Figure 4. Fbxw7 ablation combined with chemotherapy eliminates residual tumor cells in a mouse allograft model of breast cancer.
(A) RT and real-time PCR analysis of Fbxw7 mRNA abundance in E0771 breast cancer cells maintained in 2D culture as well as in primary and secondary mammospheres (day 7) formed by these cells (n = 4 independent experiments). (B) FACS analysis of E0771-derived mammospheres (day 7) stained with Hoechst 33342 and pyronin Y. (C) RT and real-time PCR analysis of Fbxw7 mRNA abundance in the cycling and quiescent cell fractions isolated as in B (n = 5 independent experiments). (D) IB analysis of Fbxw7 and c-Myc in Fbxw7-KO E0771 cells generated with the CRISPR-Cas9 system as well as in WT cells. Accumulation of c-Myc in the KO cells was consistent with Fbxw7 ablation. Asterisks indicate nonspecific bands. (E) Experimental strategy for analysis of residual tumor cells in the lung of C57BL/6J mice injected with equal numbers (2.5 × 105 each) of WT E0771 cells expressing mVenus and Fbxw7-KO cells expressing tdTomato into the fourth mammary fat pad. The primary tumors were removed at day 21, and the mice were then treated with paclitaxel (PTX) or vehicle for 5 days and analyzed at day 30. (F) FACS analysis of remaining tumor cells in the lung of mice as in E. Arrows indicate individual residual tumor cells. (G) Proportion of WT (mVenus) or Fbxw7-KO (tdTomato) chemotherapy-resistant cells remaining in the lung. (H) Survival analysis for mice (n = 8 each) injected with WT or Fbxw7-KO E0771 cells (5 × 105) separately and then treated with PTX as in E. Box-and-whisker plots show the median, the lower and upper quartiles, 1.5× the interquartile range, and outliers. *P < 0.05 by Kruskal-Wallis test (A), Mann-Whitney U test (C), or log-rank test (H).