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. 2012 Jun;11(6):M111.010744.
doi: 10.1074/mcp.M111.010744. Epub 2011 Dec 27.

CD90 is identified as a candidate marker for cancer stem cells in primary high-grade gliomas using tissue microarrays

Jintang He  1 , Yashu Liu, Thant Zhu, Jianhui Zhu, Francesco Dimeco, Angelo L Vescovi, Jason A Heth, Karin M Muraszko, Xing Fan, David M Lubman
Affiliations

CD90 is identified as a candidate marker for cancer stem cells in primary high-grade gliomas using tissue microarrays

Jintang He et al. Mol Cell Proteomics. 2012 Jun.

Abstract

Although CD90 has been identified as a marker for various kinds of stem cells including liver cancer stem cells (CSCs) that are responsible for tumorigenesis, the potential role of CD90 as a marker for CSCs in gliomas has not been characterized. To address the issue, we investigated the expression of CD90 in tissue microarrays containing 15 glioblastoma multiformes (GBMs), 19 WHO grade III astrocytomas, 13 WHO grade II astrocytomas, 3 WHO grade I astrocytomas and 8 normal brain tissues. Immunohistochemical analysis showed that CD90 was expressed at a medium to high level in all tested high-grade gliomas (grade III and GBM) whereas it was barely detectable in low-grade gliomas (grade I and grade II) and normal brains. Double immunofluorescence staining for CD90 and CD133 in GBM tissues revealed that CD133(+) CSCs are a subpopulation of CD90(+) cells in GBMs in vivo. Flow cytometry analysis of the expression of CD90 and CD133 in GBM-derived stem-like neurospheres further confirmed the conclusion in vitro. The expression levels of both CD90 and CD133 were reduced along with the loss of stem cells after differentiation. Furthermore, the limiting dilution assay demonstrated that the sphere formation ability was comparable between the CD90(+)/CD133(+) and the CD90(+)/CD133(-) populations of GBM neurospheres, which is much higher than that of the CD90(-)/CD133(-) population. We also performed double staining for CD90 and a vascular endothelial cell marker CD31 in tissue microarrays which revealed that the CD90(+) cells were clustered around the tumor vasculatures in high-grade glioma tissues. These findings suggest that CD90 is not only a potential prognostic marker for high-grade gliomas but also a marker for CSCs within gliomas, and it resides within endothelial niche and may also play a critical role in the generation of tumor vasculatures via differentiation into endothelial cells.

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Figures

Fig. 1.
Fig. 1.
Immunohistochemical staining for human CD90 in different grades of astrocytomas and normal brains. Paraffin-embedded tissue microarrays were stained using an anti-human CD90 antibody. CD90 was not detected in normal brain (A), astrocytoma WHO grade I (B) and diffuse astrocytoma WHO grade II (C). In contrast, moderate or strong staining for CD90 was observed in anaplastic astrocytoma WHO grade III (D) and GBM WHO grade IV (E). Hematoxylin counterstain was used to visualize nuclei. Scale bar, 100 μm.
Fig. 2.
Fig. 2.
Scatter plot of CD90 expression levels in n = 50 patients with different grades of astrocytomas and n = 8 normal subjects. The CD90 expression level of each tissue sample was represented by the percentage of CD90+ cells. Each point corresponds to the CD90 expression level of a single sample. CD90 expression levels were significantly elevated in patients with high-grade astrocytomas (WHO grade III and IV). Astro 1 represents astrocytoma WHO grade I; Astro 2 represents diffuse astrocytoma (WHO grade II); Astro 3 represents anaplastic astrocytoma (WHO grade III); GBM represents glioblastoma multiforme (WHO grade IV).
Fig. 3.
Fig. 3.
Double immunofluorescence staining for CD90 and CD133 in a GBM section. CD90+ cells contain a subpopulation of CD133+ cells, where the CD90+CD133+ cells are shown in yellow in the merged image. DAPI counterstain was used to visualize nuclei. As a negative control, a WHO grade II astrocytoma was double stained for CD90 and CD133 (data shown in supplemental Fig. S2). Scale bar, 100 μm.
Fig. 4.
Fig. 4.
Flow cytometry analysis of the expression of CD90 and CD133 in the stem-like neurosphere HSR-GBM1. Flow cytometry histograms showed more than 99% of HSR-GBM1 cells were CD90-positive (A) and about 80% were CD133 positive (B). C, In the flow cytometry dot plot, the HSR-GBM1 cells were classified into four cell populations based on the expression of CD90 and CD133. The percentage of each subpopulation was shown. FITC, fluorescein isothiocyanate; PE, phycoerythrin.
Fig. 5.
Fig. 5.
CD90 is a marker for GBM stem cells. A, Differential expression of CD90 and CD133 along with the differentiation of the stem-like HSR-GBM1 neurospheres. When the stem-like neurospheres were forced to differentiate, both CD90 and CD133 expression levels were dramatically reduced. Stem-like represents the stem-like HSR-GBM1 neurospheres, which are undifferentiated cells; Diff represents the differentiated HSR-GBM1 cells. B, Populations of unsorted, CD90+/CD133+, CD90+/CD133−, and CD90−/CD133− HSR-GBM1 neurospheres were analyzed for their ability of sphere formation by the limiting dilution assay. y-axis: percent of wells that did not form spheres; x-axis: the number of cells plated per well. C, The minimum number of cells required to form spheres for each population of HSR-GBM1 neurospheres. The number was similar between the unsorted, CD90+/CD133+ and CD90+/CD133− populations. Compared with these populations, the CD90−/CD133− population required the most number of cells (more than twofold) to form spheres.
Fig. 6.
Fig. 6.
Double immunofluorescence staining for CD90 and CD31 in GBM. A GBM section was stained with anti-CD90 (A) and anti-CD31 (B). DAPI counterstain (C) was used to visualize nuclei. Colocalization of CD90 and CD31 was observed in the merged image (D). An area of this merged image was selected and a higher magnification image of the area was also shown (E). Scale bar in A–D, 100 μm; Scale bar in E, 50 μm.
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