Xenograft procedure

BT474 cells (ATCC HTB-20), were plated onto 0.1% gelatin-coated T-175 culture flasks and grown to 70% confluence (exponential density). Cells were briefly harvested with 0.25% trypsin, washed with sterile PBS, counted, then injected in a volume of 200 μl as 2–5 X 10⁶ cells mixed with an equal volume of phenol red-free Matrigel™ into the fourth left mammary fat pad. Controls received sterile PBS mixed with an equal volume of phenol red-free Matrigel.™ Mice were maintained under isofluorane anesthesia (2.5 ml/min) for 5 min/injection. Mice were weighed twice each week and received cell injections once they reached 18 g The fourth mammary fat pad is large enough to be felt through the skin of the BALB/c nude mouse, so injected cells with Matrigel™ were visible as a small bump until tumor growth became palpable. Tumors were measured with calipers twice a week and volumes were calculated using the formula V = (L X W²)/2, where L is tumor length and W is tumor width, in millimeters (mm) [19]. Chemotherapy was initiated when tumor volume surpassed 100 mm³ and was maintained throughout the study. Doxorubicin (5 mg/kg) and trastuzumab (4 mg/kg) were administered by intraperitoneal injection on successive days each week, while untreated mice received injections of sterile PBS.