Xiao-hong Yao

Isolation and characterization of cancer stem cells from a human glioblastoma cell line U87

A variety of malignant cancers have been found to contain a subpopulation of stem cell-like tumor cells, or cancer stem cells (CSCs). However, the existence of CSCs in U87, a most commonly used glioma cell line, is still controversial. In this study, we demonstrate that U87 cell line contained a fraction of tumor cells that could form tumor spheres and were enriched by progressively increasing the concentration of serum-free neural stem cell medium with or without low dose vincristine. These cells possessed the ability of self-renewal and multipotency, the defined characteristics of CSCs. Moreover, the tumors formed by the secondary spheres displayed typical histological features of human glioblastoma, including cellular pleomorphism, pseudopalisades surrounding necrosis, hyperchromatic nuclei, high density of microvessels and invasion to the brain parenchyma. These results indicate that gradually increasing the concentration of serum-free neural stem cell culture medium with or without vincristine is a simple and effective method for isolation of CSCs to study the initiation and progression of human glioblastoma.

The chemokine CXCL12 and its receptor CXCR4 promote glioma stem cell‐mediated VEGF production and tumour angiogenesis via PI3K/AKT signalling

Chemokines and their receptors are actively involved in inflammation, immune responses, and cancer development. Here we report the detection of CD133+ glioma stem‐like cells (GSCs) co‐expressing a chemokine receptor CXCR4 in human primary glioma tissues. These GSCs were located in areas adjacent to tumour vascular capillaries, suggesting an association between GSCs and tumour angiogenesis. To test this hypothesis, we isolated CD133+ GSCs from surgical specimens of human primary gliomas and glioma cell lines. As compared to CD133− cells, CD133+ GSCs expressed significantly higher levels of CXCR4 mRNA and protein, and migrated more efficiently in response to the CXCR4 ligand CXCL12. In addition, CXCL12 induced vascular endothelial growth factor (VEGF) production by CD133+ GSCs via activation of the PI3K/AKT signalling pathway. Furthermore, knocking down of CXCR4 using RNA interference or inhibition of CXCR4 function by an antagonist AMD3100 not only reduced VEGF production by CD133+ GSCs in vitro, but also attenuated the growth and angiogenesis of tumour xenografts in vivo formed by CD133+ GSCs in SCID mice. These results indicate that CXCL12 and its receptor CXCR4 promote GSC‐initiated glioma growth and angiogenesis by stimulating VEGF production. Copyright

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

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. 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.

Glioblastoma stem cells produce vascular endothelial growth factor by activation of a G-protein coupled formylpeptide receptor FPR.

Glioma stem cells (GSCs), or stem cell‐like glioma cells, isolated from malignant glioma cell lines, were capable of producing vascular endothelial growth factor (VEGF). However, the exact role of such tumour cells in angiogenesis remains unknown. In this study, we isolated a small proportion of CD133+ GSCs from the human glioblastoma cell line U87 and found that these GSCs possessed multipotent differentiation potential and released high levels of VEGF as compared with CD133− tumour cells. The CD133+ GSCs also formed larger xenograft tumours that contained higher VEGF immunoreactivity and denser microvessels. Moreover, GSCs expressed a functional G protein‐coupled formylpeptide receptor FPR, which was activated by a chemotactic peptide ligand, N‐formylmethionyl‐leucyl‐phenylalanine (fMLF), to mediate calcium flux and the production of VEGF by GSCs. Our results indicate that FPR expressed by human GSCs may play an important role in glioma angiogenesis. Copyright

Isolation and characterization of stem cell-like precursor cells from primary human anaplastic oligoastrocytoma

A small population of stem cell-like precursors in solid tumors are linked to histological composition, progression, angiogenesis, metastasis, recurrence and drug resistance of a variety of malignant tumors. Oligoastrocytoma is the most common brain mixed glioma composed of mixed cells of oligodendroglial and astrocytic phenotypes. Identification and characterization of stem cell-like precursors in oligoastrocytoma may shed light on the oncogenesis of this unique type of tumor and assist in the design of novel therapeutic strategy. Here, tumor stem cell-like precursors were identified from primary human anaplastic oligoastrocytomas by labeling of the tumor sections with nestin and CD133. Tumor cells were cultured in vitro in stem cell medium with growth factors and the capacity of the surviving stem cell-like precursors to form tumor spheres was tested. The tumor spheres were further injected subcutaneously into nude mice to observe the contribution of stem cell-like precursors to histological composition and tumor progression. We found that primary human oligoastrocytoma tissues contained nestin+/CD133+ stem cell-like precursors. These cells differentiated into tumor cells with both oligodendroglial and astrocytic characteristics and formed tumor spheres in vitro, which upon implantation in nude mice, grew into tumor nodules containing nestin +/CD133+ cells at levels higher than in the primary tumor tissues. This study revealed for the first time that anaplastic human oligoastrocytomas contained stem cell-like precursors, which exhibit neural stem cell properties with tumorigenicity. These stem cell-like precursors may be responsible for the oligodendroglial and astrocytic components of human oligoastrocytoma and should be considered as therapeutic targets.

