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In vivo Matrigel Plug Angiogenesis Assay

Cancer Biology > Metastasis
Authors: Hong Lok Lung and Maria Li Lung, 9/20/2012, 9810 views, 0 Q&A

[Abstract] The matrigel plug angiogenesis assay is a simple in vivo technique to detect the newly formed blood vessels in the transplanted gel plugs in nude mice. The matrigel matrix is derived from the engelbroth-holm-swarm (EHS) mouse sarcoma, and its composition is comparable to the basement membrane proteins. The matrigel can induce differentiation of a variety of cell types such as hepatocytes, mammary epithelial cells, and endothelial cells. In our case, tumor cells are mixed with the matrigel gel and are injected into the mice. The later immunohistochemistry (IHC) staining with the endothelial marker indicates the presence of the newly formed capillaries in the sectioned gel plugs.

Materials and Reagents

  1. BALB/cAnN-nu (Nude) female mouse, 6 to 8 week-old
  2. Matrigel matrix (BD Pharmingen 354234)
  3. Formalin solution-neutral buffered 10% (Sigma HT501128-4L)
  4. Anti-rat CD34 (Santa Cruz sc-18917)
  5. Biotin goat Anti-Rat IgG (BD Pharmingen 559286)
  6. Streptavidin-peroxidase conjugate (Dako P0397)
  7. DAB (diaminobenzidine) (Dako K3467)
  8. Tumor cells
  9. Paraffin
  10. Hematoxylin and eosin

Equipment

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

Procedure

  1. Remove the medium from a T175 flask containing a 80-90% confluent monolayer of tumor cells (eg. the nasopharyngeal carcinoma HONE1 cells) and wash the cells with phosphate buffered saline (PBS).
  2. Detach the tumor cells by incubating with 3 ml trypsin for 5-10 minutes; stop trypsinization with 10 ml complete medium.
  3. Count the number of cells with a hemocytometer, spin down the cells at 1200 rpm for 5 minutes 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 a 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 hour at room temperature.
  10. Incubate the antibody with 150 μl streptavidin-peroxidase conjugate (1:200) for 1 hour at room temperature.
  11. Add the substrate DAB (diaminobenzidine) for signal detection. 
  12. Scan the slides by the Aperio ScanScope System and quantify the signal by ImageScope v10 software (Aperio, Vista, CA, USA).



    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)
    8.10 mM sodium phosphate dibasic anhydrous

References

  1. 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., Lung M.L. (2012). Anti-angiogenic and tumor-suppressive roles of candidate tumor-suppressor gene, Fibulin-2, in nasopharyngeal carcinoma. Oncogene 31(6): 728-38.
  2. 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., 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): 8390-5. 
  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., Lung M.L. (2010). Extracellular protease ADAMTS9 suppresses esophageal and nasopharyngeal carcinoma tumor formation by inhibiting angiogenesis. Cancer Res. 70(13): 5567-76.


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