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. 2013;8(3):e57188.
doi: 10.1371/journal.pone.0057188. Epub 2013 Mar 11.

"VSports注册入口" Vascular endothelial growth factor receptor 2 (VEGFR-2) plays a key role in vasculogenic mimicry formation, neovascularization and tumor initiation by Glioma stem-like cells

Xiaohong Yao  1 , Yifang Ping, Ying Liu, Kequiang Chen, Teizo Yoshimura, Mingyong Liu, Wanghua Gong, Chong Chen, Qin Niu, Deyu Guo, Xia Zhang, Ji Ming Wang, Xiuwu Bian
Affiliations

Vascular endothelial growth factor receptor 2 (VEGFR-2) plays a key role in vasculogenic mimicry formation, neovascularization and tumor initiation by Glioma stem-like cells

Xiaohong Yao et al. PLoS One. 2013.

Erratum in

  • PLoS One. 2013;8(12). doi:10.1371/annotation/aed5b555-b826-4591-8aa6-284ad888627d

Abstract

Human glioblastomas (GBM) are thought to be initiated by glioma stem-like cells (GSLCs). GSLCs also participate in tumor neovascularization by transdifferentiating into vascular endothelial cells. Here, we report a critical role of GSLCs in the formation of vasculogenic mimicry (VM), which defines channels lined by tumor cells to supply nutrients to early growing tumors and tumor initiation. GSLCs preferentially expressed vascular endothelial growth factor receptor-2 (VEGFR-2) that upon activation by VEGF, mediated chemotaxis, tubule formation and increased expression of critical VM markers by GSLCs. Knockdown of VEGFR-2 in GSLCs by shRNA markedly reduced their capacity of self-renewal, forming tubules, initiating xenograft tumors, promoting vascularization and the establishment of VM VSports手机版. Our study demonstrates VEGFR-2 as an essential molecule to sustain the "stemness" of GSLCs, their capacity to initiate tumor vasculature, and direct initiation of tumor. .

