Cell density in vitro and tumor size in vivo influenced HER-2 expression and efficacy of T-DM1. (A) Calu-3 cells were plated at low (104cells/cm2) (i) and high (8x104cells/cm2) (ii) density and after 24 h membrane HER-2 protein expression was evaluated by immunohistochemistry. (B) Calu-3 cells were plated at different density and exposed for 72 h to T-DM1 1 μg/ml and then cell number was assessed using crystal violet staining as described in Methods section. Percent inhibition of cell proliferation versus control cells was plotted as function of cell density. The experiments, repeated three times, yielded similar results. 4 × 106(C) or 8 × 106(D) Calu-3 cells were subcutaneously implanted on BALB/c-Nude mice. At the beginning of the treatments average tumor volumes were 161 ± 15 mm3 and 370 ± 50 mm3 respectively. In both settings vehicle, trastuzumab (15 mg/Kg i.p.) or T-DM1 (15 mg/Kg i.v.) were administered every six days as pointed (arrows). Tumor sizes were measured three times per week and data expressed as volume + SEM (n = 6 mice per group). (**p < 0.01, ***p < 0.001 vs control; two-way ANOVA followed by Bonferroni’s post-test). After sacrifice tumors were excised and weighted (# p < 0.05 ##p < 0.01; one-way Anova followed by Tukey’s post-test).