Significance of Glioma Stem-Like Cells in the Tumor Periphery That Express High Levels of CD44 in Tumor Invasion, Early Progression, and Poor Prognosis in Glioblastoma - PubMed (original) (raw)

. 2018 Aug 23:2018:5387041.

doi: 10.1155/2018/5387041. eCollection 2018.

Akihiro Inoue 1, Takanori Ohnishi 2, Shohei Kohno 1, Shiro Ohue 3, Shirabe Matsumoto 1, Satoshi Suehiro 1, Daisuke Yamashita 1, Saya Ozaki 1, Hideaki Watanabe 1, Hajime Yano 4, Hisaaki Takahashi 5, Riko Kitazawa 6, Junya Tanaka 4, Takeharu Kunieda 1

Affiliations

Significance of Glioma Stem-Like Cells in the Tumor Periphery That Express High Levels of CD44 in Tumor Invasion, Early Progression, and Poor Prognosis in Glioblastoma

Masahiro Nishikawa et al. Stem Cells Int. 2018.

Abstract

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumor and a subpopulation of glioma stem-like cells (GSCs) is likely responsible for the invariable recurrence following maximum resection and chemoradiotherapy. As most GSCs that are located in the perivascular and perinecrotic niches should be removed during tumor resection, it is very important to know where surviving GSCs are localized. Here, we investigated the existence and functions of GSCs in the tumor periphery, which is considered to constitute the invasion niche for GSCs in GBM, by analyzing expression of stem cell markers and stem cell-related molecules and measuring particular activities of cultured GSCs. In addition, the relationship between GSCs expressing particular stem cell markers and pathological features on MRI and prognosis in GBM patients was analyzed. We showed that GSCs that express high levels of CD44 are present in the tumor periphery. We also found that vascular endothelial growth factor (VEGF) is characteristically expressed at a high level in the tumor periphery. Cultured GSCs obtained from the tumor periphery were highly invasive and have enhanced migration phenotype, both of which were markedly inhibited by CD44 knockdown. Higher expression of CD44 in the tumor periphery than in the core was correlated with a highly invasive feature on MRI and was associated with early tumor progression and worse survival, whereas lower expression of CD44 in the tumor periphery corresponded to low invasion and was associated with longer survival. The low invasion type on MRI tended to show high levels of VEGF expression in the tumor periphery, thus presenting the tumor with high proliferative activity. These results imply the significance of GSCs with high levels of CD44 expression in the tumor periphery compared to the core, not only in tumor invasion but also rapid tumor progression and short survival in patients with GBM.

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Figures

Figure 1

Figure 1

Navigation-guided surgical method for taking tumor samples from two sites, the periphery and core in GBM. The position of the tip of the fence-post catheters that are placed along the tumor border under echo-linked image-guided navigation is determined as a target for taking samples in the tumor periphery. The target area corresponds to just outside of the Gd-enhanced area and within the positive-uptake area on Met-PET. When the tumor showed a central necrosis, tumor tissues located outside the necrotic area were collected for the samples at the tumor core (left). Otherwise, the tumor samples at the core were obtained from the center of the resected tumor.

Figure 2

Figure 2

Expression of stem cell markers and stem cell-related molecules in the core and the periphery of the tumor in four glioblastoma (GBM) patients. (a) mRNA expression of eight stem cell markers (Nestin, CXCR4, Oct3/4, CD15, CD133, Bmi-1, Sox2, and CD44) and four stem cell-related molecules (HIF-1_α_, HIF-2_α_, VEGF, and TGF-β) in the core and the periphery was determined with qRT-PCR. The values are relative expression of mRNA of stem cell markers and molecules normalized to the GAPDH. (b) Expression of eight stem cell markers shown as the P/C expression ratio for each marker in each patient. The P/C ratios for the marker were calculated by dividing the amount of mRNA expression of the marker in the tumor periphery by that in the core for each patient. (c) Expression of four stem cell-related molecules shown as the P/C expression ratio for each molecule in each patient. The P/C ratios for the molecule were calculated as described for the stem cell markers. The numbers on the _x_-axis are the patient numbers.