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Vasculogenesis and VM in xenograft tumors derived from GSLCs and primary human glioma.
(A) ECs detected with anti-human and anti-mouse CD31 antibodies in the tumors formed by U87 GBM cells (top) and GSLCs (bottom). Right panel shows quantification of human (h) and mouse (m) CD31+ cells. * Indicates statistically significant differences between tumors formed by U87 parent cells and GSLCs (* p<0.01). Nuclei were counterstained by DAPI (blue). Scale bar = 50 µm. (B) Double-immunofluorescence staining to detect human CD31+ (red) and GFAP+ (green) (top) as well as human GFAP (red) and laminin B2 (green) (bottom) in GSLC-derived xenograft tumors. Nuclei were counterstained by DAPI (blue). Scale bar = 20 µm. (C) PAS: Vascular basement membrane stained by PAS in xenograft tumor sections. Black arrow indicates blood vessel lined by ECs. Red arrow indicates VM lined by glioma cells with mitosis. Scale bar = 50 µm. IHC: Double positive staining of nestin and PAS formed tubule including red cells in GSLC xenograft tumor section, but not in U87 xenograft tumor section. Black arrow indicates blood vessel lined by ECs which were PAS-positive reaction only in the basement membrane. Red arrow indicates VM lined by glioma cells positive in nestin. Scale bar = 50 µm. TEM: Transmission electron microscopy of VM in GSLC initiated xenograft tumors. A vascular channel is lined by a thin basal lamina (red arrow) corresponding to the walls of the channel seen by conventional light microscopy. No endothelial cells line the tubule as compared to xenograft tumor formed by U87 cells (upper panel). Scale bar = 5 µm. (D) ECs in a human GBM section detected by anti-CD34 (black color). Vascular basement membrane with PAS staining (purple magenta) and tumor cells are labeled with anti-nestin antibody (brown color). a, Black arrows show tubular blood vessels stained with PAS. b, Double-staining of CD34 and PAS shows vessels (red arrows); PAS-positive tubules are lined by CD34− cells (black arrow). c, Tumor vessels containing red blood cells are positive for nestin but negative for CD34 (red arrow). Black arrow shows CD34+ blood vessels. d, PAS positive tubules containing red blood cells are lined by nestin-positive cells on the luminal surface (black arrow). Scale bar = 50 µm. (E) Tumor cell-lined vessels (red arrow) and EC-lined vessels (black arrow) are detected in the same tubule in a human GBM specimen. Scale bar = 20 µm.
Figure 2
Figure 2. Preferential expression of VEGFR-2 by GSLCs isolated from U87 GBM cells.
(A) The expression of mRNAs for VEGFR1, VEGFR-2, VE-cadherin, EphA2, and laminin 5γ2 in GSLCs and U87 GBM cells was measured by RT-PCR or real-time RT-PCR. * Indicates significantly increased expression by GSLCs. (B) Western blot of VEGFR-2 (230 and 200 KDa) in GSLCs and U87 GBM cells. β-actin was used as an internal control. (C, D) The effect of VEGF on the expression of mRNAs for VEGFR-2 (C) and VE-cadherin (D) in GSLCs or U87 GBM cells was measured by real-time RT-PCR. * Indicates significantly increased expression of genes compared to U87 cells or by VEGF treated cells (* P<0.01). (E) HE staining of human glioma specimens. IV: WHO Grade IV; III: WHO Grade III; II: WHO Grade II. Co-expression of CD133 (red) and VEGFR-2 (green) by human Grade IV GBM, Grade III anaplastic astrocytoma and Grade II astrocytoma sections is shown. Scale bar = 50 µm.
Figure 3
Figure 3. The function of VEGFR-2 in GSLCs.
(A) VEGF-induced chemotactic of GSLCs (left). * Indicates significantly increased response shown by GSLCs compared to U87 parental cells (p<0.05). A VEGFR-2 neutralizing mAb blocked GSLC response to VEGF (right). Control mAB: ctrl mAb. * Indicates significantly reduced cell response in the presence of anti-VEGFR-2 (p<0.05). (B) Tubule formation by GSLCs treated by VEGF. B, Left: Vessel formation by GSLCs; Right: Quantitative analysis of tubule formation by GSLCs. Data represent the mean ± SEM in triplicates. * Indicates significantly increased tubule formation by GSLCs compared to U87 cells and cells treated with VEGF versus untreated cells (p<0.05). (C) The effect of anti-VEGFR-2 mAb on tubule formation by GSLCs (left). The black ball indicates sphere. Right: Quantitation of tubules. * Indicates significantly reduced tubule formation by GSLCs treated with anti-VEGFR-2 mAb compared to control cells (p<0.05). (D) VEGFR-2 expression on the tubules formed by VEGF-treated GSLCs.
Figure 4
Figure 4. VEGF-induced phosphorylation of VEGFR-2, ERK, p38 and PI3K in GSLCs.
(A) IF images of phosphorylation on Y1175 in VEGFR-2 in GSLCs stimulated by VEGF (10 ng/ml) for different minutes (min). Scale bar = 20 µm. (B–D) Western blot of ERK, p38 and AKT phosphorylation in GSLCs treated with VEGF (10 ng/ml) for different times (min). (E) IF double-staining of VEGFR-2 and VEGF in GSLC-initiated xenograft tumors. Scale bar = 20 µm. VEGFR-2 (red), VEGF (green).
Figure 5
Figure 5. The effect of VEGFR-2 shRNA on the self-renewal of GSLCs and their formation of tubules.
(A) Formation of spheres by GSLCs with VEGFR-2 shRNA. * Indicates significantly reduced sphere formation by U87 cells containing VEGFR-2 shRNA (p<0.05). (B) VEGFR-2 knockdown by shRNA in GSLCs. RT-PCR analysis of VEGFR-2 mRNA (top) and Western blot of VEGFR-2 (bottom). (C) IF images of spheres formed by VEGFR-2 knockdown GSLCs (upper) or by differentiated VEGFR-2 knockdown GSLCs (lower). IF staining of CD133 (red) and Oct4 (yellow) on spheres is shown in the upper panels. IF staining of GFAP (green) and MAP-2 (red) on differentiated cells is shown in lower panels. (D) RT-PCR of mRNA for VE-cadherin in GSLCs with VEGFR-2 shRNA. * Indicates significantly reduced mRNA in VEGFR-2 knockdown GSLCs compared to Mock shRNA cells (p<0.05). (E) Tubule formation on Matrigel by GSLCs with VEGFR-2 shRNA in the presence or absence of VEGF. Spheres are indicated by white arrows; tubules are indicated by red arrows. Images were taken under light microscopy (×200). (F) Quantitative analysis of tubule formation by VEGFR-2 knockdown GSLCs. * Indicates significantly increased tubule formation by GSLCs containing Mock shRNA in response to VEGF (10 ng/ml) (* p<0.05). # Indicates significantly reduced tubule formation by GSLCs containing VEGFR-2 shRNA.
Figure 6
Figure 6. The effect of VEGFR-2 shRNA on tumorigenesis, angiogenesis, self-renewal and VM formation by GSLCs.
(A) The growth of xenograft tumors initiated by GSLCs with or without VEGFR-2 shRNA. * Indicates significantly reduced growth of tumors formed by GSLCs with VEGFR-2 shRNA (p<0.05). (B) Survival of mice with xenograft tumors formed by GSLCs with or without VEGFR-2 shRNA. * Indicates significantly prolonged survival of mice bearing tumors formed by VEGFR-2 containing GSLCs (p<0.05). (C) IF images of murine or human CD31 (red) in the xenograft tumors formed by GSLCs with or without VEGFR-2 shRNA. Nuclei were stained with DAPI (blue). Scale bar = 50 µm. (D) Self-renewal of GSLCs with VEGFR-2 shRNA. IF staining of CD133 (red) in the xenograft tumors derived from GSLCs with or without VEGFR-2 shRNA. Nuclei were stained with DAPI (blue). Scale bar = 50 µm. * Indicates significantly decreased number of CD133-positive cells in mice bearing tumors formed by VEGFR-2 knock-down GSLCs (p<0.05). (E) VM formation by GSLCs with or without VEGFR-2 shRNA. IF staining of human LamininB2 (red) or human GFAP (green) in the xenograft tumors derived from GSLCs with or without VEGFR-2 shRNA. Nuclei were counterstained with DAPI (blue). Scale bar = 20 µm. Quantitative image analysis of laminin VM immunoreactivity for glioma derived from Mock or VEGFR-2 shRNA-transfected GSLCs xenografts (n = 6 recipient mice per experimental group). Y-axis, percentage of area with reactivity (mean ±SE, * P<0.01).
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