Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

This method can help answer key questions in the cancer field about how the microenvironment supports tumor growth or whether a specific drug can effectively inhibit the breast cancer progression. The main advantages of the tumor induction technique are that no surgery is needed and it is a less invasive manner in which to establish breast cancer in situ. Using this technique, the tumor cells grow within the mammary fat pad, in the appropriate tissue environment, mimicking human breast cancer progression more closely than subcutaneously injection or xenograft to mice cells.

Ke-Xin Cao, our technician, and I, will assist Gan-Lin Zhang in demonstrating this procedure. On the day before the operation, manually restrain the recipient mouse and turn the animal belly side up to the fur to be shaved around the fourth nipples. Wipe the fur with a thin layer of depilatory cream.

Use distilled water to remove the cream after 30 to 60 seconds and dry the mouse with soft paper towels. On the day of the operation, dilute the murine breast cancer cell suspension to a two times ten to the fifth cells per milliliter of PBS concentration, in a sterile microcentrifuge tube on ice. Before each injection, load one, one milliliter syringe equipped with a 45 by 16 needle, with 50 microliters of cells per mouse.

And confirm a lack of response to toe pinch in each sedated animal. Apply ointment to the animal’s eyes and use tweezers to tent the skin around the fourth nipple of the first mouse, to create a tunnel for the syringe to follow. Hold the syringe with the bevel facing upwards and subcutaneously enter the skin at five to 10 millimeters from the nipple, following the tunnel until the needle tip almost reaches the nipple.

Using tweezers to tent the skin around the first nipple creates a tunnel for the syringe to follow. The needle should be entered between the tweezer tips and should raise up instead of depressing into the tissue. Carefully move the needle tip up into the mammary fat pad until the bevel is under the nipple, and release the skin tent to allow a slow injection of the cancer cells.

Upon insertion, carefully move the needle tip up into the mammary fat pad until the bevel is under the nipple. And you will feel resistance to ensure delivery of the cells to the correct location. When all of the cells have been injected, turn the needle slightly to the side and retract the syringe slowly.

Use a cotton swab to apply pressure to the needle wound for 10 to 20 seconds, to make there is no fluid seepage. The injected nipple should appear as a transparent, white, flat sphere, with the nipple in the center. To establish the tumor volume, restrain the mouse belly side up and use a caliper to measure the tumor length and width.

To capture the tumor bioluminescence, 10 minutes before imaging inject 150 milligrams per kilogram of D-luciferin into the back of the mouse’s neck. And capture bioluminescence images of the primary tumor and metastases under the appropriate bioluminescence imaging parameters. At the appropriate experimental time point, use scissors to harvest the tumor from the skin.

And apply the analgesic solutions to the surgical site to relieve post-operative pain. Then close the skin with surgical sutures according to standard protocols. Allow the animal to recover under a warming lamp with monitoring until full recovery.

Use a scalpel to cut the primary tumor into two equal pieces. Store one-half of the tissue in 4%paraformaldehyde for subsequent hematoxylin and eosin staining and immunohistochemistry. Freeze the other half in liquid nitrogen for western blotting or RNAi isolation.

At the appropriate experimental endpoint harvest the lungs according to standard dissection protocols and wash the organ in PBS. Then fix the lungs in 4%paraformaldehyde to verify metastasis by hematoxylin and eosin staining. Primary tumor growth can be measured by tumor volume and living tumor cell bioluminescence, as demonstrated.

Metastases are not observed during the early stages of the inoculation as either no secondary tumor has been established, or the strong bioluminescent signals of the primary tumor obstruct the detection of small metastatic foci with weak signals. After resection, the bioluminescence signal from the metastatic foci of distant organs can be detected and analyzed. Analysis of the morphology of both primary breast tumor and lung sections by hematoxylin and eosin staining reveals increased angiogenesis in the tumor cell inoculated animals as evidenced by anti-CD31 microvessel marker staining.

When attempting this procedure, it’s important to remember to make sure the needle tip is in the correct location before delivering the tumor cells. Following this procedure, atomizers, like animal ultrasound, can be performed to answer additional questions about bladder supplementation of the primary tumor in vivo. After its development, this technique paved the way for researchers in the field of tumorigenesis and the drug development in exploring the key factors and pathways involved in tumorigenesis and the mechanisms of anti-cancer drug in murine animal models.

Don’t forget that working with isoflurane or other gas anesthesia can be extremely hard to dose and that precautions such as using an activated carbon mask, should always be taken while performing this procedure.