Mouse xenograft models and toxicity study

All animal procedures were conducted under the approval of the Institutional Animal Care and Use Committee (IACUC) at MD Anderson Cancer Center (protocol number 10-14-07231). MDA-MB-231 (0.5 × 10⁶), HCC1937 (2 × 10⁶) or MCF-7 (5 × 10⁶) cells were injected into the mammary fat pads of female nude (Swiss Nu/Nu) mice of 6–8 weeks of age (Department of Experimental Radiation Oncology Breeding Core, The University of Texas MD Anderson Cancer Center). A1034 (0.5 × 10⁶) cells were injected into the mammary fat pads of female FVB/NJ mice (The Jackson Laboratory, Stock No. 001800) of 6–8 weeks of age. H1993 (0.5 × 10⁶) cells were injected subcutaneously into the right flank of female nude (Swiss Nu/Nu) mice (Department of Experimental Radiation Oncology Breeding Core, The University of Texas MD Anderson Cancer Center) of 6–8 weeks of age. When the tumor volume reached ~50 mm³, crizotinib (5 mg/kg) and foretinib (5 mg/kg), {"type":"entrez-nucleotide","attrs":{"text":"AG014699","term_id":"3649917"}}AG014699 (5 mg/kg) and ABT-888 (25 mg/kg), dissolved in aqueous 50 mM sodium acetate, pH 4, were administered to mice five times per week as single agents or in combination for the number of days specified in the figure legend. Tumor was measured at the indicated time points, and tumor volume was calculated by the formula: π/6 × length × width². For MDA-MB-231 and A1034 xenograft mouse models, mice were imaged before and after treatment using the IVIS Imaging System to assess tumor growth. Mice were injected with 100 μl of D-luciferin (Xenogen; 15 mg/ml in PBS). After 10 min, mice were anesthetized with a mixture of oxygen and isoflurane (Inhalation Anesthesia System; Matrix Medical, Orchard Park, NY) and imaged using the IVIS Imaging System. Imaging parameters were maintained across experiments for comparative analyses.

Tumor samples were collected after final treatment and analyzed by immunofluorescence staining. For toxicity assessment, mice were weighed before and after treatment (on day 21 for {"type":"entrez-nucleotide","attrs":{"text":"AG014699","term_id":"3649917"}}AG014699 and crizotinib, and on day 16 for ABT-888 and foretinib. Blood samples were collected from the orbital sinus using a microhematocrit tube after each treatment and subjected to biochemical analysis for liver marker enzymes alanine transaminase (ALT) and aspartate transaminase (AST) and kidney marker by-products creatinine and blood urea nitrogen to evaluate treatment toxicity by COSBA INTERGRA 400 plus (Roche Diagnostics, Rotkreuz, Switzerland) at The Department of Veterinary Medicine & Surgery. All in vivo experiments were conducted with 10 mice for each treatment and control group. No statistical methods were used to predetermine sample size.