Histological Characterization of the Tumorigenic “Peri-Necrotic Niche” Harboring Quiescent Stem-Like Tumor Cells in Glioblastoma

<div><p>Background</p><p>Characterization of the niches for stem-like tumor cells is important to understand and control the behavior of glioblastomas. Cell-cycle quiescence might be a common mechanism underlying the long-term maintenance of stem-cell function in normal and neoplastic stem cells, and our previous study demonstrated that quiescence induced by hypoxia-inducible factor (HIF)-1α is associated with a high long-term repopulation capacity of hematopoietic stem cells. Based on this, we examined human astrocytoma tissues for HIF-1α-regulated quiescent stem-like tumor cells as a candidate for long-term tumorigenic cells and characterized their niche histologically.</p><p>Methods</p><p>Multi-color immunohistochemistry was used to visualize HIF-1α-expressing (HIF-1α⁺) quiescent stem-like tumor cells and their niche in astrocytoma (WHO grade II–IV) tissues. This niche was modeled using spheroids of cultured glioblastoma cells and its contribution to tumorigenicity was evaluated by sphere formation assay.</p><p>Results</p><p>A small subpopulation of HIF-1α⁺ quiescent stem-like tumor cells was found in glioblastomas but not in lower-grade astrocytomas. These cells were concentrated in the zone between large ischemic necroses and blood vessels and were closer to the necrotic tissues than to the blood vessels, which suggested that a moderately hypoxic microenvironment is their niche. We successfully modeled this niche containing cells of HIF-1α⁺ quiescent stem-like phenotype by incubating glioblastoma cell spheroids under an appropriately hypoxic condition, and the emergence of HIF-1α⁺ quiescent stem-like cells was shown to be associated with an enhanced sphere-forming activity.</p><p>Conclusions</p><p>These data suggest that the “peri-necrotic niche” harboring HIF-1α⁺ quiescent stem-like cells confers a higher tumorigenic potential on glioblastoma cells and therefore may be a therapeutic target to control the behavior of glioblastomas.</p></div

SOX2⁺ (or NANOG⁺) HIF-1α⁺ RNApII-S2P^-/low tumor cells are not found in zones around small pseudopalisading necroses or in areas showing no necrotic change.

<p><b>These cells are found in glioblastomas (WHO grade IV), but not in diffuse astrocytomas (grade II) and anaplastic astrocytomas (grade III). a, b:</b> Serial sections of glioblastoma tissue containing small pseudopalisading necroses. <b>c, d:</b> Serial sections of glioblastoma tissue showing no necrotic changes. H-E staining (a, c) and triple immunostaining for SOX2/HIF-1α/RNApII-S2P (b, d) are shown. Although SOX2⁺ and/or HIF-1α⁺ tumor cells were found, they were RNApII-S2P⁺. Therefore, SOX2⁺ HIF-1α⁺ RNApII-S2P^-/low cells were not observed. N_S, small pseudopalisading necrosis; V, blood vessel. Scale bars, 50 μm. <b>e:</b> Triple immunostaining for SOX2/HIF-1α/RNApII-S2P in astrocytomas of WHO grade II and III. No SOX2⁺ HIF-1α⁺ RNApII-S2P^-/low cells were observed. Scale bars, 25 μm. <b>f, g:</b> Frequency of SOX2⁺ HIF-1α⁺ RNApII-S2P^-/low cells (f) and NANOG⁺ HIF-1α⁺ RNApII-S2P^-/low cells (g) in cases of astrocytic tumors of WHO grade II–IV. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01 (grade IV <i>vs</i>. combined group of grades II and III).</p

Triple immunofluorescent staining for SOX2, HIF-1α, and RNApII-S2P in zones around small pseudopalisading necroses or in areas showing no necrotic changes.

<p><b>a:</b> Glioblastoma tissue containing small pseudopalisading necrosis. Although SOX2⁺ and/or HIF-1α⁺ tumor cells were found, they were RNApII-S2P⁺. Therefore, SOX2⁺ HIF-1α⁺ RNApII-S2P^-/low cells were not observed. <b>b:</b> Glioblastoma tissue showing no necrotic changes. SOX2⁺ HIF-1α⁺ RNApII-S2P^-/low cells were not found. Insets at lower left of the panels a and b show higher magnification of the boxed areas in the merged images. Ns, small pseudopalisading necrosis; V, blood vessels; DIC, differential interference contrast image. Scale bars, 25 μm.</p

A schematic model illustrating the main findings of this study.

<p>The SOX2⁺ (or NANOG⁺) HIF-1α⁺ RNApII-S2P^-/low cells identified in this study represent hypoxic quiescent stem-like tumor cells, and the emergence of these cells is associated with an increased sphere-forming activity. Thus, the most common locations of these cells, namely, the neighborhood of large ischemic necroses in glioblastoma tissues (“peri-necrotic niche”) and the zone of intermediate depth from the surface of the spheroids cultured under 5% O₂, indicate a microenvironment supporting tumorigenicity. Since a gradient of O₂ concentration is present within the glioblastoma tissues and spheroids, this microenvironment is formed under an appropriate level of hypoxia.</p

Significance of emergence of NANOG⁺ HIF-1α⁺ RNApII-S2P^-/low cells in spheroid cultures of T98G glioblastoma cells.

<p><b>a:</b> 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>b–j:</b> 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. <b>k:</b> 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; *, <i>P</i> < 0.05.</p

Tumor cells with a SOX2⁺ (or NANOG⁺) HIF-1α⁺ RNApII-S2P^-/low phenotype are found in human glioblastoma tissues.

