5.4. Endometrial priming for receipt of tumor cells and in utero cancer cell injection

Female mice (average age 12.5 weeks) were injected with 100 ng of E2 per day for three days prior to cell engraftment. On the day of cellular injection (considered experimental Day 0), a small incision was made on the left flank of the animal anterior to the femur and lateral to the spine. The ovary was visualized through the inner pelvic fascia and a deeper cut into the pelvic cavity was made to expose the ovary. Forceps were used to pull the left uterine horn and ovary outside of the body cavity. A blunted 25G needle was inserted into the uterine lumen and used to mechanically abrade the luminal mucus layer along the full length of the anti-mesometrial 500,000 MECPK-GFP cells were suspended in 50 μl of a 1:1 mixture of Matrigel (Corning. Corning, NY USA) and PBS and injected into the abraded uterine lumen using a 25G needle. We confirmed that our abrasion technique was not directly seeding the injected cancer cells into the blood by comparing abraded animals to tail vein injected animals and looking for GFP labeled cancer cells in the lungs (Supplemental Figure 1). Animals were bilaterally ovariectomized (OVX) at the time of cell injection and a 20 μg estrogen beeswax pellet (replaced every 4 weeks) was placed under the skin at the base of the posterior neck between the shoulder blades. The right uterine horn was left intact as a sham control after OVX. The mice were sacrificed at 1 month for short-term experiments and at a humane endpoint in long-term disease course experiments (as determined by palpable primary tumor volume, external signs of animal distress, or death). Following death, mouse uteri were then excised, weighed and fixed in 4% paraformaldehyde for histological analysis.