Fig. 2: In vitro establishment of T-DM1-resistant cells requires prior induction of epithelial–mesenchymal transition.

A HME2 cells were left untreated (parental) or were stimulated with TGF-β1 and allowed to recover (post-TGF-β) as described in the “Materials and methods”. These two cell populations were subsequently treated with T-DM1 (250 ng/ml) every 3 days for a period of 5 weeks. Representative wells were stained with crystal violet at the indicated time points to visualize viable cells. B Brightfield microscopy of crystal-violet-stained HME2 parental and post-TGF-β cells following 3 weeks of continuous T-DM1 treatment. C The T-DM1-resistant (TDM1R) cells that survived 5 weeks of treatment were further expanded and cultured for a period of 4 weeks in the absence of T-DM1. These cells along with passage-matched parental HME2 cells were subjected 96 h treatments with the indicated concentrations of T-DM1 and assayed for cell viability. Data are the mean ± SE of three independent experiments resulting in the indicated P value. D Parental, post-TGF-β, and TDM1R HME2 cells were stained with Alexafluor 647-labeled trastuzumab and antibody binding was quantified by flow cytometry. The percentage of cells in each quadrant with reference to forward scatter (FSC) is indicated. Also shown is the mean, ±SD, percentages of low trastuzumab binding (Trastuzumab^Low) cells of three independent experiments resulting in the indicated P values.