CXCL12 Mediates Trophic Interactions between Endothelial and Tumor Cells in Glioblastoma Shyam Rao1., Rajarshi Sengupta2., Eun Joo Choe2, B. Mark Woerner2, Erin Jackson3,4, Tao Sun2, Jeffrey Leonard5, David Piwnica-Worms3,4,6,7, Joshua B. Rubin2,8* 1 Department of Radiation Oncology, Washington University School of Medicine, St. Louis, Missouri, United States of America, 2 Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, United States of America, 3 Bridging Research with Imaging, Genomics and High-Throughput Institute, Washington University School of Medicine, St. Louis, Missouri, United States of America, 4 Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, Missouri, United States of America, 5 Department of Neurosurgery, Washington University School of Medicine, St. Louis, Missouri, United States of America, 6 Department of Cell Biology & Physiology, Washington University School of Medicine, St. Louis, Missouri, United States of America, 7 Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri, United States of America, 8 Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri, United States of America Abstract Emerging evidence suggests endothelial cells (EC) play a critical role in promoting Glioblastoma multiforme (GBM) cell proliferation and resistance to therapy. The molecular basis for GBM-EC interactions is incompletely understood. We hypothesized that the chemokine CXCL12 and its receptor CXCR4 could mediate direct interactions between GBM cells and tumor-associated endothelial cells and that disruption of this interaction might be the molecular basis for the anti-tumor effects of CXCR4 antagonists. We investigated this possibility in vivo and in an in vitro co-culture model that incorporated extracellular matrix, primary human brain microvascular ECs (HBMECs) and either an established GBM cell line or primary GBM specimens. Depletion of CXCR4 in U87 GBM cells blocked their growth as intracranial xenografts indicating that tumor cell CXCR4 is required for tumor growth in vivo. In vitro, co-culture of either U87 cells or primary GBM cells with HBMECs resulted in their co-localization and enhanced GBM cell growth. Genetic manipulation of CXCL12 expression and pharmacological inhibition of its receptors CXCR4 and CXCR7 revealed that the localizing and trophic effects of endothelial cells on GBM cells were dependent upon CXCL12 and CXCR4. These findings indicate that the CXCL12/CXCR4 pathway directly mediates endothelial cell trophic function in GBMs and that inhibition of CXCL12-CXCR4 signaling may uniquely target this activity. Therapeutic disruption of endothelial cell trophic functions could complement the structural disruption of anti-angiogenic regimens and, in combination, might also improve the efficacy of radiation and chemotherapy in treating GBMs. Citation: Rao S, Sengupta R, Choe EJ, Woerner BM, Jackson E, et al. (2012) CXCL12 Mediates Trophic Interactions between Endothelial and Tumor Cells in Glioblastoma. PLoS ONE 7(3): e33005. doi:10.1371/journal.pone.0033005 Editor: Shu-ichi Okamoto, Sanford-Burnham Medical Research Institute, United States of America Received August 4, 2011; Accepted February 8, 2012; Published March 12, 2012 Copyright: 2012 Rao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Pilot Study Funding from the Molecular Imaging Center at Washington University School of Medicine(mic.wustl.edu/ index.htmland the National Institutes of Health (NIH) P50 CA94056 (DP-W), NIH Neuroscience Blueprint Core Grant P30 NS057105 to Washington University (Viral Vectors Core), National Cancer Institute/NIH RO1CA118389 (JBR) and The Josie Foundation (JBR)(JosieFoundation.org). The St Louis Children’s Hospital Pediatric Brain Tumor Bank is supported by a grant from The Children’s Discovery Institute (JL)(childrensdiscovery.org). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rubin_j@kids.wustl.edu . These authors contributed equally to this work. Introduction Tissue architecture, including cell-cell and cell-extracellular matrix interactions, is an essential determinant of cellular behavior in normal and pathological states. The potential of three- dimensional tissue relationships to influence the phenotype of cancer cells has been appreciated since the time of Virchow and directly demonstrated more recently (reviewed in [1]). Increasing- ly, malignant behavior is being redefined as a consequence of not only mutational events in cancer cells but also specific tumor- stroma interactions that promote tumor formation, progression, and resistance to therapy [2]. The earliest histopathological descriptions of glioblastoma multiforme (GBM) recognized the involvement of vascular endothelial cells (ECs) in tumor architecture. The close apposition of glioblastoma cells to the abluminal surface of endothelial cells, a cardinal feature of GBM, was originally described by Scherer, who suggested this was a route by which tumor cells invaded surrounding brain [3]. In GBMs, there is an abundance of vascular endothelial cells, which was initially assumed to maintain the high metabolic rates observed in these tumors through the formation of perfusing blood vessels [4]. However, it is now clear that the vasculature within GBM is abnormal. Despite a redundancy in microvessels, tumors exist in a relatively hypoxic state in which they utilize anaerobic metabolism to a greater degree than normal brain tissue [5]. These observations call into question the role of the abundant vasculature in GBM. Scherer suggested that the perivascular space possessed specialized properties important for the maintenance and spread PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e33005