Figure 5. A blockade of Wnt/β-catenin signaling suppresses tumorigenesis in a murine xenograft model.

A schematic representation of the experimental protocol, as described in the Materials and Methods section (A). Mice were implanted with 4T1 cells (5 × 10⁴ cells/mouse) by orthotopic injection into the thoracic mammary fat pads. Tumor tissues were isolated from mice bearing 4T1 or MDA-MD-435 cells transfected with Wnt1 shRNA or control shRNA. Tumor volumes were measured, as described in the Materials and Methods section (B,C). The ALDH-positive subpopulation, as a proportion of the total cell population in the tumor xenografts, was assessed by immunohistochemistry (D). The relative expression of downstream components of Wnt/β-catenin signaling, such as Wnt1, LEF1, and β-catenin, in bulk tumors was assessed by immunohistochemistry (E). Wnt1 knockdown-mediated apoptotic DNA fragmentation in tumor xenografts was visualized by TUNEL assay (F). Tumorigenesis promoted by Wnt/β-catenin signaling was further confirmed by performing proliferating cell nuclear antigen (PCNA) immunohistochemistry to assess tumor xenografts (G). DAPI staining was carried out to label the nuclei within each field. The results are presented as the mean ± SD, as determined from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.