In vivo Matrigel Plug Angiogenesis Assay   

Materials and Reagents

1. BALB/cAnN-nu (Nude) female mouse, 6 to 8 week-old
   
2. Tumor cells
   
3. Matrigel matrix (BD Biosciences, Falcon^®, catalog number: 354234 )
   
4. 10% formalin solution-neutral buffered (Sigma-Aldrich, catalog number: HT501128-4L )
   
5. Anti-rat CD34 (Santa Cruz, catalog number: sc-18917 )
   
6. Biotin goat Anti-Rat IgG (BD Biosciences, catalog number: 559286 )
   
7. Streptavidin-peroxidase conjugate (Dako, catalog number: P0397 )
   
8. Diaminobenzidine (DAB) (Dako, catalog number: K3467 )
   
9. Paraffin
   
10. Potassium chloride
    
11. Potassium phosphate monobasic
    
12. Dodium chloride (NaCl)
    
13. Sodium phosphate dibasic anhydrous
    
14. Hematoxylin and eosin (H&E)
    
15. Trypsin
    
16. Complete medium
    
17. Plain medium
    
18. Phosphate buffered saline (PBS) (see Recipes)
    

Software

1. Aperio ScanScope System
   
2. ImageScope v10 software (Aperio, Vista)
   

Equipment

1. Aperio Scanscope CS-S microscopic slide scanning system
   
2. Tumor cell culture set up
   
3. Hemocytometer
   
4. Centrifuges
   
5. 24G syringe
   
6. T175 flask
   

Procedure

1. Remove the medium from a T175 flask containing an 80-90% confluent monolayer of tumor cells (e.g. the nasopharyngeal carcinoma HONE1 cells) and wash the cells with PBS.
   
2. Detach the tumor cells by incubating with 3 ml trypsin for 5-10 min; stop trypsinization with 10 ml complete medium.
   
3. Count the number of cells with a hemocytometer, spin down the cells at 1,200 rpm for 5 min and wash once with PBS.
   
4. Mix a total of 5 × 10⁶ to 1 × 10⁷ cells (cell type-dependent) with 50 μl plain medium and 250 μl ice-cold matrigel (the matrigel maintains as liquid form at 2-8 °C and solidifies rapidly at 22 -37 °C).
   
5. Subcutaneously inject the 300 μl cell matrigel mixture into a flank of five female athymic nude mice (one injection site per mouse) with an ice-cold syringe with a 24G one inch needle.
   
6. Polymerize the matrigel mixed with the cells to form a solid gel plug, which allows cell growth and blood vessel formation.
   
7. After inoculation for 7 days, excise the matrigel, fix with formalin overnight, embed in paraffin, and section onto slides.
   
8. Stain the slides with hematoxylin and eosin (H&E) for histological observation.
   
9. Stain the blood vessels formed with 150 μl endothelial cell marker CD34 monoclonal antibody (1:40) overnight at 4 °C and 150 μl secondary biotin-conjugated goat anti-Rat IgG antibody (1:100) for 1 h at room temperature (RT).
   
10. Incubate the antibody with 150 μl streptavidin-peroxidase conjugate (1:200) for 1 h at RT.
    
11. Add the substrate DAB for signal detection.
    
12. Scan the slides by the Aperio ScanScope System and quantify the signal by ImageScope v10 software.
    
    
    Figure 1. Representative results from matrigel plug assay. The endothelial cells were stained with anti-CD34 antibody, as indicated by the brown stain (indicated by arrows). A. Plenty of blood capillaries are formed in the gel plug section with growing tumor cells. B. Representative image of cell necrosis observed in center region of tumor nodules formed with lack of blood capillaries (indicated by asterisks).
    

Recipes

1. PBS (pH 7.4)
   2.67 mM potassium chloride
   1.47 mM potassium phosphate monobasic
   137.93 mM sodium chloride (NaCl)
   8.1 mM sodium phosphate dibasic anhydrous
   

Acknowledgments

The study was financed by the Research Grants Council of the Hong Kong Special Administrative Region, People’s Republic of China: Grant numbers HKU6617/08M and HKU6415/06M to MLL; the NIH award AR49930 to SSA; and the Swedish Cancer Society, the Swedish Research Council, the Swedish Institute, Cancer Research Institute in New York/Concern Foundation in Los Angeles and Karolinska Institute to ERZ.

References

1. Cheung, A. K., Ko, J. M., Lung, H. L., Chan, K. W., Stanbridge, E. J., Zabarovsky, E., Tokino, T., Kashima, L., Suzuki, T., Kwong, D. L., Chua, D., Tsao, S. W. and Lung, M. L. (2011). Cysteine-rich intestinal protein 2 (CRIP2) acts as a repressor of NF-kappaB-mediated proangiogenic cytokine transcription to suppress tumorigenesis and angiogenesis. Proc Natl Acad Sci U S A 108(20): 8390-8395.
   
2. Law, E. W., Cheung, A. K., Kashuba, V. I., Pavlova, T. V., Zabarovsky, E. R., Lung, H. L., Cheng, Y., Chua, D., Lai-Wan Kwong, D., Tsao, S. W., Sasaki, T., Stanbridge, E. J. and Lung, M. L. (2012). Anti-angiogenic and tumor-suppressive roles of candidate tumor-suppressor gene, Fibulin-2, in nasopharyngeal carcinoma. Oncogene 31(6): 728-738.
   
3. Lo, P. H., Lung, H. L., Cheung, A. K., Apte, S. S., Chan, K. W., Kwong, F. M., Ko, J. M., Cheng, Y., Law, S., Srivastava, G., Zabarovsky, E. R., Tsao, S. W., Tang, J. C., Stanbridge, E. J. and Lung, M. L. (2010). Extracellular protease ADAMTS9 suppresses esophageal and nasopharyngeal carcinoma tumor formation by inhibiting angiogenesis. Cancer Res 70(13): 5567-5576.