Figure 1.

Synergistic anti-proliferation of DB-1310 plus trastuzumab in in vitro models with high HER2 and HER3 expression. (A) The cell surface HER2 and HER3 on BC cell lines was measured by FACS. The fluorescence signal was detected and analyzed using FACSC. The translation into the HER2 and HER3 copy number was calibrated with BD Quantibrite™ PE beads. (B) BC cells were treated with PBS control, 100 nM DB-1310, 100 nM trastuzumab, or a combination of the two for 7 d. Cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay. Proliferation inhibition was compared with the single-agent group. (C) DB-1310 (100 nM) or human IgG1 isotype control was conjugated with IncuCyte^® Human Fabfluor-pH Red Antibody Labeling Reagent followed by the indicated cell treatment. DB-1310-HER3 complex internalization was analyzed using the IncuCyte^® Live-Cell Analysis System and quantified using the IncuCyte software. The top panel represented the internalization results 24 h after treatment with the labeled complex. PathHunter cells with HER2 and HER3 expression were treated with DB-1310 and trastuzumab alone (D) or in combination (E) at the indicated concentrations in the presence of 0.3 μM NRG-1. Inhibition of ligand-induced HER2/HER3 dimerization was analyzed using the PathHunter^® Kinase Dimerization Assay Kit. The results are representative of three different experiments and expressed as the mean ± SEM. *P < 0.05, **P < 0.01. HER2, human epidermal growth factor receptor 2; HER3, human epidermal growth factor receptor 3; BC, breast cancer; FACS, fluorescence-activated cell sorting; MFI, mean fluorescence intensity; IC₅₀, half-maximal inhibitory concentration; hIgG1, human immunoglobulin G1; SEM, standard error of the mean; PBS, phosphate-buffered saline.