The overall goal of the following experiment is to develop a mouse model of breast cancer that eventually metastasizes in the presence of a healthy immune system. This is achieved first by administering a Cree expressing adenovirus into the mammary ductal canal of the experimental animals. The mammary gland is then palpated regularly to monitor tumor progression.
Ultimately, tumor metastasis and leukocyte infiltration to the axillary lymph node can be evaluated by flow cytometric analysis. The main advantage of this technique over existing breast tumor models is that it allows the precise initiation of tumors that can develop in the presence of a mature immune system, and that can be tracked by YFP expression. Visual demonstration of this method is critical as the introductional injection steps are difficult to learn.
The correct placement of the needle requires precision and practice. On the day of the experiment, store Eloqua of four times 10 of the eighth plaque forming units of adenovirus Cree on dry ice, thaw the virus set room temperature and mix it with sufficient 3%sucrose in sterile water to bring the final volume up to 10 microliters. Next gently mix 34 microliters of mem and four microliters of freshly prepared calcium chloride into the virus and incubate the solution at room temperature.
After 15 to 20 minutes, the media will appear cloudy indicating the formation of the adenovirus precipitates. Gently flick the tube to make sure the virus particles are evenly distributed throughout the suspension, and then to inject the virus particles draw up three microliters of the virus into a 10 microliter syringe. Next place an anesthetized mouse onto its back on the illuminated stage of a clean dissection microscope.
Illuminate the abdomen with an extra light source and locate the left fourth or the right ninth inguinal mammary gland by the small white patches of fur surrounding each nipple. Now rub the nipple gently with a sterile ethanol soaked cotton tipped applicator. Then secure the nipple with fine surgical forceps and pull up with light force.
To remove the keratin plug, stabilize the nipple between the forceps and gently insert the needle between the tips. Cannulating the duct canal at a 90 degree angle to ensure the proper depth of injection, gently pull the needle up after inserting it into the lumen of the duct, drawing the nipple up along the edges of the needle as it is pulled up. When the needle is appropriately placed, gently plunge the entire contents of the syringe.
The nipple should inflate slightly as the liquid is added. After the injection, place the mouse back onto the heating pad until it begins to recover from the anesthesia. Then place the animal back into a clean cage and monitor it until full recovery is observed.
Palpate the injected memory gland at day 30 for assessment of the enlargement and swelling of the gland. Monitor the tumor progression every five to seven days. Once a swollen and enlarged mammary gland is observed, then measure the tumor volumes every three to four days for tumor growth kinetics.
Once palpable tumors appear successful targeting of the mammary ductal tree can be visualized by preparing whole mounts of the mammary gland after injection of trip and blue to verify proper injection or after injection of an adenovirus expressing M cherry to verify proper viral preparation and infection of ductile epithelial cells. When tumors are induced in F Fluxed P 53 LSLK RAs transgenic mice, initial tumors will not become apparent until around day 40 when the mammary glands become enlarged and swollen. Beginning around day 56, the tumors will begin to grow exponentially.
At this point, it is critical to measure the tumor volumes every three days. If kinetic studies are desired, as there will be some normal mouse to mouse variability in tumor progression, large abdominal masses will be apparent by day 80 after which mice should be euthanized if the tumors exceed more than 10%of the animal's body weight. CDNA analysis of three cell lines derived from breast tumors in flux P 53 LSL KRAS transgenic mice revealed that the tumors express mesothelin cytokeratin eight, HER two new and estrogen receptor alpha, similar to the cellular microenvironment in human breast cancer infiltration of alpha, beta and gamma delta T cells, as well as myeloid derived suppressor cells and macrophages into the tumors is observed.
The vasculature draining to the axillary lymph node will begin to engorge before the tumors have grown and eventually encompass the entire memory tissue where the injection was performed. There will be evident engorgement of the superficial epigastric vein between the inguinal and axillary lymph nodes as well. After seven to eight weeks, the axillary lymph node will become enlarged due to the lymphovascular invasion of the tumor cells.
The lymphovascular invasion and metastasis of the tumor cells can be tracked by crossing Flocked P 53 LSLK RAs transgenic mice with L-S-L-E-Y-F-P mice. After pre mediad excision cells expressing both high and low levels of YFP are detected in the tumor and their metastasis can be traced to the draining axillary lymph node. Once mastered, this technique can be performed on an experimental cohort of 20 mice in two to three hours if performed properly.
Following this procedure, other tumor models can be developed by breeding mice with additional locks, p transgenes, to answer questions like, how do different oncogenes affect the pathology, immune microenvironment and metastatic invasion of breast cancer.
