A Novel Mouse Skin Graft Model of Vascular Tumors Driven by Akt1

Materials and Reagents

1. Sterile surgical gloves
   
2. 10 mm skin punch biopsy instrument (Acu-Punch Biopsy Punch 10 mm) (Acuderm, catalog number: P1025 ) (Figure 2)
   
   
   Figure 2. A 10-mm punch biopsy instrument
   
   
3. Ethilon non-absorbable nylon suture, 5-0 PC-3 (Ethicon, catalog number: 1965G )
   
4. Sterile surgical towel drape (Steri-Drape) (3M, catalog number: 1010 )
   
5. Sterile surgical gauze (4 x 4 Gauze 8 Ply) (COVIDIEN, Dermacea^TM, catalog number: 441001 )
   
6. Alcohol pads (WebCol Alcohol Prep) (COVIDIEN, catalog number: 6818 )
   
7. Donor mice (females, 6-8 weeks old transgenic mice, see reference for the generation of transgenic mice: Sun JF, Phung T, Shiojima I, Felske T, Upalakalin JN, Feng D, Kornaga T, Dor T, Dvorak AM, Walsh K, Benjamin LE. Microvascular patterning is controlled by fine-tuning the Akt signal. Proc Natl Acad Sci U S A 2005; 102: 128-33)
   
8. Recipient mice (females, 6-8 weeks old nu/nu mice from Charles River Laboratories, catalog number: 490 )
   
9. Isoflurane (Sigma-Aldrich, catalog number: CDS019936 )
   
10. Phosphate buffered saline (PBS), 1x sterile solution (AMRESCO, catalog number: K812 )
    

Equipment

1. Needle holder (Halsey serrated jaws) (Roboz Surgical Instrument, catalog number: RS-7841 )
   
2. Fine single-toothed forceps (Micro dissecting tweezers Pattern 7) (Roboz Surgical Instrument, catalog number: RS-5047 )
   
3. Surgical scissors (Micro dissecting scissors) (Roboz Surgical Instrument, catalog number: RS-5910 )
   
4. Electric hair shaver (Wahl Battery Operated Compact Travel Trimmer)
   
5. Warming plate (Ala Science, catalog number: TCW220x120 )
   

Procedure

Important: Appropriate Institutional Animal Care and Use Committee (IACCU) approval must be obtained prior to performing the skin graft procedure in animals.

1. Anesthetize donor and recipient mice with isoflurane anesthetic gas. The donor animal is typically sedated for 5-10 min while the punch biopsy and removal of donor skin is performed. The recipient animal is typically sedated for 20-30 min while the two skin grafts are sutured in place. Animals are adequately sedated when they do not display a response to pinching of the foot pad.
   
2. For the donor mouse, chose a skin area from where the skin will be taken. In our study, we selected the back skin because the skin would be grafted at the same anatomic site on the back of the recipient mouse.
   
3. Shave the hair over the skin area from where the skin graft will be taken. Be careful to avoid scratching or injuring the skin surface (Figure 3 and Video 1).
   
   
   Figure 3. Procedure for removing a punch of skin from donor mouse. A-B. Shave the hair over the skin area from where the skin graft will be taken. C. Hold the skin stretched slightly and position the 10-mm punch instrument vertically on top of the stretched skin. D. Gently rotate the punch instrument until the punch cuts through the skin. E. Once the punch cuts through the skin, remove the punch. F-G. Cut out the piece of punched skin and underlying subcutaneous tissue, and place the skin on a piece of sterile gauze soaked in sterile phosphate buffered saline to keep the skin moist. A close-up photo of the host skin punch with the skin dermis side up is shown in panel G.
   
   
   
   Video 1. Skin graft procedure
   
   
4. Place the animal onto a clean surface covered with a sterile surgical drape.
   
5. Clean the shaved skin of the donor animal thoroughly with 70% alcohol wipe pads.
   
6. Hold the skin stretched slightly, and position the 10-mm punch instrument vertically on top of the stretched skin. Gently rotate the punch instrument with a smooth 360° turn motion while applying downward pressure until the punch cuts through the skin (also see video demonstration of the technique by Levitt et al., 2013).
   
