Orthotopic Xenograft Tumor Model

Animal experiments were conducted in accordance with accepted standards of humane animal care and approved by the Animal Care and Use Committee at North Carolina Central University. Xenografts were generated as we previously described [33 (link)]. Briefly, 5-week-old female Hsd:Athymic Nude-Foxn1^nu mice (Envigo, Dublin, VA) were injected orthotopically into the right abdominal mammary gland with 5 × 10⁵ of the indicated cell model suspended in 30.0 μl of 50/50 PBS:Matrigel. Weekly tumor growth was measured via calipers and tumors excised when volume neared 400 mm³. Whole tumors were homogenized and RNA extracted using the Qiagen RNeasy kit as instructed. Three independent experiments were performed to ensure repeatability. Mice that did not form tumors were euthanized 5 months post-injection. Detection of metastatic xenograft cells in liver and lung was conducted as previously described [33 (link)].