My contemporaries in 20^<th> century.

De novo copy number variations (CNVs) identified in the 1210B2 iPSC-derived NSPCs. (XLSX 39 kb

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

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

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

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

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

Effects of tumor necrosis factor-α, vascular endothelial growth factorand SU1498 on ADAM15 expression

<p><b>Copyright information:</b></p><p>Taken from "Expression of ADAM15 in rheumatoid synovium: up-regulation by vascular endothelial growth factor and possible implications for angiogenesis"</p><p>Arthritis Research & Therapy 2005;7(6):R1158-R1173.</p><p>Published online 5 Aug 2005</p><p>PMCID:PMC1297561.</p><p>Copyright © 2005 Komiya et al.; licensee BioMed Central Ltd.</p> Effect of tumor necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF)on ADAM15 expression. After starvation, rheumatoid arthritis (RA) synovial fibroblasts (SFs) were treated with 10 ng/ml TNF-α and/or 40 ng/ml VEGFfor 24 h, and then the expression of ADAM15, vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2 and neuropilin-1 was examined by RT-PCR at 25 cycles (ADAM15 and β-actin) or 30 cycles (VEGFR-1, VEGFR-2 and neuropilin-1) as described in Materials and methods. Note that treatment with TNF-α induces VEGFR-2 and co-treatment with TNF-α and VEGFstimulates the mRNA expression of ADAM15 in RA SFs. Effect of SU1498 on ADAM15 expression. After starvation for 24 h, RA SFs were stimulated with 10 ng/ml TNF-α for 24 h to induce VEGFR-2. Then, the cells were treated with SU1498 (0, 1 and 10 μM) for 30 minutes and stimulated with 40 ng/ml VEGFfor 24 h. The mRNA expression of ADAM15 was determined by RT-PCR at 25 cycles as described in Materials and methods. Note that the stimulated expression of ADAM15 mRNA is inhibited by the treatment with SU1498, while the expression of VEGFR-2 mRNA is not affected by the treatment

Bendamustine-treated MM cells remaining at the metaphyseal region express VE-cadherin and show hypoxic phenotypes.

<p><b>A.</b> A single dose of bendamustine (20 mg/kg) or an equivalent amount of PBS was administered intraperitoneally to engrafted mice 5 days after U266-EGFP cell transplantation. Four to six weeks after administration, mice were sacrificed for analysis. <b>B.</b> Over the course of a week, two sequential doses of bendamustine (20 mg/kg) or equivalent amounts of PBS were administered intraperitoneally to engrafted mice 5 days after U266-EGFP cell transplantation. These mice were sacrificed for analysis when the mice had developed disease symptoms. <b>C, D.</b> IHC of human CD138 (gray) at the diaphyseal region (<b>C</b>) or the metaphyseal region (<b>D</b>) at 4 weeks after treatment in bendamustine-treated mice (right panel) or non-treated (PBS-treated) mice (left panel). Representative data from five fields of the BM of two mice per group in two independent experiments are shown. In A and B, the left bars = 200 µm, and in B, the right bar = 100 µm. <b>E.</b> Double IHC of human CD138 (gray) and VE-cadherin (red) at 4 weeks after treatment in bendamustine-treated mice. The arrows indicate human CD138-positive MM cells. Representative data from five fields of the BM of two mice in two independent experiments are shown. Bar = 50 µm. <b>F–H.</b> Quantification of HIF-2α (<b>F</b>) or HIF-1α (<b>H</b>) protein levels in cultured U266-EGFP cells, or VE-cadherin⁺ or VE-cadherin⁻ fractions, 6 weeks after U266-EGFP cell transplantation and quantification of pimonidazole retention (<b>G</b>) in VE-cadherin⁺ or VE-cadherin⁻ fractions at 6 weeks after transplantation. The experiment was performed twice with similar results. <b>I.</b> Human CD138⁺ mouse CD45⁻ MM cells were sorted from non-treated or bendamustine-treated mice, and VEGFA, IL-6 or FGF-2 transcripts were examined by qRT-PCR. Each value was normalized to β-actin expression (mean ± SD, **<i>P</i><0.01, n = 4). The experiment was performed twice with similar results.</p

Bendamustine suppresses MM cells <i>in vivo</i>.

<p><b>A.</b> Serum levels of human IgE. Human IgE levels in the serum of NOG mice 4 weeks after treatment with bendamustine were analyzed by ELISA. Non-treated mice were injected with PBS (n = 5, mean ± SD). The experiment was performed three times with similar results. <b>B.</b> Histological studies of the thoracic vertebrae of NOG mice 4 weeks after treatment with bendamustine (right panel) or non-treatment (left panel). Massive infiltration of U266-EGFP cells was observed in non-treated mice. However, an overall reduction of U266-EGFP cells and maintenance of normal hematopoiesis were observed in bendamustine-treated mice. HE staining: T, tumor; m, marrow; arrow, megakaryocyte. Representative data from five fields from the BM of five mice per group from two independent experiments are shown. Upper panel bars = 200 µm. Lower panel bars = 100 µm. <b>C.</b> FACS analysis of U266-EGFP cells at 4 weeks after treatment in bendamustine-treated or non-treated mice (n = 5, mean ± SD). Representative FACS plots of at least 10 mice from two independent experiments are shown.</p