Wnt1 expression decreased subcutaneous GL261 tumor growth and increased animal survival. (A) Western blots showing Wnt1 and Dkk1 expression +/−DOX in GL261 cells. (B) sTOP-FLASH assay on human embryonic kidney (HEK293)/GL261 co-cultures without DOX. Monoculture of transfected HEK293 cells served as baseline. Wnt1 and Dkk1 cells were cultured without (−) or with (+) Wnt3aCM (one experiment in triplicate). (C) In vitro proliferation of the control-, Wnt1-, and Dkk1-GL261 line cultured +/−DOX. (D, left) Representative pictures of NUDE mice with subcutaneous tumors −DOX. (right) Tumor volumes (n = 7/group) of the transplanted glioma cell lines −DOX (*, P < 0.05; **, P < 0.01). (E) H&E-stained paraffin sections revealed reduced necrotic areas for Dkk1-expressing tumors (N). (F, left) Pimonidazole (brown) immunohistochemistry staining revealed tumor hypoxia, hematoxylin counterstaining (blue). (right) Hypoxia in Dkk1^−D compared with the control^−D (*, P < 0.05) and Wnt1^−D tumors (**, P < 0.01; n = 4 tumors/group, slices from the center of similar sized tumors). (G, left) Representative vessels, stained for nuclear β-catenin (brown) and hematoxylin (blue) of control^−D, Wnt1^−D, and Dkk1^−D tumors. Arrowheads point to β-catenin⁺ nuclei. (right) Quantification of β-catenin⁺ nuclei (n = 4 tumors/group, 20 vessels/tumor; ***, P < 0.001). Bars: (D) 1 cm; (E and F) 400 µm; (G) 35 µm. Error bars indicate SEM.