ORIGINAL PAPER Journal of Pathology J Pathol 2011; 224: 344–354 Published online 27 May 2011 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/path.2908 The chemokine CXCL12 and its receptor CXCR4 promote glioma stem cell-mediated VEGF production and tumour angiogenesis via PI3K/AKT signalling Yi-fang Ping, 1 Xiao-hong Yao, 1 Jian-yong Jiang, 1 Lin-tao Zhao, 1 Shi-cang Yu, 1 Tao Jiang, 2 Marie CM Lin, 1,3 Jian-hong Chen, 1 Bin Wang, 1 Rong Zhang, 1 You-hong Cui, 1 Cheng Qian, 1 Ji Ming Wang 4 and Xiu-wu Bian 1 * 1 Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing 400038, China 2 Department of Neurosurgery, Tiantan Hospital, Beijing Capital Medical University, Beijing 100050, China 3 Brain Tumour Centre, Division of Neurosurgery, Deparment of Surgery, PWH, The Chinese University of Hong Kong, Shatin, Hong Kong, China 4 Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD 21702, USA *Correspondence to: Xiu-wu Bian, Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing 400038, China. e-mail: bianxiuwu@263.net Abstract 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  2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Keywords: glioma; cancer stem cells; angiogenesis; CXCR4; cell signalling Received 6 May 2010; Revised 19 January 2011; Accepted 21 March 2011 No conflicts of interest were declared. Introduction Cancer stem cells (CSCs) from various solid tumours including brain tumours have been identified [1–8]. Although CSCs account for only a small fraction of total cancer cells, due to their ability of self- renewal/tumour initiation and their resistance to che- motherapy/radiotherapy [9–11], they are considered to be the most important cells responsible for can- cer recurrence and metastasis, hence the critical tar- gets for cancer therapy. We have previously isolated glioma stem-like cells (GSCs) from a human glioblas- toma (GBM) cell line, U87, using the stem cell marker CD133 and demonstrated that these CD133 + cells are capable of self-renewal, forming neurosphere-like spheroids, and generating copies of tumour cells from which they derived [12,13]. CSCs have also been shown to promote tumour angiogenesis by secret- ing high levels of vascular endothelial growth factor (VEGF) [12–16]. Angiogenesis is crucial for the pro- gression of an established tumour. However, the clin- ical relevance, mechanisms, and signal transduction pathways for human CSC-mediated VEGF production and tumour angiogenesis remain elusive. Tumour cells express increased levels of cell surface receptors for their growth advantage. These receptors include growth factor receptors such as EGFR and FGFR. In addition, chemokine receptors have been implicated in promoting tumour cell migration, growth, and metastasis [17,18]. We and others have previously reported elevated expression of a chemokine receptor, CXCR4, in glioma cells [19–21]. CXCR4 expression correlated directly with the degree of malignancy of the human glioma cell lines and primary tumours [19]. We have also found that GSCs express CXCR4 and pro- duce the angiogenic factor VEGF [13]. However, the precise role of CXCL12/CXCR4 in glioma progression, Copyright  2011 Pathological Society of Great Britain and Ireland. J Pathol 2011; 224: 344–354 Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com