Protocol

In Vitro Assays for the Extracellular Matrix Protein-Regulated Extravasation Process

Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA

¹Corresponding author (wang0011{at}mc.duke.edu)

INTRODUCTION

Extravasation, the process by which circulating tumor cells pass through the blood vessel wall, is a critical step of metastasis. Extracellular matrix (ECM) proteins secreted by the cancer cells are likely to play an interactive role in the dynamic interaction between cancer cells and endothelial cells during the extravasation process. This protocol describes two in vitro assays, the transendothelial cell migration (TEM) assay and the vascular permeability assay, which are used to demonstrate the involvement of ECM proteins in cancer cell extravasation. Both assays employ primary human umbilical vein endothelial cells (HUVEC) to reconstitute a vessel wall (HUVEC monolayer) on a porous filter membrane within a Transwell chamber. The TEM assay examines the efficiency of cancer cells to migrate through the vessel by co-culturing the cancer cells with the endothelial monolayer. The vascular permeability assay allows the study of the impact of secreted ECM proteins on the permeability of the vessel wall by applying conditioned medium from cancer cells to the endothelial monolayer.

MATERIALS

Reagents

5-(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (5 mM) (CellTrace; Invitrogen C34554)

Immediately before use, dilute the 5 mM stock to 50 µM with 1X PBS containing 5% (v/v) FBS.

Cancer cells of interest

Endothelial growth medium-2 (EGM-2; Lonza) (prewarmed to 37°C)

Fetal bovine serum (FBS) (Mediatech)

Fibronectin (1 mg/mL) (BD Biosciences)

FITC-dextran (25 mg/mL; m.w. 4 x 10⁴) (Sigma FD40S)

Growth Factor Reduced (GFR) Basement Membrane Matrigel Matrix (1 mg/mL) (BD Biosciences 356230)

HUVEC (passage 5-9)

Medium for cancer cells (growth medium and serum-free medium)

Paraformaldehyde (4% [w/v] in 1X PBS)

PBS (1X) containing 5% (v/v) FBS (prewarmed to 37°C)

Phosphate-buffered saline (PBS) (1X, pH 7.4)

Versene (Invitrogen)

Equipment

Centrifuge

Cotton swabs

Coverslips

Fluorescence plate reader

Ice

Incubator preset to 37°C, 5% CO₂

Laminar flow hood

Microscope (fluorescence) (Zeiss Axio)

Plates (96-well, solid black) (Corning 3915)

Slides

Syringe (10-mL)

Syringe filter (0.22-µm)

Transwell chambers for the TEM assay (24-well, 8-µm pore size) (Corning 3422)

Transwell chambers for the vascular permeability assay (24-well, 0.4-µm pore size) (Corning 3413)

METHOD

TEM Assay

A schematic diagram of the TEM assay is shown in Figure 1 .

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Figure 1. A schematic diagram of the TEM assay. (A) HUVEC cells form a monolayer on a Matrigel-coated membrane filter. (B) CFSE-labeled tumor cells (1 x 105 cells) are seeded into the upper chamber of the Transwell chamber in 100 µL of culture medium. The chamber is incubated at 37°C in an atmosphere of 5% CO2. (C) After 4 h of incubation, transmigrated cells have passed through the endothelial monolayer and Matrigel layer onto the basolateral side of the membrane. (D) Cells on the apical side are removed. The membranes are washed, fixed, and mounted. Migrated cells are counted for quantification.

Generation of HUVEC Monolayer

1. For Matrigel coating, thaw Matrigel on ice and dilute with ice-cold H₂O to 0.125 mg/mL. Apply 150 µL of diluted Matrigel to the membrane filter (8-µm pore size) of each Transwell insert, and immediately remove 50 µL. Dry filters overnight in a laminar flow hood.
To establish a uniform horizontal layer of Matrigel, it is essential to add extra Matrigel and then remove it.

2. To reconstitute the Matrigel layer, gently add prewarmed EGM-2 medium (200 µL) to the inserts and incubate at 37°C for 1 h. Aspirate the medium carefully.

3. Plate HUVEC cells (5 x 10⁴ in 100 µL of EGM-2 medium) on the Matrigel-coated insert in the chambers and maintain overnight in this medium to allow the endothelial cells to form a monolayer.

4. Determine the confluency of the HUVEC monolayer:

i. Add high-molecular-weight FITC-dextran (4 µL) to the insert. Add EGM-2 medium (600 µL) to the lower chamber. Every 30 min, collect samples (50 µL) from the lower chamber and replace immediately with the same volume of EGM-2 medium to maintain hydrostatic equilibrium.

ii. Dilute the samples to 1 mL with 1X PBS. Transfer 100 µL of each diluted sample into 96-well black plates and measure the fluorescent content at 492/520 nm absorption/emission wavelengths for FITC-dextran.
No fluorescent intensity is detected in the lower chamber once the HUVEC monolayer reaches confluency.

