Estradiol pellet

Five-week-old female nonobese diabetic (NOD)/SCID mice were purchased from the Korea Research Institute of Bioscience & Biotechnology (KRIBB, Daejeon, Korea) and kept under specific pathogen-free conditions. MDA-MB-231-Luc cells infected with lentiviruses encoding an empty vector or Id1 cDNA (1 × 10⁶ cells) suspended in Matrigel (BD Biosciences) were injected into the mammary fat pads on the left and right sides, respectively, of the same animal. One week after injection, mice were given a solution of D-luciferin (150 mg/kg in PBS; Caliper Life Sciences, Hopkinton, MA) and anesthetized with 1% isoflurane/oxygen. The imaging and quantification of light emitted from the bioluminescent tumors were conducted using the IVIS camera system (Xenogen Corporation) and Living Image analysis software (Xenogen Corporation). Tumor volume was calculated using as 1/2 × long diameter × short diameter². Tumor growth was measured twice weekly. For xenografts with MCF7 cells, BALB/c-nude mice were supplemented with estradiol pellets (0.72 mg/pellet, 60 day release; Innovative Research of America, Sarasota, FL) 1 week before cell implantation. The stable Id1-overexpressing MCF7 cells or control cells (2 × 10⁶ cells) were suspended in Matrigel and then injected into the mammary fat pads of the nude mice. Tumor volume was monitored as described above for 28 days.

The PDX models used in this study WHIM20 (isolated from a patient who developed the Y537S mutation) and WHIM43 (D538G mutation) were obtained from Washington University in St. Louis (Horizons Discovery Group, Waterbeach UK). All animal procedures were reviewed and approved by Tulane University IACUC. SCID/beige (CB17.Cg-Prkdc^scidLyst^bg-1/Crl) 4–6-week-old female mice were obtained from Charles River Laboratory. Intact tumor pieces were removed and sliced with a scalpel to 3mm x 3mm and coated with 100 uL phenol-free growth factor reduced Matrigel (BD Biosciences). Control groups had PDX tumor coated in Matrigel and implanted bilaterally in the mammary fat pads under isoflurane and oxygen. In ASC groups, 10⁶ pooled donors (n = 6) of lnASCs or obASCs were resuspended in Matrigel and coated the tumor. Where indicated, estradiol pellets were implanted subcutaneously in the lateral area of the neck (0.72 mg, 60-day release; Innovative Research of America). Tumors were implanted into the fifth mammary fat pad bilaterally under isoflurane and oxygen anesthesia delivered by nose cone and animals were given 5mg/kg/day meloxicam for three days post-surgery. Tumors were measured by digital caliper every three to four days. At endpoint (tumors reach 750–1000 mm³) blood and lungs were collected for analysis. PDX derived cells were cloned out from PDX tumors.

The 6-week-old female nude mice were purchased from Vital River, Beijing. MCF-7 cells at limiting dilution (1 × 10⁵, 1 × 10⁴ and 1 × 10³) were injected with Matrigel into the mammary fat pads of the mice. The mice were supplemented with estradiol pellets (0.72 mg, released over 60 days) (Innovative Research of America, Sarasota, FL, USA). Tumor volume was measured every 3 days using a Vernier caliper and calculated according to the formula: Length × Width²/2. The mice killed after 30 days, and the tumors were excised and photographed.

All animal care and experimental procedures were approved by the Jilin University Institutional Animal Care and Use Committee. 6–7 week old athymic nude (Nu/Nu) mice (Vital River, Beijing, China) were used in xenograft experiments. The subcutaneous xenograft tumor model established by the injection of T47D (5 × 10⁶ cells/200 μL) or MCF-7 (5 × 10⁶ cells/200 μL) into the flank of mice. The athymic nude Mice were surgically implanted with estradiol pellets (0.72 mg, released over 60 days; Innovative Research of America, Sarasota, FL, United States). Implantation of the estrogen pellet was performed before mice was injected with T47D cells or MCF-7 cells. Tumors size were measured by using digital caliper, and tumor volumes were estimated by using the formula: V = [(D + d)/2]³, where D and d were the larger and smaller diameters, respectively. After injection of breast cancer cells, once the tumor volume reached approximately 40–55 mm³ the mice are randomized into groups of four–six mice per group, and the mice were treated with vehicle, IgG1 (isotype control), or H53. The tumor size was measured every 4 days using calipers. After the experiments are finished, Tumors were then harvested, fixed with 10% buffered formalin, embedded in paraffin, and subjected to pathological and immunohistochemical examinations.