Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

This protocol will help researchers generate a new type of mouse model to recapitulate human breast cancer development from different subsets of mammary epithelial cells. This technique allows researchers to generate breast cancer mouse models in developmental stage tissue on a cell type specific manner and without a need for extensive mouse breeding. To begin this procedure, generate, maintain, and anesthetize floxed mice as described in the text protocol.

Bring the mouse to an area separate from where the surgery is to be performed. After confirming anesthesia by performing a toe pinch, inject Meloxicam as analgesia subcutaneously at a dose of five milligrams per kilogram to prepare for the surgical procedure. Expose the nipple surgical site by applying several drops of hair removal cream.

Remove excessive cream and loose hair using soft paper towels. Shaving is not recommended to avoid damage to the nipples. Disinfect the surgical site with iodophors first followed by 70%alcohol and end with a final application of scrub iodophors.

Do this in a circular motion from the center of the work area toward the periphery using a gauze sponge or a cotton-tipped applicator. Repeat the cycle three to four times. Use aseptic techniques throughout the surgical procedure.

Make an incision site on the skin at a length of approximately one centimeter between the two fourth inguinal mammary glands. Carefully separate the skin flap from the parietal peritoneum so as to visual the mammary ductal tree. Carefully hold the nipple with watchmakers'forceps to remove the exterior nipple without cutting any nearby skin using a microdissection scissor.

Load approximately three to five microliters of Cre-adenovirus injection mixture into a 25 microliter Hamilton syringe with a 33 gauge metal hub needle affixed. Estimate the volume of the injection mixture in the syringe based on the blue dye included in the mixture. Gently hold the edge of the skin flap with a fine curved tweezer and inject the Cre-adenovirus injection mixture slowly into the nipple while monitoring the spreading of blue dye into the mammary ductal tree.

Maintain the injection rate at slow as possible to avoid damage to the ductal lumen. A successful intraductal injection is indicated by injected fluids spreading throughout the entire ductal tree without leaking into the stroma compartment. Gently withdraw the needle from the nipple to avoid any leakage of the injected fluid.

Examine the distal side of the mammary gland or the surrounding area of the injected nipple. Dye diffusing into the nearby stroma as shown here indicates a mammary fat pad injection rather than a successful intraductal injection. Close the surgical wounds in the skin with wound clips.

Remove the mouse from the anesthesia and place it on a heating pad inside a clean cage for recovery. Monitor the development of the mammary tumor as described in the text protocol. The youngest female mice for which intraductal injection can be successfully performed are those at approximately three weeks of age.

Although for most mammary tumor induction experiments, young adult female mice are used. Intraductal injection with a larger volume of the injection mixture can be performed in female mice during early to mid gestation to target alveolar cells. To target different mammary epithelial cell subpopulations for mammary tumor induction, Cre-adenoviruses under the control of different mammary epithelial cell subset specific promoters were used for injection.

For example Cre-adenovirus under the control of keratin 8 promoter was used to target luminal mammary epithelial cells. Cre-adenovirus under the control of keratin 5 promoter targeted the basal lineage leading to genetic marking of only basal mammary epithelial cells. For a female P53 floxed homozygous mice with a ROSA26-YFP reporter, the intraductal injection of keratin 8 Cre-adenovirus led to the development of mammary tumors several months after the injection.

Due to the inclusion of the conditional R26-Y reporter, tumor epithelial cells were typically marked by YFP and could be detected by flow cytometry. The cells can be enriched by the flow sorting of YFP positive cells for further analysis. The most important things to remember when attempting this procedure are to use a small injection volume, inject slowly, and also withdraw the needle slowly.

Following this procedure, one can monitor progression of the targeted YFP positive mammary epithelial cells to pre-malignant and cancer stages by flow cytometry or co-immunofluorescence staining of the injected gland. This technique will pave the way for researchers to study cells of origin of breast cancer, how they respond to different autogenic events and how they progress to cancer cells. Both adenovirus and the needle for injection are potentially hazardous.

Therefore, one should always wear gloves and use safety needles and syringes when performing intraductal injection.