7. Once the punch cuts through the skin and reaches its maximum depth of approximately 1-2 mm through the dermis and subcutaneous tissue, remove the punch. Cut out the piece of punched skin and underlying subcutaneous tissue with sterile fine surgical scissors.
   
8. Gently remove the skin fragment with sterile fine forceps. The forceps are sterilized by autoclaving. Please note, when removing the skin, be careful to avoid crushing it by picking up the tissue piece at the edge with fine single-toothed forceps. Save the punched-out skin in order to graft it onto the back of the recipient mouse. Keep the skin graft moist by placing it onto a piece of sterile gauze soaked in 1x sterile phosphate buffered saline (PBS) solution.
   
9. Hemostasis can be achieved by applying gentle pressure over the cut area with a dry sterile surgical gauze. Once this step is completed, the donor animal can be immediately euthanized per approved Institutional Animal Care and Use Committee (IACCU) protocol.
   
10. Repeat the same punch biopsy procedure for the recipient mouse. Punch out a 10-mm circular piece of skin from the back of the recipient mouse. Cut out the skin fragment and the underlying subcutaneous tissue and discard it (Figure 4 and Video 1).
    
    
    Figure 4. Procedure for suturing donor skin onto recipient mouse. A-B. Remove a piece of skin from the back of the recipient mouse using a punch device as described in Figure 3. C. Take the donor skin fragment and put it into place in the punched out area on recipient mouse. D-G. Suture donor skin fragment into place.
    
    
11. Apply gentle pressure over the cut area with a sterile surgical gauze to stop any bleeding.
    
12. Take the donor skin fragment and put it into place in the punched out area on the back of the recipient mouse.
    
13. To help position the skin graft in place, use the circular skin punch as a clock face. First, place a suture stitch at 12, 3, 6 and 9 o’clock positions. Then add a suture stitch at 1:30, 4:30, 7:30 and 10:30 o’clock positions. Suture the skin fragment in place with non-absorbable nylon suture. Perform double tie knots to secure suture thread in place.
    
14. When completed, clean the sutured area by gently wiping it with a sterile gauze soaked in sterile PBS solution.
    
15. Keep the animal under observation on a warming plate at 37 °C until it is fully awake and moving, which typically takes about 10-15 min. Animal usually awakens and starts moving soon after anesthesia is withdrawn, so there needs to be constant observation until the animal is fully awake. Once the animal is fully awake and can move, it can be placed back into the cage with clean cage bedding, water and food (place a few food pellets on the cage floor so the animal can feed easily during the first 1-2 days post procedure).
    
16. Monitor the animal daily for signs of wound infection and for evidence of healing and wound closure (Figure 5D).
    
17. Sutures can be removed 10-14 days after the surgical procedure, which is approximately the time frame needed for complete skin wound healing.
    
18. Successful skin engraftment is evident as the grafted donor skin becomes pink with hair growth in the skin graft (Figure 5D).
    Note: Detailed demonstration of the entire procedure can be viewed in our video that is included with this paper. Additional information on how to perform a skin punch biopsy can also be obtained from the video reference in Levitt et al., 2013.
    

Data analysis

Using this skin graft model to study the effects of endothelial Akt1 on vascular tumor formation, we have found that overexpression of constitutively activated Akt1 for 4 weeks resulted in the development of red masses at the skin graft sites that measured 0.39 ± 0.14 cm³ in size and had histologic features of a vascular tumor (Figures 5 and 6) (also see Figure 3 in Phung et al., 2015). Immunofluorescence staining of tumor sections shows that tumor vessels express the endothelial marker CD31 (Figure 6D). For immunofluorescence staining, fresh tissues are fixed in cold 4% paraformaldehyde, blocked in 5% goat serum for 1 h, and incubated with anti-CD31 antibody (BD Biosciences MEC13.39) overnight at 4 °C, followed by incubation for 1 h with Dylight^TM 488-conjugated secondary antibody (Jackson ImmunoResearch Labs). More detailed information on the immunofluorescence staining technique and analysis of skin graft tumors is presented in the original publication (adapted from Phung et al., 2015).


