Glial progenitor-like phenotype in low-grade glioma and enhanced CD133-expression and neuronal lineage differentiation potential in high-grade glioma

Abstract

Background: While neurosphere- as well as xenograft tumor-initiating cells have been identified in gliomas, the resemblance between glioma cells and neural stem/progenitor cells as well as the prognostic value of stem/progenitor cell marker expression in glioma are poorly clarified.

Methodology/principal findings: Viable glioma cells were characterized for surface marker expression along the glial genesis hierarchy. Six low-grade and 17 high-grade glioma specimens were flow-cytometrically analyzed for markers characteristics of stem cells (CD133); glial progenitors (PDGFRalpha, A2B5, O4, and CD44); and late oligodendrocyte progenitors (O1). In parallel, the expression of glial fibrillary acidic protein (GFAP), synaptophysin and neuron-specific enolase (NSE) was immunohistochemically analyzed in fixed tissue specimens. Irrespective of the grade and morphological diagnosis of gliomas, glioma cells concomitantly expressed PDGFRalpha, A2B5, O4, CD44 and GFAP. In contrast, O1 was weakly expressed in all low-grade and the majority of high-grade glioma specimens analyzed. Co-expression of neuronal markers was observed in all high-grade, but not low-grade, glioma specimens analyzed. The rare CD133 expressing cells in low-grade glioma specimens typically co-expressed vessel endothelial marker CD31. In contrast, distinct CD133 expression profiles in up to 90% of CD45-negative glioma cells were observed in 12 of the 17 high-grade glioma specimens and the majority of these CD133 expressing cells were CD31 negative. The CD133 expression correlates inversely with length of patient survival. Surprisingly, cytogenetic analysis showed that gliomas contained normal and abnormal cell karyotypes with hitherto indistinguishable phenotype.

Conclusions/significance: This study constitutes an important step towards clarification of lineage commitment and differentiation blockage of glioma cells. Our data suggest that glioma cells may resemble expansion of glial lineage progenitor cells with compromised differentiation capacity downstream of A2B5 and O4 expression. The concurrent expression of neuronal markers demonstrates that high-grade glioma cells are endowed with multi-lineage differentiation potential in vivo. Importantly, enhanced CD133 expression marks a poor prognosis in gliomas.

Figure 1. Low-grade glioma cells concurrently express multiple cell surface markers characteristic of adult human glial progenitors.

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.

Figure 2. Neuronal marker expression was not detected in low-grade gliomas, but in high-grade gliomas.

Staining patterns of GFAP and NSE expression from representative low-glioma (patient ID: #2) and high-grade glioma ((patient ID: #12 and #19) are shown. Original magnification: ×100

Figure 3. Maintenance of glial progenitor-like phenotype, but enhanced CD133 expression in high-grade gliomas.

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.

Figure 4. Vessel or glioma origin of CD133 expressing cells.

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.

Figure 5. Glioma cells are compromised in downstream differentiation at the O4 stage.

Glioma cells were stained with APC conjugated anti-CD45 and anti-CD31 mAbs in combination with staining for A2B5, or O4 or O1. Data shown are the ratio of the percentages between O4⁺/CD45⁻CD31⁻ and A2B5⁺/CD45⁻CD31⁻ phenotype or between O1⁺/CD45⁻CD31⁻ and A2B5⁺/CD45⁻CD31⁻ phenotype in each patient. The ependymoma patient was not included in this analysis.

Figure 6. CD133 expression correlates inversely with grade II to IV glioma patient survival time.

The survival time calculated from the day of operation was plotted against the percentage of CD133⁺ cells in the CD45⁻ cell fraction from the specimens of each patient. UD: undetectable CD133 expression. Bold black bars indicate the median survival time for patients in groups with CD133⁺ cells either lower or higher than 30% of total CD45⁻ cells.