Fig. 5
Inhibition of RT-induced subpopulations sensitized tumor response to RT. A 6–7-week-old BALB/c female mice were subcutaneously injected with 4T1 cells. When tumor volumes reached 80–100 mm3, mice were treated with brachytherapy RT on two alternate days (12 Gy). B Tumor volumes in the control group (red) versus the RT group (black). *p < 0.01 and ±SD are shown. C Fold change in the abundancy of subpopulations b and f as compared to untreated tumors. A significant expansion due to RT in subpopulation b harboring Her2+/EGFR+ and subpopulation f harboring cMet+/MUC1+ is detected. D Mice were subcutaneously injected with 4T1 cells and treated with RT. Trastuzumab (T), 5 mg/kg, and Crizotinib (C), 25 mg/kg, were administrated IP 2d/week and by gavage 5d/week respectively from d0 (3 days prior to RT) until the end of the experiment (d17). E C+T sensitized TNBC to RT in the 4T1 model, ± S.E., and *p < 0.01 are shown. F In vivo fold changes in subpopulations b and f showed optimal reduction when T and C were used in combination with RT. These results were consistent 6 and 12 days after RT. For C and F: quantification of subpopulations was performed using at least ~30,000 cells from each condition, which were obtained from at least three independent experiments for each time point, fold change is relative to control of each time point. For B, C mice used for each condition: control n=12, RT n= 11. On day 6 post-RT, we euthanized 7 control mice and 5 RT mice. On day 12 post-RT the experiment was completed and all remaining mice were euthanized. For E, F mice for each condition: control n= 12, RT n= 11, RT+T+C n= 7, T+C n= 10, RT+T n=12. On day 6 post-RT, 7 control mice, 5 RT mice, 4 RT+T+C mice, 6 RT+T, and 5 T+C mice were euthanized. On day 12 post-RT, the experiment was completed and all remaining mice were euthanized. For B, E, and F, statistically significant differences compared to cells treated with RT (B) and cells treated with RT+T+C (E, F) were determined using a two-tailed Student’s t test (*P < 0.01)