Figure 5. Mouse model of endothelial myrAkt1-driven vascular tumor. A. Schematic of myrAkt1 skin graft model of hemangioma. Transgenic donor mice in FVB background express inducible expression of myristoylated activated Akt1 (myrAkt1) in endothelial cells. Skin from myrAkt1 mice (green circles) was used as skin graft donors in nu/nu recipient mice. 10-mm biopsy punches of skin from donor mice were grafted onto the back skin of host (recipient) mice. Grafts were allowed to heal for 2 weeks, at which time half of the recipient mice were taken off tetracycline (given plain drinking water) to turn on myrAkt1 and the other half were kept on tetracycline to turn off myrAkt1 for 4 weeks. B. Photographs of recipient nu/nu mice showing fresh skin grafts with suture on day 0 (arrows). C. Photographs of mice 6 weeks later showing the presence of successful skin engraftment with hair growth in the skin graft (black arrow indicates tuft of white hair in graft site). There is no tumor mass at the skin graft sites in recipient mouse with myrAkt1 OFF, but there are red tumor masses (see red arrow) in the skin graft sites in recipient mouse with myrAkt1 ON. D. Successful skin engraftment is evident by healing of the donor skin graft and the grafted skin becomes pink with hair growth in the graft site (Reference: Phung, T. L., Du, W., Xue, Q., Ayyaswamy, S., Gerald, D., Antonello, Z., Nhek, S., Perruzzi, C. A., Acevedo, I., Ramanna-Valmiki, R., Rodriguez-Waitkus, P., Enayati, L., Hochman, M. L., Lev, D., Geeganage, S. and Benjamin, L. E. (2015). Akt1 and akt3 exert opposing roles in the regulation of vascular tumor growth. Cancer Res 75(1): 40-50).


Figure 6. Endothelial myrAkt1 activation drives vascular tumor formation in vivo. A. Donor mouse skin engrafted in nu/nu recipient mouse as seen by the presence of tuffs of white hair at the graft site. Vascular tumor development in the skin graft was monitored following myrAkt1 induction. Tumor developed in the skin graft 4 weeks following myrAkt1 induction. Magnification X1. B. Microscopic features of the tumor showing a tumor mass (arrows). C. The tumor consists of numerous blood vessels filled with red blood cells in the dermis (arrows). The vessels have variable sizes and irregular lumen with thin vascular wall. D. The tumor is composed of numerous blood vessels as shown in CD31 immunofluorescence stain (green). Cell nuclei are stained with Hoechst dye (blue).

Notes

In our experience, the skin graft procedure works successfully almost all the time as evident by the successful engraftment of the donor skin. We have not observed animal death or skin infection at the graft site. Because immunodeficient nu/nu recipient mice are used as graft recipients, there is no evidence of graft rejection in our study.

Acknowledgments

This work was supported in part by NIH grants R01 CA106263 and P01 CA09264401 (to L. E. Benjamin), American Heart Association 11BGIA5590018, NIH K08 HL087008, NIH R03 AR063223, American Cancer Society 122019-RSG-12-054-01-CSM, and the Baylor Clinical and Translational Research Program (to T. L. Phung).

References

1. Levitt, J., Bernardo, S., Whang, T. (2013). Videos in clinical medicine. How to perform a punch biopsy of the skin. N Engl J Med 369(11): e13.
   
2. Phung, T. L., Du, W., Xue, Q., Ayyaswamy, S., Gerald, D., Antonello, Z., Nhek, S., Perruzzi, C. A., Acevedo, I., Ramanna-Valmiki, R., Rodriguez-Waitkus, P., Enayati, L., Hochman, M. L., Lev, D., Geeganage, S. and Benjamin, L. E. (2015). Akt1 and akt3 exert opposing roles in the regulation of vascular tumor growth. Cancer Res 75(1): 40-50.
   
3. Sun, J. F., Phung, T., Shiojima, I., Felske, T., Upalakalin, J. N., Feng, D., Kornaga, T., Dor, T., Dvorak, A.M., Walsh, K. and Benjamin, L. E. (2005). Microvascular patterning is controlled by fine-tuning the Akt signal. Proc Natl Acad Sci U S A 102(1): 128-33.