Image1_From AVATAR Mice to Patients: RC48-ADC Exerted Promising Efficacy in Advanced Gastric Cancer With HER2 Expression.TIFF

RC48-ADC is a novel humanized antibody specific for human epidermal growth factor receptor 2 (HER2)in conjugation with a microtubule inhibitor via a cleavable linker. This study was to evaluate the antitumor activity and mechanism of RC48-ADC in gastric cancer (GC) and explore the population that may benefit from RC48-ADC treatment. Four human GC cell lines and nine patient-derived xenograft (PDX) models were exploited to evaluate the antitumor effect of RC48-ADC or trastuzumab treatment in vitro and in vivo. The expression and phosphorylation of HER2 were assessed by immunohistochemistry (IHC) staining. Critical molecules of downstream PI3K/AKT and cell cycle and apoptosis signaling pathways were detected and quantified by immunoblotting. Combined with preliminary results of preclinical research, three patients with IHC3+, IHC2+/FISH+, and IHC2+/FISH- of HER2 were enrolled to verify the efficacy of RC48-ADC treatment in advanced GC. In vitro, RC48-ADC had superior antiproliferative effects in a dose-dependent manner on GC cells, especially on HER2-positive cells. In vivo, RC48-ADC exceeded trastuzumab in GC PDX models with HER2 expression, even in models with moderate to low expression of HER2. Further exploration of mechanism showed that RC48-ADC exerted the antitumor effect by inhibiting phosphorylation of HER2, inducing G2/M phase arrest and cell apoptosis in HER2-expressed PDX models. In clinical practice, RC48-ADC had satisfactory efficacy in HER2-positive and HER2 moderately expressed GC patients and demonstrated promising efficacy in HER2-positive patients who have progressed after anti-HER2 therapy. In conclusion, RC48-ADC exerted promising antitumor activity in HER2-positive as well as score of 2+ in IHC and ISH-negative AGC patients after progression of systematic treatment.</p

Additional file 5: of Mouse avatar models of esophageal squamous cell carcinoma proved the potential for EGFR-TKI afatinib and uncovered Src family kinases involved in acquired resistance

Figure S3. Pathway enrichment by RNA-Seq. Dot plots showing the enrichment results of KEGG pathway analysis for KYSE450-R versus KYSE450-P (A), EC109-R versus EC109-P (B), and PDX03-R versus PDX03-P (C), as detected by RNA-seq. The size of the dots indicates the number of genes enriched in the corresponding pathways. The color of the dots indicates the significance level of the enriched pathways, as represented by the value of Padj. (DOCX 549 kb

Additional file 3: of Mouse avatar models of esophageal squamous cell carcinoma proved the potential for EGFR-TKI afatinib and uncovered Src family kinases involved in acquired resistance

Figure S1. Efficacy of different EGFR blockers on ESCC cell lines and PDX models. (A) Six ESCC cell lines were treated with different EGFR blockers (gefitinib, afatinib, osimertinib, cetuximab, and nimotuzumab) at the indicated concentrations (from 0 to 10 μM for TKIs and 0–1000 μg/mL for mAbs). After treatment for 72 h, cell growth inhibition was detected using CCK-8 assays. Cell viability at different doses relative to vehicle-treated controls is shown (means ± SD; three independent assays). (B) Curves plots the growth of PDX03 and PDX06 treated with vehicle control, gefitinib (50 mg/kg/day, oral gavage), afatinib (15 mg/kg/day, oral gavage), osimertinib (15 mg/kg/day, oral gavage), cetuximab (0.5 mg per mouse, twice a week, i.p.), or nimotuzumab (0.5 mg per mouse, twice a week, i.p.). Mice were sacrificed after 21 days of treatment and xenografts were isolated. Pictures of the xenografts are shown and the corresponding TGI is listed in the tables. Data are presented as means ± SDs; n = 5. (DOCX 1320 kb

Additional file 2: of Mouse avatar models of esophageal squamous cell carcinoma proved the potential for EGFR-TKI afatinib and uncovered Src family kinases involved in acquired resistance

Table S1. Efficacy of different EGFR blockers on ESCC cell lines. (DOCX 17 kb

Additional file 1: of Mouse avatar models of esophageal squamous cell carcinoma proved the potential for EGFR-TKI afatinib and uncovered Src family kinases involved in acquired resistance

Materials and Methods. (DOCX 22 kb

Additional file 4: of Mouse avatar models of esophageal squamous cell carcinoma proved the potential for EGFR-TKI afatinib and uncovered Src family kinases involved in acquired resistance

Figure S2. Effects of afatinib on cell cycle and apoptosis in EC109 cells. EC109 cells were treated with 0, 10 nM, 100 nM, and 1 μM afatinib after serum-starvation for 12 h. After 48 h treatment, the cells were harvested and assayed as described below. The effects of afatinib on cell cycle distribution were assessed using flow cytometry after PI/RNase staining (A). The distribution of cells in the cell cycle is depicted (B). G1 phase-associated proteins (P21, P27, CDK4, CDK6, and CCND1) were assessed using western blotting (C).Flow cytometry showed the apoptosis induced by afatinib treatment using PE-annexin V and 7-AAD staining (D). The percentage of cells in early apoptosis (Q3) and late apoptosis (Q2) was calculated as the total apoptosis ratio (E). Apoptosis-related proteins (c-PARP, c-caspase8, BCL2, and BAX) were measured by western blotting after afatinib treatment (F). Data are presented as means ± SDs of three independent assays. P values were calculated using one-way ANOVA or unpaired two-tailed t-tests.*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns = not significant. (DOCX 570 kb

Additional file 7: of Characterization and validation of potential therapeutic targets based on the molecular signature of patient-derived xenografts in gastric cancer

Table S4. The clinicalpathological characteristics of xenografts. (DOCX 14 kb

Additional file 3: of Characterization and validation of potential therapeutic targets based on the molecular signature of patient-derived xenografts in gastric cancer

Supplementary method. The synthesis details of BK011. (DOCX 13 kb

Additional file 2: of Characterization and validation of potential therapeutic targets based on the molecular signature of patient-derived xenografts in gastric cancer

Figure S1. The work flow of gene variation calling. (DOCX 195 kb

Additional file 9: of Characterization and validation of potential therapeutic targets based on the molecular signature of patient-derived xenografts in gastric cancer

Table S6. The expression of PD-L1 in EBV-positive and EBV-negative xenografts. (DOCX 12 kb