Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

This method can help answer key questions in the breast cancer field about how to mimic human breast cancer progression and treatment in a mouse model. The main advantage of this mouse breast cancer model is that it has a high tumorigenesis rate after orthotopic implantation and a low local recurrence rate after mastectomy. Begin by confirming a lack of response to toe pinch in an anesthetized female mouse and taping the animal's limbs to a surgical board.

Using sterile cotton swabs sterilize the surgical area with chlorhexidine, iodine and 75%ethanol. And make a five-millimeter skin incision in the middle of the anterior chest wall. Next lift the right side of the skin adjacent to the incision and use sterile microdissection scissors to detach the skin from the chest wall.

Invert the skin to expose the right number two mammary fat pad and use a one-milliliter insulin syringe equipped with a 28.5-gauge needle to carefully inject 20 microliters of cancer cell suspension through the wound into the fat pad. Hold the needle within the fat pad for five seconds to allow the gelatinous cancer cell protein mixture to solidify before removing the needle, and closing the incision with sterile 5-0 nonabsorbable sutures. Then place the animal in a clean cage with monitoring until full recumbency.

To perform the mastectomy eight days after cancer inoculation prepare the mouse for surgery as just demonstrated, and make a five-millimeter skin incision two millimeters to the left of the surgical scar from the cancer cell inoculation. Extend the incision toward the root of the forelimb to remove the tumor, and the skin, including the surgical scar, and the lesion in contact with the tumor, and the axillary lymph node basin in which typically no visible lymph nodes are present at the time of the mastectomy. Then close the skin defects with sterile 5-0 nonabsorbable sutures in the shape of a Y.And return the animal to its cage with monitoring until full incumbency.

For lung metastasis quantification 21 days after inoculation inject 150 milligrams per kilogram of D-Luciferin IP 15 minutes before euthanasia. And use curved Mayo scissors to open the skin and peritoneum at the mid-abdomen. Press down the liver until the diaphragm can be visualized, and cut the diaphragm and the bilateral ribs from caudad to cephalad.

Flip the anterior thorax wall to expose the lungs and lift the lungs to identify the thoracic esophagus, which looks like a cord connecting the lungs to the spine. Cut the esophagus with microdissection scissors and use forceps to pull the bilateral lungs and heart toward the caudad. Then cut the trachea and major vessels to the lung apex at the cephalad and place the lungs into a 10-centimeter Petri dish for bioluminescent imaging.

Use the scissors to remove the heart from the lung tissue. Beginning at about eight days after mammary fat pad cancer cell inoculation the bioluminescence in the lungs is deeper and smaller than in the primary lesion reflecting the primary tumor burden in live mice. Measurement of the tumor size with calipers allows estimation of the tumor volume in situ.

This mastectomy model also allows serial metastatic tumor burden monitoring without having to euthanize the animals. The tumor burden of the lung metastases can also be quantified by ex vivo imaging, but the limitation is that the mice must be euthanized. Mouse survival after surgical treatment can also be monitored as a translatable clinical endpoint.

After its development this technique paved the way for researchers in the field of breast cancer to explore not only new drug development, but also breast cancer research in general.