Cancer Cell Labeling

5. Dissociate the cancer cells with Versene, resuspend as single cells in growth medium for cancer cells, and count.
See Troubleshooting.

6. Centrifuge the cells (1 x 10⁶) and resuspend the cell pellet in 900 µL of prewarmed 1X PBS containing 5% FBS.

7. Immediately mix the cancer cells with 100 µL of diluted CFSE (50 µM) and incubate for 10 min at 37°C. Wash the cells three times with 1X PBS containing 5% FBS and resuspend in EGM-2 medium at 1 x 10⁶/mL.
The final working concentration for CFSE is 5 µM.

Co-culture and Determination of the Transendothelial Migration Efficiency

8. Carefully aspirate the culture medium from the inserts. Gently seed cancer cells (1 x 10⁵ in 100 µL of EGM-2 medium) on top of the HUVEC monolayer. Place growth medium for cancer cells (600 µL) in the lower chamber as a chemoattractant.

9. Incubate the co-cultures for 4 h.
This time may vary with the cell line used.

10. Remove the cells on the apical side of each insert using cotton swabs. Wash the membranes three times with 1X PBS, fix with 4% paraformaldehyde for 10 min at room temperature, and mount onto slides.
Accurate quantitation of cell transmigration requires the careful removal of nonmigrated cells from the surface of the Matrigel layer using a cotton swab. If all of the nonmigrated cells are not removed, cell clusters can be observed under an immunofluorescent microscope. In this case, the insert should be discarded.

11. Visualize migrated cells with an immunofluorescent microscope at 20X magnification at a 488-nm excitation wavelength. Within each well, count five randomly selected microscopic fields.
See Troubleshooting.

Vascular Permeability Assay

A schematic diagram of the vascular permeability assay is shown in Figure 2 .

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Figure 2. A schematic diagram of the vascular permeability assay. (A) HUVEC cells form a monolayer on a fibronectin-coated membrane filter. (B) Conditioned medium and FITC-dextran (100 µL) are added into the upper chamber of the Transwell chamber. The chamber is incubated at 37°C in an atmosphere of 5% CO2. (C) FITC-dextran passes through the endothelial monolayer. Samples are taken from the lower chamber periodically to quantify the fluorescence intensity in the lower chamber. The permeability of the endothelial monolayer correlates with the fluorescence intensity in the lower chamber.

Generation of HUVEC Monolayer

12. For fibronectin coating, dilute fibronectin (1 mg/mL) with 1X PBS to 50 µg/mL. Apply 50 µL of diluted fibronectin to the membrane filter (0.4-µm pore size) of each Transwell insert and incubate for 30 min at room temperature. Carefully aspirate any excessive liquid. Wash inserts once with 1X PBS.

13. Plate HUVEC cells (2.7 x 10⁴) on the fibronectin-coated insert and culture with 100 µL of EGM-2 medium in the upper chamber and 600 µL of EGM-2 medium in the lower chamber. Grow the cells for 3 d without medium change (until they have reached confluence).

14. Determine the confluency of the HUVEC monolayer by examining the permeability of high-molecular-weight FITC-dextran through the monolayer as described in Step 4.

Generation of Conditioned Medium

15. Grow the cancer cells in growth medium for cancer cells until they reach 80% confluency. Wash twice with 1X PBS, and culture in serum-free medium for cancer cells (5 mL for each 10-cm plate) for 24 h.
The volume used is smaller than regular culture volumes, so that the molecule of interest is concentrated.

16. Collect the conditioned medium with a 10-mL syringe and filter through a 0.22-µm syringe filter.
Filtration removes cell debris and cells suspended in the medium.

Co-culture and Determination of the Permeability of the Monolayer

17. Carefully aspirate the culture medium from the inserts. Add 96 µL of the conditioned medium from Step 16 and 4 µL of FITC-dextran to the upper chamber.
The final concentration of FITC-dextran is 1 mg/mL.

18. Every 30 min, collect samples (50 µL) from the lower chamber and replace immediately with the same volume of growth medium for cancer cells to maintain hydrostatic equilibrium.

19. Dilute the samples to 1 mL with 1X PBS. Transfer 100 µL of each diluted sample into 96-well black plates and measure the fluorescent content at 492/520 nm absorption/emission wavelengths for FITC-dextran.
The permeability of the HUVEC monolayer correlates with the fluorescent intensity in the lower chamber.
See Troubleshooting.

TROUBLESHOOTING

Problem: A single cancer cell suspension cannot be generated with Versene treatment.

[Step 5]

Solution: The use of Versene minimizes damage to cell surface proteins, because Versene contains EDTA but no trypsin (see Discussion). Seed cancer cells sparsely onto the culture plates 24 h before the dissociation; short culturing time and low-density seeding help to avoid cell-cell adhesion.

Problem: High background is observed in the control untreated group, or there is low readout in the experimental group.

[Steps 11 and 19]

Solution: Consider the following:

• The HUVEC monolayer may not be intact. Be extremely careful when aspirating or adding samples onto the monolayer.
  
