CXCR4 increases in-vivo glioma perivascular invasion, and reduces radiation induced apoptosis: A genetic knockdown study

// Viveka Nand Yadav 1, 2 , Daniel Zamler 1, 2 , Gregory J. Baker 1, 2, 3 , Padma Kadiyala 1, 2 , Anat Erdreich-Epstein 4 , Ana C. DeCarvalho 5 , Tom Mikkelsen 5 , Maria G. Castro 1, 2 , Pedro R. Lowenstein 1, 2 1 Department of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA 2 Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA 3 Current address: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA 4 Department of Neurosurgery, at the Saban Research Institute at Children’s Hospital Los Angeles, CA 90027, USA 5 Departments of Neurology and Neurosurgery, Henry Ford Hospital, Detroit, MI 48202, USA Correspondence to: Pedro R. Lowenstein, email: pedrol@umich.edu Keywords: CXCR4 knockdown, autovascularization, perivascular invasion, glioma radiotherapy resistance, combination therapies Received: August 31, 2016 Accepted: October 17, 2016 Published: November 11, 2016 ABSTRACT Glioblastoma (GBM) is a highly invasive brain tumor. Perivascular invasion, autovascularization and vascular co-option occur throughout the disease and lead to tumor invasion and progression. The molecular basis for perivascular invasion, i.e., the interaction of glioma tumor cells with endothelial cells is not well characterized. Recent studies indicate that glioma cells have increased expression of CXCR4. We investigated the in-vivo role of CXCR4 in perivascular invasion of glioma cells using shRNA-mediated knock down of CXCR4. We show that primary cultures of human glioma stem cells HF2303 and mouse glioma GL26-Cit cells exhibit significant migration towards human (HBMVE) and mouse (MBVE) brain microvascular endothelial cells. Blocking CXCR4 on tumor cells with AMD3100 in-vitro , inhibits migration of GL26-Cit and HF2303 toward MBVE and HBMVE cells. Additionally, genetic down regulation of CXCR4 in mouse glioma GL26-Cit cells inhibits their in-vitro migration towards MBVE cells; in an in-vivo intracranial mouse model, these cells display reduced tumor growth and perivascular invasion, leading to increased survival. Quantitative analysis of brain sections showed that CXCR4 knockdown tumors are less invasive. Lastly, we tested the effects of radiation on CXCR4 knock down GL26-Cit cells in an orthotopic brain tumor model. Radiation treatment increased apoptosis of CXCR4 downregulated tumor cells and prolonged median survival. In summary, our data suggest that CXCR4 signaling is critical for perivascular invasion of GBM cells and targeting this receptor makes tumors less invasive and more sensitive to radiation therapy. Combination of CXCR4 knock down and radiation treatment might improve the efficacy of GBM therapy.