Hypoxia potentiates Wnt signaling and supports clonogenic activity. (A) Flow cytometric analysis of pimonidazole staining, a marker of hypoxia. Mice were transplanted with 7TGP-transduced leukemia cells and, after developing clinically morbid disease, were injected by tail vein with pimonidazole (60 mg/kg). One hour later, mice were euthanized and BM cells harvested, fixed, and stained with anti-pimonidazole antibody. Gated NGFR⁺ leukemia cells are shown. The marrow contained >90% tumor cells (of which 40% to 70% were GFP⁺). Results depicted are representative of 2 different leukemic animals. (B) Immunohistochemical staining for GFP in BM sections of mice following transplantation with 7TGP-transduced leukemia cells. Mice were clinically morbid with disease at the time they were euthanized and contained >90% tumor cells (of which 40% to 70% were GFP⁺) within the marrow. Red arrowheads indicate individual GFP⁺ cells. Images depicted are representative of 2 different leukemic animals. Scale bar = 60 μm. (C-D) Analysis of β-catenin protein expression level in NOTCH1-ΔE mouse leukemia cells cultured in vitro under reduced oxygen conditions. Cells were cultured in complete growth media under 20% or 5% oxygen for 3 days in plastic tissue culture dishes, then harvested for analysis by western blot (C) and flow cytometry (D). The numbers below the β-catenin blot panel in (C) indicate relative band intensities after normalization to the β-actin loading control. Gated leukemia cells are depicted in (D). Results from at least 2 independent leukemias are shown. (E-F) CFC assay for in vitro clonogenic activity. Primary leukemia cells were plated in methylcellulose-containing media supplemented or not with Wnt3a ligand or Dickkopf-related protein 1 and cultured in either 20% or 5% oxygen. Each colored data point depicted represents an individual culture dish. Mean ± SD values are indicated by horizontal lines. Results in (F) are representative of 2 different leukemias. *P < .05; **P < .01; ****P < .0001 (Student t test in [E], 2-way analysis of variance [ANOVA] with Bonferroni multiple comparisons test in [F]). ns, not significant.