Maintenance of glial progenitor-like phenotype, but enhanced CD133 expression in high-grade gliomas.

<p>In addition to multiple glial progenitor cell markers, a high proportion of glioma cells co-expressing CD133 was detected in most of the high-grade glioma specimens. Dot-plot profiles of glial progenitor cell surface markers and CD133 expression on high-grade glioma cells from representative patients (#14 (A) and #22 (B)) are shown. Freshly isolated glioma cells were simultaneously stained with the indicated antibodies. The hematopoietic cells were distinguished with anti-CD45 staining. The numbers in each quadrate represent the percentages of the cells stained positively or negatively by the respective antibodies.</p

Low-grade glioma cells concurrently express multiple cell surface markers characteristic of adult human glial progenitors.

<p>Dot-plot profiles of glial progenitor cell surface markers and CD133 expression on low-grade glioma cells from two representative patients (#1 (A) and #4 (B)) are shown. Freshly isolated glioma cells were simultaneously stained with the indicated antibodies. The hematopoietic cells were distinguished with anti-CD45 staining. The numbers in each quadrate represent the percentages of the cells stained positively or negatively by the respective antibodies.</p

Karyotype analysis of short-term cultured glioma cells

<p>Freshly isolated glioma cells were cultured and analyzed for karyotype between passage 3 to 4. The number in brackets represents the number of the indicated karyotype.</p

Differential expression of PDGFRA in glioma subtypes and its association with patient survival time.

<p>In both GSE16011 (<b>A, B</b>) and Rembrandt (<b>C, D</b>) data sets, PDGFRA expression (Mean ± 2 SEM, SEM: standard error of the mean) was highly variable among the samples of each morphological subtype (<b>A</b> and <b>C.</b> NT: non-tumor; AII, astrocytoma grade II; AIII: astrocytoma grade III; GBM, glioblastoma; OD: oligodendroglioma; OA: oligoastrocytoma). Low-grade gliomas showed significantly higher levels of PDGFRA expression compared to high-grade gliomas (<b>B</b> and <b>D</b>). Kaplan-Meier plots showed that in both GSE16011 (<b>E</b>) and Rembrandt (<b>F</b>) data sets, the survival of patients with the upper 25% PDGFRA expression (PDGFRA-high) in glioma samples irrespective of their morphological diagnosis was significantly longer compared with those glioma patients with PDGFRA expression at the lowest 25% (PDGFRA-low) and the intermediate 50% (log-rank test).</p

Co-expression of neuronal and glial markers in high-grade glioma cells

<p>The GFAP and NSE staining was performed in the fixed glioma specimens from the same surgical procedures as those used for generating viable cells. The staining was semi-quantitatively evaluated. +++: markedly positive staining; ++: moderately positive staining; +: low positive; −: no positive staining in the tumor.</p

Expression of neural stem cell and glial progenitor surface markers in low- and high-grade glioma specimens analyzed.

<p>Freshly prepared or thawed glioma cells were first incubated with non-specific mouse IgG1 mAb, cells were subsequently incubated with APC conjugated anti-CD45 mAb in combination with FITC conjugated anti-CD44, or anti-CD24 mAbs or PE conjugated anti-PDGFRα, anti-EGFR, anti-CD133 mAbs. For A2B5 and O4 staining, cells were incubated with unconjugated A2B5 or O4 mAb respectively and subsequently stained with PE conjugated rat anti-mouse IgM. Cells negatively stained with 7-AAD were analyzed for cell surface marker expression. Data shown are the percentages of CD45 negative cells positively stained for indicated cell surface markers.</p>*<p>: secondary GBM. UD: undetectable; ND: not done. PNET: primitive neuroectodermal tumor.</p

Vessel or glioma origin of CD133 expressing cells.

<p>In contrast to high-grade glioma specimens, CD133 expressing cells detected in low-grade glioma specimens are predominantly derived from blood vessel endothelial cells. Dot-plot profiles of CD133 expression versus CD45 and/or CD31 expression of cells from glioma specimens of indicated patients are shown. The numbers in each quadrate represent the percentages of the cells stained positively or negatively by the respective antibodies.</p

Immunohistochemical staining results for PDGFRA and FGF2 in glioma samples - Predominant staining intensity in each sample.

<p><b>++:</b> Markedly positive staining. <b>+:</b> Low-moderately positive staining. <b>−:</b> No positive staining.</p

Enriched FGF2 expression in low-grade gliomas.

<p>The relative expression levels of FGF2 (Mean ± 2SEM) were analyzed in the GSE16011 (<b>A</b> and <b>B</b>) and the Rembrandt (<b>C</b> and <b>D</b>) data sets according to the morphological diagnosis of gliomas (<b>A</b> and <b>C</b>). Low-grade gliomas showed significantly higher levels of FGF2 expression compared to high-grade gliomas (<b>B</b> and <b>D</b>, <i>t</i> test).</p

Concordant patterns of FGF2 and PDGFRA immunohistochemical staining in glioma samples.

<p>Consecutive sections for each glioma samples were used for the staining of FGF2 and PDGFRA. Staining results of representative glioma samples are shown. In all samples, a correlation between the extent of FGF2 and PDGFRA staining was observed. The scale bars denote 20 µm.</p