The study design is outlined in
Supplemental Figure 1. All mice were maintained and treated in accordance with the National Institutes of Health Guide for the Care and Use of Experimental Animals with approval from the Baylor College of Medicine Institutional Animal Care and Use Committee. A detailed surgical protocol was published elsewhere [19 ].
Pretreatment biopsy and post-treatment surgical specimens were received within an hour after excision. For fragment transplantation, samples were minced into ~1 mm
3 fragments and transplanted directly into epithelium-free “cleared” fat pads [20 (
link)] of recipient SCID/Beige (Charles River Laboratories, Wilmington, MA), or NSG mice (Jackson Laboratories, Bar Harbor, ME) (n=2 per patient). Transplants were performed under the following conditions: Condition 1: unmanipulated host mice, Condition 2: 17β-Estradiol supplementation (60-day release, 0.36 mg/pellet, Innovative Research of America, FL, Cat.#SE-121), or Condition 3: 17β-Estradiol supplementation with inclusion of 5 × 10
4 immortalized normal human fibroblasts (passages 35-41) (1:1 unirradiated:irradiated cells (4 Gy), as described previously [21 (
link)] (fibroblasts generously provided by Dr. Charlotte Kuperwasser). Mice were palpated weekly, and tumor growth measured using calipers. Conditions found to be optimal for SCID/Bg mice were then tested using NSG mice (Condition 4).
We were also able to obtain a few samples from either pleural effusion or metastatic ascites. Fluid was centrifuged, and cells resuspended in a volume of 10-50ul, and injected into the cleared mammary fat pad using a Hamilton syringe. Xenografts derived from such samples were not included in the statistical analyses, but are included here for completeness of the collection.
Regardless of the source of tumor cells, when primary outgrowths reached 10mm in diameter, or if glands were suspected of carrying small primary outgrowths, fragments were re-transplanted into new hosts (n=3-4) as secondary xenografts. If no overt tumor formation was observed by 30 weeks, glands were harvested and processed for histological evaluation. Primary outgrowth take was defined as a surviving tissue fragment >1mm in diameter and demonstrated to be proliferative (Ki67 positivity). A xenograft line was defined as stable upon growth at transplant generation 3 (TG3).
Zhang X., Claerhout S., Pratt A., Dobrolecki L.E., Petrovic I., Lai Q., Landis M.D., Wiechmann L., Schiff R., Giuliano M., Wong H., Fuqua S.W., Contreras A., Gutierrez C., Huang J., Mao S., Pavlick A.C., Froehlich A.M., Wu M.F., Tsimelzon A., Hilsenbeck S.G., Chen E.S., Zuloaga P., Shaw C.A., Rimawi M.F., Perou C.M., Mills G.B., Chang J.C, & Lewis M.T. (2013). A Renewable Tissue Resource of Phenotypically Stable, Biologically and Ethnically Diverse, Patient-derived Human Breast Cancer Xenograft (PDX) Models. Cancer research, 73(15), 4885-4897.