Connexin 43 Reverses Malignant Phenotypes of Glioma Stem Cells by Modulating E-Cadherin†‡§

Malfunctioned gap junctional intercellular communication (GJIC) has been thought associated with malignant transformation of normal cells. However, the role of GJIC‐related proteins such as connexins in sustaining the malignant behavior of cancer stem cells remains unclear. In this study, we obtained tumorspheres formed by glioma stem cells (GSCs) and adherent GSCs and then examined their GJIC. All GSCs showed reduced GJIC, and differentiated glioma cells had more gap junction‐like structures than GSCs. GSCs expressed very low level of connexins, Cx43 in particular, which are key components of gap junction. We observed hypermethylation in the promoter of gap junction protein α1, which encodes Cx43 in GSCs. Reconstitution of Cx43 in GSCs inhibited their capacity of self‐renewal, invasiveness, and tumorigenicity via influencing E‐cadherin and its coding protein, which leads to changes in the expression of Wnt/β‐catenin targeting genes. Our results suggest that GSCs require the low expression of Cx43 for maintaining their malignant phenotype, and upregulation of Cx43 might be a potential strategy for treatment of malignant glioma. STEM CELLS 2012; 30:108‐120.

Contribution of cancer stem cells to tumor vasculogenic mimicry

Vasculogenic mimicry (VM), a newly-defined pattern of tumor blood supply, provides a special passage without endothelial cells and is conspicuously different from angiogenesis and vasculogenesis. The biological features of the tumor cells that form VM remain unknown. Cancer stem cells (CSCs) are believed to be tumor-initiating cells, capable of self-renewal and multipotent differentiation, which resemble normal stem cells in phenotype and function. Recently CSCs have been shown to contribute to VM formation as well as angiogenesis. These findings challenge the previous understanding of the cellular basis of VM formation. In this review, we present evidence for participation of CSCs in VM formation. We also discuss the potential mechanisms and possible interaction of CSCs with various elements in tumor microenvironment niche. Based on the importance of VM in tumor progression, it constitutes a novel therapeutic target for cancer.

The active contribution of Toll-like receptors to allergic airway inflammation

Epithelia lining the respiratory tract represent a major portal of entry for microorganisms and allergens and are equipped with innate and adaptive immune signaling receptors for host protection. These include Toll-like receptors (TLRs) that recognize microbial components and evoke diverse responses in cells of the respiratory system. TLR stimulation by microorganism-derived molecules activates antigen presenting cells, control T helper (Th) 1, Th2, and Th17 immune cell differentiation, cytokine production by mast cells, and activation of eosinophils. It is clear that TLR are involved in the pathophysiology of allergic airway diseases such as asthma. Dendritic cells (DCs), a kind of antigen presenting cells, which play a key role in the induction of allergic airway inflammation, are privileged targets for pathogen associated molecular patterns (PAMPs). During the allergic responses, engagement of TLRs on DCs determines the Th2 polarization of the T cells. TLR signaling in mast cells increases the release of IL-5, and TLR activation of airway epithelial cells forces the generation of proallergic Th2 type of cytokines. Although these responses aim to protect the host, they may also result in inflammatory tissue damage in the airway. Under certain conditions, stimulation of TLRs, in particular, TLR9, may reduce Th2-dependent allergic inflammation by induction of Th1 responses. Therefore, understanding the complex regulatory roles of TLRs in the pathogenesis of allergic airway inflammation should facilitate the development of preventive and therapeutic measures for asthmatic patients.

Production of angiogenic factors by human glioblastoma cells following activation of the G-protein coupled formylpeptide receptor FPR

Activation of the formylpeptide receptor (FPR), a G-protein-coupled receptor, by its chemotactic peptide ligand N-formylmethionyl-leucyl-phenylalanine (fMLF) promotes the directional migration and survival of human glioblastoma cells. fMLF also stimulates glioblastoma cells to produce biologically active VEGF, an important angiogenic factor involved in tumor progression. In this study, we examined the capacity of FPR to regulate the production of another angiogenic factor, the chemokine IL-8 (CXCL8), in addition to its demonstrated ability to induce VEGF secretion by malignant glioma cells. We showed that the human glioblastoma cell line U87 secreted considerable levels of IL-8 (CXCL8) upon stimulation by the FPR agonist peptide fMLF. Tumor cells transfected with small interference (si)RNA targeting FPR failed to produce IL-8 as well as VEGF in response to fMLF. Glioblastoma cells bearing FPR siRNA exhibited reduced rate of tumorigenicity in nude mice and tumors formed by such tumor cells showed less active angiogenesis and lower level expression of both IL-8 and VEGF. These results suggest that FPR plays an important role in the angiogenesis of human malignant gliomas through increasing the production of angiogenic factors by FPR positive tumor cells.

Endothelial cells promote stem-like phenotype of glioma cells through activating the Hedgehog pathway

Microenvironmental regulation of cancer stem cells (CSCs) strongly influences the onset and spread of cancer. The way in which glioma cells interact with their microenvironment and acquire the phenotypes of CSCs remains elusive. We investigated how communication between vascular endothelial cells and glioma cells promoted the properties of glioma stem cells (GSCs). We observed that CD133+ GSCs were located closely to Shh+ endothelial cells in specimens of human glioblastoma multiforme (GBM). In both in vitro and in vivo studies, we found that endothelial cells promoted the appearance of CSC‐like glioma cells, as demonstrated by increases in tumourigenicity and expression of stemness genes such as Sox2, Olig2, Bmi1 and CD133 in glioma cells that were co‐cultured with endothelial cells. Knockdown of Smo in glioma cells led to a significant reduction of their CSC‐like phenotype formation in vitro and in vivo. Endothelial cells with Shh knockdown failed to promote Hedgehog (HH) pathway activation and CSC‐like phenotype formation in co‐cultured glioma cells. By examination of glioma tissue specimens from 65 patients, we found that the survival of glioma patients was closely correlated with the expression of both Shh by endothelial cells and Gli1 by perivascular glioma cells. Taken together, our study demonstrates that endothelial cells in the tumour microenvironment provide Shh to activate the HH signalling pathway in glioma cells, thereby promoting GSC properties and glioma propagation.