Figure 3

Figure 3

Immunohistochemical expression of the stem cell markers CD44 and Nestin. (a) Immunostaining for CD44 in the core and the periphery of the tumor in four GBM patients. The number of tumor cells showing positive staining for CD44 in the periphery was much higher than that in the core in tumors with a high P/C ratio for CD44 expression (patient numbers 1 and 4) (scale bar: 100 _μ_m) (×400). (b) Double-labeling immunofluorescence. CD44 (green) and Nestin (red) were colocalized (M: merge) at the cell membrane in some GBM cells in the tumor periphery. Nuclei were labeled with Hoechst (blue).

Figure 4

Figure 4

Characterization of sphere-forming cells (SFCs) as glioma stem-like cells. (a) Both SFC-1 and SFC-2 expressed much more CD133 and Nestin mRNA than those parent cells (PC-1 and PC-2). SFC-1 expressed CD44 at a significantly higher level than SFC-2. ∗ p < 0.01. (b) CD44 (green) and Nestin (red) were colocalized at the cell membrane in both SFC-1 and SFC-2. (c) Both SFC-1 and SFC-2 showed the capacity for multilineage differentiation into neurons (TUJ1; green) and astrocytes (GFAP; red). The nuclei are labeled with Hoechst (blue). (d) Both SFCs transplanted in the mouse brain generated invasive tumors (HE × 400), in which tumor cells in the periphery were positively stained for CD44 (bar 100 _μ_m) (×400).

Figure 5

Figure 5

Invasive and migratory activities of cultured SFCs and PCs and inhibitory effect of CD44 knockdown with CD44 siRNA on their activities. In both invasion (a) and migration (b), cultured SFCs (SFC-1 and SFC-2) showed much higher activity than each PCs (PC-1 and PC-2). These high activities of invasion and migration of SFCs were significantly inhibited by knockdown of CD44 with the siRNA. ∗ p < 0.001 and ∗∗ p < 0.005.

Figure 6

Figure 6

Expression of the stem cell marker, CD44, and the stem cell-related molecule, VEGF, in 13 GBM patients. (a) mRNA expression of CD44 in the tumor core and periphery was determined with qRT-PCR. The values are relative expression of mRNA normalized to the GAPDH. (b) Expression of CD44 represented by the P/C ratio. The P/C ratios for CD44 were calculated by dividing the amount of mRNA expression of CD44 in the tumor periphery by that in the core for each patient. (c) mRNA expression of VEGF in the tumor core and periphery. The expression and the level were determined as described for CD44. (d) Expression of VEGF represented by the P/C ratio. The P/C ratios for VEGF were determined as described for CD44.

Figure 7

Figure 7

Representative combined images of MRI and PET-computed tomography for four groups of features with different intensities representing invasion and proliferation in the tumor periphery. Inv: invasion, prol: proliferation, CN: central necrosis. In all images, the upper left is T1-Gd, the upper right is a fluid-attenuated inversion recovery image, and the lower image is Met-PET. Values at the bottom are the mean ± SD of the P/C ratio for CD44 expression (upper row) and the Ki-67 SI in the tumor periphery (lower row). n: number of patients.

Figure 8

Figure 8

(a) Relationship between the P/C ratio for CD44 expression and tumor invasiveness as seen on MRI. Patients with a high P/C ratio showed a significant, more highly invasive type on MRI than those with a low P/C ratio (p = 0.0441, _t_-test). (b) Relationship between the level of CD44 mRNA expression in the tumor periphery and tumor invasiveness as seen on MRI. The level of CD44 mRNA expression in the tumor periphery did not correlate with invasiveness on MRI (p = 0.285, _t_-test). (c) Relationship between the level of VEGF mRNA expression in the tumor periphery and tumor invasiveness as seen on MRI. The low invasive type on MRI tended to have higher amounts of VEGF expression in the tumor periphery (p = 0.201, _t_-test). (d, e) Kaplan-Meier survival curves for the P/C ratio for CD44 expression predicting PFS (d) and OS (e). Patients with a high P/C ratio for CD44 expression showed significantly earlier tumor progression and worse survival than those with a low P/C ratio (p = 0.0359 and 0.0215, respectively, log-rank test).

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