is best for imaging to avoid aberrations close to the sides of the wells.
14. Acquire time-lapse images for 60 h with a time-lapse interval of 20 min.
    
    
    Figure 1. Steps in 3D migration setup. A. Schematic overview depicting the steps involved in the protocol. B. Exemplary time-lapse images of the MDA-MB-231 cells that are sandwiched between two collagen-fibronectin gels, depicting the cell morphology changes during its 3D migration. Scale bar: 40 µm. For the exemplary depiction of the 3D migration video, refer to Visweshwaran et al., 2018, Movie EV2.
    

Data analysis

1. Once the video acquisition is finished, assign the image properties. Open the video/image stack file in Fiji and assign the image properties by selecting ‘Image’ → ‘Properties…’ and fill in ‘Unit of length’ (example-µm), ‘Pixel width’ (pixel size in µm), ‘Pixel height’ (pixel size in µm) and ‘Frame interval’ (20 min). Save the video/image stack. A similar assignment can also be done by selecting ‘Analyze’ → ‘Set scale’ option.
2. Manual tracking and analysis of cell trajectories are done as described in Dang and Gautreau, 2018, and Gorelik and Gautreau, 2014. Briefly, manual tracking performed with the help of Fiji software plugin 'MTrackJ', which gives the trajectory data. These data are then copied to MS Excel file containing DiPer suite of custom-made macros for quantifying migration parameters. By running the DiPer macros, various migration parameters like cell speed, directional persistence (direction autocorrelation), and mean squared displacement (MSD) are calculated and plotted, which are used to make publication-quality figures (Figure 2).
3. Automated tracking and analysis of cell trajectories are also possible for this set-up. Visweshwaran and Maritzen, 2019 describes a suitable procedure to quantify 3D cell migration automatically with minimal input by the experimenter.
   Note: MS Excel file loaded with DiPer suite of macros are available as supplementary file: Excel_loaded_with_DiPer. Detailed description about DiPer and the migration parameters like cell speed, directional persistence (direction autocorrelation) and mean squared displacement (MSD) available in Gorelik and Gautreau, 2014.
   
   
   Figure 2. Representative 3D migration quantification of HSBP1 depleted MDA-MB-231 cells. Please refer to Visweshwaran et al. (2018) for details. Briefly, top panels display single-cell trajectories. Mean Square Displacement (MSD) analysis shows that HSBP1 depleted cells explore a smaller territory than control cells. This effect might be accounted for by decreased speed and directional persistence. At least 10 cells were tracked per condition, in 3 independent experiments; data are mean ± S.E.M; ANOVA **P < 0.01; ****P  <  0.0001.
   

Notes

1. When MDA-MB-231 cells are cultured in Leibovitz’s L-15 medium, no CO₂ should be used for pH control. The L-15 medium formulation was devised for use with atmospheric air. Incubation in an atmosphere enriched with CO₂ would be detrimental to cells.
2. For running macros to analyze the cell trajectories, ‘Developer’ tab in the MS Excel must be enabled. The ‘Developer’ tab is enabled by going to ‘Excel Options’ → ‘Customize Ribbon’ → ‘Customize the Ribbon’ → ‘Main tab’ → checking ‘Developer’.
3. The effects of drugs/inhibitors on 3D migration of the cells could be investigated in this set-up. For a long-term effect analysis, the drug could be added while making the collagen-fibronectin gels and in the medium that is added at the final stage to prevent the gel drying. In this method, cells are exposed constantly to the drug for a full period of observation. On the other end, the addition of drugs/inhibitors during 3D migration is also possible. In this method, a high concentration of drug should be added (1-10 µl) gently to the medium that is present on the top of the gels to prevent the gel drying. Here, the drug will diffuse into the gels. In this case, the experimenter initially would record a 3D cell migration for a period where there is no drug treatment and after addition of the drug, a period of 3D cell migration under drug treatment. If the drugs/inhibitors are toxic, a brief pre-treatment of the cells with the drug and sandwiching them between gels for migration analysis are also an alternative method.
   

Recipes

1. MDA-MB-231 culture medium
   L-15 medium
   10% FBS
   1x Penicillin-Streptomycin
2. Collagen-Fibronectin gel mix
   2 mg/ml Collagen I
   10 µg/ml FN
   25 mM HEPES
   10% FBS
   1x MEM completed with sterile water
   For 1 ml gel volume:
   

   10x MEM	100 µl
   HEPES (1 M)	25 µl
   Collagen I (4.41 mg/ml)	452 µl
   FBS	100 µl
   FN	10 µl
   Sterile water	313 µl

Acknowledgments

This protocol was adapted from Dang et al. (2013) with modifications. The establishment of this protocol was funded by AG’s group grant: the Agence Nationale de la Recherche (ANR ANR-15-CE13-0016-01), the fondation ARC pour la Recherche sur le Cancer (PGA120140200831), and Institut National du Cancer (INCA_6521). SPV was supported by PhD fellowships from Ministère de l'Enseignement Supérieur et de la Recherche for the first 3 years and from Ligue Nationale contre le Cancer for the 4th year.

Competing interests

The authors declare that they have no conflict of interests.

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