Figure 6

HIF-1α promotes tumorsphere formation in cultured cells and in freshly isolated tumor cells independent of oxygen tension. (A) Representative experiment in which the mean sphere formation efficiency (SFE) ± SEM was determined when wild-type (WT) and knockout (KO) cells were cultured in tumorsphere culture conditions. Tumorspheres ≥ 100 μm were scored positive, and the mean SFE (the percentage of cells capable of forming spheres per total number of single cells plated) per experiment (n ≥ 12 wells/genotype, P < 0.05, unpaired Student's t-test) was determined at each of the primary, secondary and tertiary generations of spheres. A summary presenting the mean fold enrichment in WT SFE observed among biological replicates is included in Additional file 2 Figure S6. (B) A representative experiment in which the mean primary and secondary SFE ± SEM was determined when single cells were isolated from mammary tumors of Hif1a DF, MMTV-PyMT+ mice, followed by immediate adenoviral transduction (n ≥ 12 wells/genotype, P < 0.05, unpaired Student's t-test). (C) Comparison of SFE when WT or KO cultured cells were subjected to normoxia or chronic hypoxia. Immediately following plating of single cells, dishes were cultured under normoxic or hypoxic conditions for the duration of the experiment; therefore, cells were exposed to chronic hypoxia. The graph presents mean SFE obtained from the secondary generation of spheres (n ≥ 12 wells/genotype). Differences among columns were assessed by one-way ANOVA, *P < 0.05. (D) The mean fold change in gene expression observed in KO tumorspheres was evaluated by qRT-PCR. The grand mean fold change ± SEM was determined on the basis of three independent experiments. cDNA was derived from three preparations of WT and KO secondary spheres grown in six-well format and pooled at the experiment end point to prepare total RNA (n = 3 pools of spheres/genotype). The mean expression of Notch 4 (N4), Dll1, Hey1, Hey2 and Prom1 (CD133) was reduced in KO tumorspheres by > 1.5-fold, whereas the mean expression of Notch1, Notch2 and Notch3 (N1 to N3) or Vegf did not vary by > 50% between genotypes. The decreased expression in KO tumorspheres is indicated as a negative fold change.