Histological Characterization of the Tumorigenic “Peri-Necrotic Niche” Harboring Quiescent Stem-Like Tumor Cells in Glioblastoma
Fig 6
Significance of emergence of NANOG+ HIF-1α+ RNApII-S2P-/low cells in spheroid cultures of T98G glioblastoma cells.
a: Scheme of the experiments. Spheroids were generated under normoxic conditions (20% O2), and then divided into 2 groups (normoxic and hypoxic). After normoxic or hypoxic (5% O2) culture for 9 or 24 h, histological analysis and sphere formation assay under normoxic conditions were performed. Data for the normoxic and hypoxic groups sampled after the same culture time were compared. b–j: Histological analysis of the spheroids cultured under normoxic or hypoxic conditions. Since the spheroids of normoxic group cultured for 9 and 24 h showed similar results, only the spheroids cultured for 24 h are shown. H-E staining (b–d), chromogenic double immunostaining for HIF-1α/RNApII-S2P (e–g), and triple immunostaining for NANOG/HIF-1α/RNApII-S2P (h–j) are presented (NANOG, red; HIF-1α, blue; RNApII-S2P, brown). NANOG+ HIF-1α+ RNApII-S2P-/low cells (purple cells) were detected in spheroids cultured in hypoxia for 24 h (j, arrows). The inset in j shows a higher magnification of these cells. These data are representative of at least 3 independent experiments. Scale bars, 50 μm. k: Sphere-forming efficiency of the cells derived from the spheroids of normoxic or hypoxic groups. Values are relative ones in which the results for the normoxic spheroids sampled after the same culture time were set to 1. The results are expressed as the mean ± SD of at least 7 independent experiments. N.S., not significant; *, P < 0.05.