Xenograft models and treatment protocol

Four-to-six-week-old female BALB/c nude mice, purchased from Shanghai Slac Laboratory Animal Corporation (Shanghai, China), were housed with regular 12/12-hour light-dark cycle for at least three days before the study. Animal care was carried out in accordance with the Principles of Laboratory Animal Care (NIH publication#85-23, revised in 1985). All experimental protocols were approved by the Institutional Animal Care and Use Committee of Zhejiang University (approval ID: SYXK[ZHE]2005-0072). Tumor specimens were obtained at initial surgery from patients after written informed consent as mentioned above. PDX gastric carcinoma xenograft models were established as previously described [8, 12]. The tumors were subcutaneously implanted into the flanks of mice under anesthesia with isoflurane; xenograft growth was monitored at least twice weekly by Vernier caliper measuring of the tumor length (a) and width (b).At about 1500 mm³, tumors were extracted for serial transplantation. Numerous samples from early passages were stored in the tissue bank, cryopreserved in liquid nitrogen, and used for further experiments. Third generation xenografts (i.e. the second mouse-to-mouse passage) were used for experiments at tumor volumes of about 100-200 mm³. Totally, 136 mice were used in this research. Mice with third generation xenografts were randomized divided into several groups (n = 5), including i) vehicle (DMSO as vehicle); ii) AZD4547, daily 6.25 mg/kg oral administration; iii) crizotinib, daily 50 mg/kg oral administration; iv) trastuzumab, weekly 20 mg/kg intraperitoneal injection; v) daily 6.25 mg/kg AZD4547 + 50 mg/kg crizotinib per os. All treatments were administered for 4 weeks, and the dosages were selected according to previous reports [21, 25, 26]. Mouse weights and tumor volumes were assessed daily, with tumor volume derived as (length × width 2)/2. Relative tumor growth inhibition (TGI) was determined by the following formula: TGI = 1 - T/C, where T/C represents the relative tumor growth of compound-treated mice divided by that of control mice. For tumor regression, in which the tumor volume after treatment was smaller than the initial value before dosing, the following equation was used: regression % = 100 × (T0-Ti)/T0. T0 and Ti are tumor volumes in the same group, measured at different time points, with T0 representing the tumor volume on the day preceding the first treatment and Ti the tumor volume at the last measurement day after treatment.