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. 2015 Jul 23;5:12465. doi: 10.1038/srep12465

Figure 2. Constitutive activation of the Wnt/β-catenin signaling pathway is a hallmark of tumorigenicity and maintenance of BCSCs.

Figure 2

67NR cells form primary tumors readily, although the tumor cells do not intravasate. On the other hand, 4T1 cells have full metastatic properties (A). The percentages of LEF1, cyclin D1, TCF-4, and β-catenin-positive cells in both Aldefluor-positive (B) and Sca-1-positive (C) subpopulations of non-invasive 67NR cells and highly invasive 4T1 cells were evaluated by flow cytometric analysis (B,C). Wnt3a-induced Wnt/β-catenin signaling in ALDH1-positive BCSC subpopulations was assessed using a TOP Flash luciferase reporter. Wnt3a treatment induced transcriptional activity to a greater extent in the ALDH1-positive BCSC subpopulations compared with that in the ALDH1-negative subpopulations (D). Wnt1 knockdown inhibited the tumor sphere formation of 4T1 cells. Spheres that were greater than 100 μm in size were enumerated, and a representative image of a tumor sphere is shown. The averages of three independent experiments are shown (E). Wnt1 knockdown led to a decrease in the percentage of CD44+/CD24− cells as a proportion of the total cancer cells (F). Abbreviations: TSFE, tumor sphere-forming efficiency. The results are presented as the mean ± SD, as determined from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.