  
• As primary cells, HUVEC have variable growth rates according to their condition. Thus, the time required to form a monolayer may be different for different cells. It is always safest to test the permeability of the monolayer before conducting experiments (Steps 4 and 14).
  
  
• Different cancer cells may have different properties in terms of their transendothelial migration activity. The co-culture incubation time (Step 9) should be tested to find the best working window for a particular cell line.
  
  

DISCUSSION

Extravasation is a highly regulated process. It requires a self-activated signaling that increases the adhesion and motility of the cancer cells and a forward signaling that induces breaches in the endothelium layer (Bernstein and Liotta 1994; Chambers et al. 2002; MacDonald et al. 2002; Weis and Cheresh 2005). The TEM and vascular permeability assays are simple and effective methods for demonstrating the functional involvement of molecules in the extravasation process in vitro (Lampugnani et al. 1992; Voura et al. 2001; Tremblay et al. 2006; Gupta et al. 2007; Karnoub et al. 2007; Ma et al. 2008; Welm 2008). The two protocols described here were designed to study the ECM proteins in particular (Ma et al. 2008). In the TEM assay, cancer cells are dissociated from the culture plates using Versene (which contains EDTA but not trypsin) instead of trypsin/EDTA. This modification helps to preserve the transmembrane and membrane-bound proteins on the cell surface, which are important players in the co-culture system. The TEM assay provides direct evidence for protein-regulated extravasation, because it mimics dynamic interaction between cancer cells and endothelial cells. However, counting the number of transmigrated cells is time consuming and sometimes subjective. In the vascular permeability assay, conditioned medium containing the secreted factors from cancer cells is used to treat the endothelium monolayer. Macromolecules (FITC-dextran) are used to examine the permeability of the endothelium monolayer. Although the readout of this assay is not sufficient to draw conclusions, it is relatively objective and high-throughput. The vascular permeability assay is ideal for drug and inhibitor screening (Tremblay et al. 2006; Ma et al. 2008). Combining the results from both assays should provide a comprehensive understanding of the ECM protein-regulated extravasation process.

REFERENCES

Bernstein, L.R. and Liotta, L.A. 1994. Molecular mediators of interactions with extracellular matrix components in metastasis and angiogenesis. Curr. Opin. Oncol. 6: 106–113.[Medline]

Chambers, A.F., Groom, A.C., and MacDonald, I.C. 2002. Dissemination and growth of cancer cells in metastatic sites. Nat. Rev. Cancer 2: 563–572.[Medline]

Gupta, G.P., Nguyen, D.X., Chiang, A.C., Bos, P.D., Kim, J.Y., Nadal, C., Gomis, R.R., Manova-Todorova, K., and Massague, J. 2007. Mediators of vascular remodelling co-opted for sequential steps in lung metastasis. Nature 446: 765–770.[Medline]

Karnoub, A.E., Dash, A.B., Vo, A.P., Sullivan, A., Brooks, M.W., Bell, G.W., Richardson, A.L., Polyak, K., Tubo, R., and Weinberg, R.A. 2007. Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature 449: 557–563.[Medline]

Lampugnani, M.G., Resnati, M., Raiteri, M., Pigott, R., Pisacane, A., Houen, G., Ruco, L.P., and Dejana, E. 1992. A novel endothelial-specific membrane protein is a marker of cell-cell contacts. J. Cell Biol. 118: 1511–1522.[Abstract/Free Full Text]

Ma, C., Rong, Y., Radiloff, D.R., Datto, M.B., Centeno, B., Bao, S., Cheng, A.W., Lin, F., Jiang, S., Yeatman, T.J., et al. 2008. Extracellular matrix protein βig-h3/TGFBI promotes metastasis of colon cancer by enhancing cell extravasation. Genes & Dev. 22: 308–321.[Abstract/Free Full Text]

MacDonald, I.C., Groom, A.C., and Chambers, A.F. 2002. Cancer spread and micrometastasis development: Quantitative approaches for in vivo models. Bioessays 24: 885–893.[Medline]

Tremblay, P.L., Auger, F.A., and Huot, J. 2006. Regulation of transendothelial migration of colon cancer cells by E-selectin-mediated activation of p38 and ERK MAP kinases. Oncogene 25: 6563–6573.[Medline]

Voura, E.B., Ramjeesingh, R.A., Montgomery, A.M., and Siu, C.H. 2001. Involvement of integrin _vβ₃ and cell adhesion molecule L1 in transendothelial migration of melanoma cells. Mol. Biol. Cell 12: 2699–2710.[Abstract/Free Full Text]

Weis, S.M. and Cheresh, D.A. 2005. Pathophysiological consequences of VEGF-induced vascular permeability. Nature 437: 497–504.[Medline]

Welm, A.L. 2008. TGFβ primes breast tumor cells for metastasis. Cell 133: 27–28.[Medline]