Fig. 6

Increased phosphorylation of Src family kinases leads to acquired afatinib resistance in ESCC. a Increased pSFKs levels were observed in KYSE450-R and PDX03-R resistant models, but total SFKs levels were unchanged. Cells were harvested after treatment with 200 nM afatinib for 48 h. The PDX lysates used were the same as those described in Fig. 4d. All assays were repeated three times independently. b Resistant cells were treated with the indicated concentrations of afatinib in the presence or absence of 100 nM dasatinib for 72 h, and CCK-8 assays were performed to assess cell viability. Data are presented as the means ± SDs of three independent assays. c KYSE450-R and EC109-R cells were treated with 200 nM afatinib alone or in combination with 100 nM dasatinib for 48 h. d, e Curves showing the xenografts growth of KYSE450-R (d) and PDX03-R (e) treated with vehicle control, afatinib (15 mg/kg), crizotinib (25 mg/kg), afatinib (15 mg/kg) plus crizotinib (25 mg/kg), dasatinib (15 mg/kg), or afatinib (15 mg/kg) plus dasatinib (15 mg/kg). Data are presented as means ± SDs; n = 5. Mice were sacrificed after 21 days of treatment, and xenografts were isolated. Pictures of the xenografts are shown with the corresponding TGI listed in the tables. f, g Lysates were extracted from KYSE450-R (f) and PDX03-R (g) xenografts after 21 days of treatment with the corresponding inhibitors and analyzed by western blotting to explore the downstream signaling responses. The lysates were then probed with the indicated antibodies. All experiments were repeated three times independently. h A schematic of the molecular mechanisms of acquired resistance revealed in this study