<p><b>a:</b> Hematoxylin-eosin (H-E) staining and single-color immunostaining for SOX2, NANOG, HIF-1α, and RNApII-S2P (serine 2 phosphorylation of the C-terminal domain of RNA polymerase II) in tumor tissue from a representative case of glioblastoma. Suppression of RNApII-S2P immunoreactivity was noted around the necrotic area (N). <b>b:</b> Triple immunofluorescent staining for SOX2, HIF-1α, and RNApII-S2P. SOX2⁺ HIF-1α⁺ RNApII-S2P^-/low cells (arrows) were found around the necrotic area (N). <b>c:</b> Triple immunofluorescent staining for NANOG, HIF-1α, and RNApII-S2P. NANOG⁺ HIF-1α⁺ RNApII-S2P^-/low cells (arrows) were found around the necrotic area (N). Insets at lower left of the panels b and c show higher magnification of the boxed areas in the merged images. N, necrotic area; V, blood vessels; DAPI, 4′,6-diamidino-2-phenylindole (nuclear stain); DIC, differential interference contrast image. Scale bars, 25 μm. <b>d:</b> Summary of the staining methods used in b and c. Sections were incubated with goat anti-SOX2 (b) or anti-NANOG (c) antibody and then with Alexa Fluor 546-conjugated donkey anti-goat IgG secondary antibody. Next, rabbit anti-RNApII-S2P antibody and then Alexa Fluor 635-conjugated goat anti-rabbit IgG secondary antibody were applied. The sections were reacted with mouse anti-HIF-1α antibody and then with biotinylated donkey anti-mouse IgG secondary antibody and Alexa Fluor 488-conjugated streptavidin.</p

Inhibition of Fatty Acid Synthase Decreases Expression of Stemness Markers in Glioma Stem Cells

<div><p>Cellular metabolic changes, especially to lipid metabolism, have recently been recognized as a hallmark of various cancer cells. However, little is known about the significance of cellular lipid metabolism in the regulation of biological activity of glioma stem cells (GSCs). In this study, we examined the expression and role of fatty acid synthase (FASN), a key lipogenic enzyme, in GSCs. In the <i>de novo</i> lipid synthesis assay, GSCs exhibited higher lipogenesis than differentiated non-GSCs. Western blot and immunocytochemical analyses revealed that FASN is strongly expressed in multiple lines of patient-derived GSCs (G144 and Y10), but its expression was markedly reduced upon differentiation. When GSCs were treated with 20 μM cerulenin, a pharmacological inhibitor of FASN, their proliferation and migration were significantly suppressed and <i>de novo</i> lipogenesis decreased. Furthermore, following cerulenin treatment, expression of the GSC markers nestin, Sox2 and fatty acid binding protein (FABP7), markers of GCSs, decreased while that of glial fibrillary acidic protein (GFAP) expression increased. Taken together, our results indicate that FASN plays a pivotal role in the maintenance of GSC stemness, and FASN-mediated <i>de novo</i> lipid biosynthesis is closely associated with tumor growth and invasion in glioblastoma.</p></div

<i>De novo</i> lipogenesis using glucose and acetate as carbon source in GSCs and differentiated non-GSCs.

<p>G144, Y10 and G179 cells were incubated with [¹⁴C]-glucose (A) or [¹⁴C]-acetate (B) for 24 or 8 h to measure glucose or acetate incorporation into total lipid, respectively. Data are represented as means ± SEM for three independent experiments for each cell line. * P < 0.01.</p

Effects of cerulenin on stemness and differentiation status.

<p>(A) qPCR results showing the expression of nestin, CD133, Sox2 and FABP7 in G144 GSC lines before and after administration of cerulenin. * P < 0.05, ^# P < 0.01 (B) The protein level of nestin, CD133, Sox2 and FABP7 was down-regulated by cerulenin. * P < 0.05, ^# P < 0.01 C. Expression of GFAP and NeuN in GSC lines before and after administration of cerulenin. * P < 0.01.</p

Expression of FASN in human glioblastoma cells and GSC lines.

<p>(A) Human glioblastoma cells show the strong expression of FASN (blue) and the neural stem cell marker, Sox2 (red). Sox2 expression is confined to the nuclei, whereas FASN is expressed in the cytosol. The white arrows show FASN⁺Sox2⁺ cells. (B) Western blotting showing the expression of FASN, CD133, Sox2, and FABP7 in G144, Y10, G179, Y02, Y04 and Y14 GSC lines. Upon differentiation in the presence of FBS, FASN expression, similar to that of CD133, Sox2, and FABP7, was down-regulated. Expression of β-actin was used as an internal control. (C) Expression of FASN in GSC lines before and after differentiation. GSCs show strong expression of FASN and other neural stem cell markers, Sox2, nestin and CD133. Upon differentiation in the presence of FBS, FASN expression is down-regulated, similar to that of Sox2, nestin, and CD133. Immunofluorescence micrographs showing the co-expression of FASN with Sox2 (a, e, a’) and nestin (b, f, b’) in G144 and Y10 GSC lines. Phase contrast micrographs showing the morphology of GSC lines (G144 and Y10) in the presence of EGF and FGF (d, h) or after differentiation in the presence of FBS (d’). Bars in a-c, e-g, a’-c’ = 50 μm, Bars in d, h, d’ = 20 μm.</p