Abstract 368: In vivo vessel co-option by glioma cells following chronic anti-angiogenic treatment
Authors: Kirkpatrick Nathaniel D. , Jain Rakesh K. 
Published: February 2011

Abstract
Introduction: Anti-vascular endothelial growth factor (VEGF) therapy using monoclonal antibodies or multitargeted tyrosine kinase inhibitors (TKIs) has shown modest efficacy in recurrent Glioblastoma Multiforme (rGBM). In some orthotopic murine models, increased survival with these agents can primarily be attributed to a reduction in edema rather any direct anti-tumor effect (W. Kamoun et al, JCO 2009). Furthermore, reports have suggested gliomas may respond to anti-angiogenic therapy with an increased invasive phenotype although direct evidence has not yet been established. Here, we used intravital multiphoton laser scanning microscopy (MPLSM) and several orthotopic invasive tumor models to test the invasive tumor response of glioblastoma cells to two anti-angiogenic agents: cediranib and sunitinib.
Materials and Methods: We transfected CNS1 (rat glioma) and GL261 (mouse glioma) with DsRed fluorescent protein and maintained the cells in culture as neurospheres. Using mice with surgically implanted cranial windows, we injected 10,000 tumor cells into the cerebrum of SCID (CNS1 cells/xenograft) or C57/b6 (GL261/syngeneic) mice. When tumors had established a primary lesion as determined by intravital microscopy, we treated animals with 6 mg/kg cediranib or 40 mg/kg sunitinib (daily oral gavage) with control groups treated with the corresponding drug vehicle. Tumor cell dynamics relative to brain microvessels were measured with MPLSM using a fluorescence angiography. Following treatment and imaging, we assessed brain sections with immunohistochemistry (IHC).
Results: We found that chronic treatment (2 week) of CNS1 tumors with cediranib led to a marked increase of vessel co-option (direct migration of tumor cells along microvessels) compared to control determined both in vivo (+67%) and with IHC (+80%). In vivo imaging revealed perivascular tumor invasion deep into the cortex allowing for time-lapse imaging of tumor cell motility. Using IHC, we measured co-opted regions extended over 1 mm from the primary lesion. Co-opted vessels appeared relatively normal although astrocyte/endothelial cell contact seemed to be disrupted. We confirmed this increase in vessel co-option during anti-angiogenic therapy using sunitinib treatment in CNS1 tumors (5/5 treated) and using cediranib in GL261 tumors (6/8 treated, 2/8 controls).
Discussion: With the recent US approval of bevacizumab for treatment of rGBM, anti-VEGF therapy in these tumors may become the standard of care. It is therefore critical to understand the tumor response to this therapy. Here, we find that long-term treatment of established GBMs with anti-angiogenic TKIs leads to an increase in tumor invasion along existing brain vessels. This model provides a foundation for dynamic imaging of this process and for further investigation of the mechanisms governing tumor co-option in the brain.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 368.