临床前小鼠模型的外科手术和方法

We have developed a new murine model of de novo mammary cancer metastasis that closely mimics the course of human breast cancer, providing a preclinical platform for evaluating the biology, underlying metastatic progression of disease, and the efficacy of therapies delivered in the adjuvant setting. Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee at Oregon Health and Science University. Our murine model is established by harvesting enzymatically dissociated primary tumors from transgenic 100-day old mouse mammary tumor virus polyoma middle T or MMTV-PyMT mice.

One times 10 to the sixth tumor cells are orthotopically injected into syngeneic recipient female mice and primary tumors develop over a 38 to 60-day period. Mice bearing tumors 170 to 450 millimeters cubed are enrolled for surgical resection of the primary tumor. Analysis at 115 days postsurgery resulted in mice that have histologically detectable metastatic foci in the lungs with 45%penetrance.

This video depicts surgical resection of the primary mammary tumor, a crucial component of the preclinical murine model. Mice are administered 2%isoflurane and surgery can commence once the foot pinch reflex is absent. Spray the skin overlying the primary tumor with 70%ethanol.

Betadine is then applied to the surgical area beginning on the skin overlying the tumor followed by the area surrounding the primary tumor using a sterile cotton swab. Using blunt-tip scissors, an initial incision is made in the skin medial caudal to the primary tumor. The incision is extended in a superior direction medial to the primary tumor.

Identify and cauterize potential vessels feeding the tumor from the overlying skin. If bleeding occurs, further cauterization should be performed. When making a lateral skin incision inferior to the primary tumor, make sure to avoid cutting the underlying mammary gland as vessels may be present.

The skin incision is then extended circumferentially around the primary tumor. Thin adhesions in the mammary gland medial to the tumor are then carefully dissected separating the tumor from the abdominal wall. Blood vessels may be present within these adhesions.

Cauterize any vessels identified within the adhesions prior to cutting. Arrows indicate the fourth and fifth right mammary glands, shown above and below the tumor. The fifth mammary gland is then cauterized and excised as it may contain small vessels that are difficult to visualize.

After the tumor has been separated from the abdominal wall, larger vessels can be identified. The vessel running through the fourth mammary gland is shown by the top arrow. The underlying femoral artery is shown by the middle arrow.

And the large vessel feeding the tumor between the fourth and fifth mammary gland is indicated by the bottom arrow. Cauterization along the large vessel running between the fourth and fifth mammary gland is then performed, making sure to avoid cauterization of the underlying femoral artery on the leg. After cauterization of the large vessel is complete, cut through the vessel up to the fourth mammary gland.

Locate and cauterize the second large vessel running through the fourth mammary gland. Cut through the vessel without cutting through the entire mammary gland so that if bleeding occurs, the vessel can be easily identified and further cauterized. The remainder of the fourth mammary gland is then excised.

The overlying skin is then closed with wound clips using an AutoClip wound clip applier. Make sure the overlying skin is sufficiently separated from the abdominal wall to avoid clipping the underlying muscle. Bring the lateral and medial edges of the wound together, lifting the skin in an anterior direction to apply the wound clip.

Make sure to avoid clipping the urogenital tract or underlying muscle of the right leg. Mobilization of the right leg by removing tape may allow for easier application of wound clips. Wound clips should be removed 10 days following surgery.

The surgical resection of the primary tumor displayed in this video allows for disseminated cancer cells to develop in the metastatic foci in the lung 115 days postsurgery where a representative of each in the image of a metastatic foci is shown. Given that a large percentage of women diagnosed with breast cancer undergo surgical removal of the tumor and can succumb to metastatic disease postsurgery, we established a preclinical murine model that mimics the course of patient disease and treatment. By recording the methodology for surgical removal of the primary mammary tumor, this key component of our murine model of de novo mammary cancer metastasis can be easily reproduced in order to assess metastatic disease biology post tumor resection and adjuvant therapy efficacy.