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. 2019 May 10;43:211–224. doi: 10.1016/j.ebiom.2019.04.061

Fig. 4.

Fig. 4

T-DM1 treatment-induced ROR1+ cells enriched with CSC features show higher self-renewal efficiency and increased resistance with enriched CSC features. (a) Treatment naïve HER2+ BC patients tumor cells were enzymatically dissociated and cultured in supplemented CSC medium, treated with either vehicle (DMSO) and 5 nM T-DM1 and incubated for 10 days (follow the schematic diagram). Spheres were dissociated for single cells and subjected to FACS sorting for ROR1. Extreme limiting dilution assay (ELDA) was performed by plating sorted cells with increasing cell numbers and analyzed for stemness frequencies by ‘R' statistical package “statmod” (p < .0427; one-way ANOVA). Table 1 (right) showed the confidence interval for 1/stemness frequency. (b) Bulk cells were isolated and treated as described in (a) and analyzed for sphere forming efficiency (Bar graph represents the mean +/− SE, n = 3, run in triplicate). (c) HER2+ patient tumor cells treated with either vehicle or T-DM1 for 5 days, harvested and FACS sorted for ROR1+ and ROR1− populations, and then sorted cells were cultured in supplemented CSC medium [follow the schematic diagram] for 10 days and analyzed for the sphere forming efficiency from each cell population (black arrowhead; p = .001 for T-DM1 treatment; p = .05 for vehicle, unpaired two-sided t-test, Scale bar: 100 μM). (d) HCC1954 cells were treated and analyzed as described in (c) [black arrowhead; p = .01 for T-DM1 treatment; p = ns (not significant) for vehicle, unpaired two-sided t-test; Scale bar: 100 μM]. (e) HCC1954 cells were either treated with vehicle or T-DM1 and sorted as described in (c) and quantitatively analyzed for Bmi1, Nanog, Oct3/4. Sox2 mRNA in ROR1+ and ROR1− populations. (Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .05; p = .01; p = .001; one-way ANOVA). (f) ROR1+ cells were more significantly resistant than ROR1− cells at individual T-DM1 treatment and showed higher EC50 in response to T-DM1 as compared to ROR1− cells. (Bar graph represents the mean +/− SE, n = 3; run in triplicate; p = .05, p = .01).