Fig. 2

Effects of the combined treatment of osimertinib and T-DM1 on 2D and 3D cell growth, cell death, signal transduction pathways and ADCC. a PC9 and PC9-T790M cells were treated with increasing concentrations of T-DM1 for 72 h and then cell proliferation was assessed using crystal violet staining. b PC9 and PC9-T790M cells were treated with increasing concentrations of osimertinib in absence or presence of T-DM1 2.5 μg/ml or 1.5 μg/ml, respectively. After 72 h cell proliferation was assessed by MTT assay and the effect of drug combination was evaluated using the Bliss interaction model. Data in a e b are expressed as percent inhibition of cell proliferation versus control cells and are means ±SD of three separate experiments. c cells were treated with the drugs (osimertinib 30 nM, T-DM1 5 μg/ml in PC9, 2.5 μg/ml in PC9-T790M) for 72 h and then cell death was quantitated by fluorescence microscopy analysis on Hoechst 33342 and propidium iodide-stained cells. Data, expressed as percent values, are means ±SD of three independent experiments. d spheroids from PC9 and PC9-T790M cells were treated with 30 nM osimertinib, 5 μg/ml (PC9) 2.5 μg/ml (PC9-T790M), T-DM1 or with both drugs and the fold increase (FI) versus T0 was calculated after 6 days of treatment. Representative images of tumor spheroids are shown. Data are from a representative experiment. The experiment, repeated twice, yielded similar results. e PC9 and PC9-T790M cells were treated with 30 nM osimertinib in absence or in presence of T-DM1 5 μg/ml or 2.5 μg/ml respectively, and after 6 h the cells were lysed and Western blot analysis was performed to detect the indicated proteins. The immunoreactive spots were quantified by densitometric analysis, ratios versus Actin were calculated and values, expressed as fold increase versus control (control value = 1) are reported. f PC9-T790M cells were treated with 30 nM osimertinib for 24 h, then 2.5 μg/ml T-DM1 was added to tumor cells (in absence or in presence of osimertinib) seeded with activated-NK cells at the ratio of 1:25. After 4 h LDH release was determined as described in the Methods section. Data in e and f are representative of two independent experiments. (***p < 0.001, ****p < 0.0001 vs ctrl; #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 vs osimertinib; $$$ < 0.001, $$$$ < 0.0001 vs T-DM1; one-way ANOVA followed by Bonferroni’s post-test)