Establishing xenografts 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 h light/12 h dark cycles for at least 3 days before use. Ambient temperature was 20 ~ 22 °C, kept at constant humidity of 40 ~ 60%. PDX models were established as we previously reported^71,93. We monitored xenograft growth at least twice weekly by a vernier caliper measuring the length (L) and width (W) of the tumor, and then removed them for serial transplantation after the volume reached about 1500 mm³. The tumor volume (V) was calculated according to the following formula: V = L × W²/2.

We used xenografts from the third generation (the second mouse-to-mouse passage) for the experiments once the tumor volume reached about 150–200 mm³. Mice were randomly assigned to different groups (five to seven mice/group)as follows: (i) vehicle; (ii) Trastuzumab (SelleckChem) 10 mg/kg twice weekly of intraperitoneal injection; (iii) AZD5438 (SelleckChem) 20 mg/kg daily by oral gavage; (iv) AZD5438 + Trastuzumab, for 3 weeks. Experiments were ended once the tumor volume surpassed 1500 mm³ or mouse weight loss reached 20%. The percentage of tumor growth inhibition (TGI) was calculated according to the following formula: TGI = (1 − T/C) × 100%, where T/C represents the relative tumor volume of treatment group and control group. After the mice had been killed, we conducted immunoblot to assess the expression of various markers. Animal care and experiments were performed under the approval and supervision of the Animal Experimental Ethical Inspection of the First Affiliated Hospital, College of Medicine, Zhejiang University (number 2018-378).