Fig. 3.

T-DM1 treatment alters ROR1 expression without significant changes in HER2 expression: (a) Immunostaining of HER2 expression from treatment naïve (n = 5), sensitive (n = 5) and resistant (n = 5) HER2⁺ BC patient's tumor samples (Scale bar: 50 μM). (b) HCC1954 and MCF-7-HER2+ cells were cultured on a glass cover slip in 24-well plates and treated either with vehicle (DMSO) or 5 nM of T-DM1 for 5 days. Cells were fixed and immunofluorescence stained images captured for HER2 (red) and nucleus DAPI (blue) (Scale bar: 50 μM). (c) HCC1954 and MCF-7-HER2+ cells were cultured, treated and fixed as (b), and immunofluorescently stained for ROR1 (red) and nuclear DAPI (blue) expression (Scale bar: 50 μM). (d) Treatment naïve BC patient tumor cells, (e) HCC1954(HER2+), and (f) MCF-7-HER2+ cells were treated with T-DM1 (5 nM) for 5 days, trypsinized and cultured in supplemented CSC medium in sphere culture for 10 days (10 days is denoted as first generation). After 10 days, spheres were dissociated and re-cultured in CSC medium for an additional 10 days (20 days denoted as second generation) followed by dissociation of spheres and then re-cultured again in CSC medium (30 days denoted as third generation). In every 10-day culture cycle, sphere forming efficiency was examined in ROR1⁺ and ROR1⁻ populations by counting the number and size of spheres. (Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = ns [not significant]; p = .05; p = .01, p = .001; one-way ANOVA). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)