Fig. 4

Distinct differences are presented in afatinib-refractory ESCC models compared with its parental sensitive models. a The cartoon depicts the process of generating two afatinib-resistant ESCC cell lines in vitro (a1) and one afatinib-resistant ESCC PDX in vivo (a2). b Dose-response curves were generated to confirm the resistant phenotype of KYSE450-R and EC109-R after 72 h of afatinib treatment. Data are presented as means ± SDs of three independent assays. In vivo xenograft experiments were conducted to confirm the resistant phenotype of PDX03-R. PDX03-P and PDX03-R were treated with afatinib for 21 days by oral gavage at a dose of 15 mg/kg/day (n = 5). Data are presented as means ± SDs. c Representative images of resistant and parental cells or PDX (× 100 magnification for cells; × 200 magnification for PDX; scale bars = 100 μM). The morphology of the resistant PDX model was detected by H&E staining. d Responses of the EGFR downstream signaling to afatinib treatment in parental and resistant cells or PDX. Cells were harvested after 200 nM afatinib treatment for 48 h. Tissue lysates were extracted from PDX03-R and PDX03-P xenografts after 21 days of afatinib treatment. All experiments were repeated three times independently. e Volcano plots showed the distinct differential gene profiles after the acquisition of a resistant phenotype, as detected by RNA-seq. Red dots represented upregulated genes and green dots indicated downregulated genes in resistant models compared with their counterparts. DEG, differential genes; UP, upregulated genes; DOWN, downregulated genes