Clear cell renal cell carcinoma or CCRCC, is the most common subtype of kidney cancer. Localize CCRCC has favorable outcome with surgery. However, one third of CCRCC patients will develop distant metastasis to the lung.
Which leads to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage. As the molecular mechanism of metastasis remains allusive.
The purpose of this protocol is to introduce and compare the two animal models recently established for metastatic CCRCC. The first model is the mouse of the topic implantation. In this mouse model, heterogeneous CCRCC is implanted to the renal capsule of the mouse.
Tumor growth and the metastasis are asset after 10 weeks. The second model is the chicken chorioallantoic membrane model, or the CAM model. In this chicken model the heterogeneous CCRCC is implanted in the egg.
Tumor growth and circulating tumor cells are asset after 10 days of implantation before the embryo hatches. Lung metastasis is asset after 24 days of the implantation. Which is two weeks after hatching.
These two novel models will be useful to further study the molecular mechanism behind the metastasis in CCRCC. Please note that the CAM procedure is not featured in this video. The detailed protocol for the CAM model is described and filmed in another Trove article by Sharrow et al.
After anesthetizing a the mouse, disinfect the back of the mouse entirely with povidone-iodine for three times. Follow by 70%ethnol once, and wipe it dry with sterile cotton swabs. Then apply three sterile medical dressing sequentially covering the whole back in order to create a surgical field as well as to immobilize the mouse.
Before the operation, the operators should disinfect their fingers with povidone-iodine. Place the mouse in the prone position. And, use fingers to locate the left kidney just under the left flank.
Use a pair of blunt forceps and scissors to cut open the skin and muscle layer under the specified location. Use sterile cotton swabs and forceps to partially exteriorize the left kidney out of the abdomen. Load either an insulin syringe or a customized Hamilton syringe with the tumor cell suspension prepared separately.
Inject 20 microliters of cell suspension under the kidney capsule. Slowly pull out needle in order to solidify the extra cellular matrix solution. As well as to prevent hemorrhage or tumor cell leakage.
And then, use a sterile cotton swab to push the kidney back into the abdomen. Use suture to stitch the muscle layer. Disinfect the skin with povidone-iodine.
And, close the skin with wound auto-clips. Orthotopic laying planted tumor cells successfully grew on mice kidney as confirmed by BLI. Although there was no difference in the primary growth, only the heterogeneous metastatic tumor had robust metastasis to the lung.
The metastasis was also confirmed by immunohistochemistry. Similarly, the circulating tumor cell counts were higher in the mice bearing metastatic tumor than those bearing non-metastatic tumor. In concordance with the mouse model the CAM model also showed no growth difference between metastatic and non-metastatic tumors.
On the other hand, the number of circulating tumor cells was significantly higher in the eggs with metastatic tumors. Histologically detectable metastasis was observed in the lungs of hatched chicken. In conclusion, this protocol illustrated two novel animal models for metastatic CCRCC.
Both the mouse and the chicken models faithfully recapitulated the lung metastasis observed in the clinic. These two pre-clinical models will be useful to expand the understanding of the molecular mechanism behind the metastasis. As well as to discover novel treatments for metastatic CCRCC.
