Magnetic resonance imaging-based detection of glial brain tumors in mice after antiangiogenic treatment An Claes 1 , Giulio Gambarota 2 , Bob Hamans 2 , Olaf van Tellingen 3 , Pieter Wesseling 1 , Cathy Maass 1 , Arend Heerschap 2 and William Leenders 1 * 1 Department of Pathology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands 2 Department of Radiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands 3 Department of Clinical Chemistry, Dutch Cancer Institute, Amsterdam, The Netherlands Proper delineation of gliomas using contrast-enhanced magnetic resonance imaging (CE-MRI) poses a problem in neuro-oncology. The blood brain barrier (BBB) in areas of diffuse-infiltrative growth may be intact, precluding extravasation and subsequent MR-based detection of the contrast agent gadolinium diethylene- triaminepenta-acetic acid (Gd-DTPA). Treatment with antiangio- genic compounds may further complicate tumor detection as such compounds can restore the BBB in angiogenic regions. The increasing number of clinical trials with antiangiogenic com- pounds for treatment of gliomas calls for the development of alter- native imaging modalities. Here we investigated whether CE-MRI using ultrasmall particles of iron oxide (USPIO, Sinerem 1 ) as blood pool contrast agent has additional value for detection of gli- oma in the brain of nude mice. We compared conventional T1- weighted Gd-DTPA-enhanced MRI to T2*-weighted USPIO- enhanced MRI in mice carrying orthotopic U87 glioma, which were either or not treated with the antiangiogenic compound van- detanib (ZD6474, ZACTIMA TM ). In untreated animals, vessel leakage within the tumor and a relatively high tumor blood vol- ume resulted in good MRI visibility with Gd-DTPA- and USPIO- enhanced MRI, respectively. Consistent with previous findings, vandetanib treatment restored the BBB in the tumor vasculature, resulting in loss of tumor detectability in Gd-DTPA MRI. How- ever, due to decreased blood volume, treated tumors could be readily detected in USPIO-enhanced MRI scans. Our findings sug- gest that Gd-DTPA MRI results in overestimation of the effect of antiangiogenic therapy of glioma and that USPIO-MRI provides an important complementary diagnostic tool to evaluate response to antiangiogenic therapy of these tumors. ' 2007 Wiley-Liss, Inc. Key words: glioblastoma; angiogenesis; MRI; USPIO; vandetanib; blood brain barrier Glioblastoma multiforme (GBM) is the most frequent and most malignant glial brain tumor with dismal prognosis and median sur- vival of less than 2 years, despite development of novel chemo- therapeutic compounds. 1 GBMs are characterized by extensive diffuse infiltrative growth in the brain parenchyma making cura- tive treatment by surgery or radiotherapy impossible. 2,3 One of the hallmarks of GBMs is the regional occurrence of prominent angio- genesis. 4 The disrupted blood brain barrier (BBB) in such regions allows magnetic resonance (MR)-based detection of these tumors by extravasation and accumulation in the interstitial spaces of con- trast agents like gadolinium diethylenetriaminepenta-acetic acid (Gd-DTPA). 4–6 Importantly, however, the blood vessels in espe- cially diffuse-infiltrative tumor regions may not be leaky, causing difficulties in radiological delineation of these tumors. 7 This is of critical importance since neuro-oncologists depend on MRI for staging of tumors, monitoring response to therapy, distinguishing recurrent tumor from radiation necrosis and planning stereotactic biopsies and surgical resections. 8 Because of the occurrence of angiogenesis in GBM, the propo- sition is that this tumor type may be candidate for antiangiogenic therapy. 9 Several agents have been developed that target angio- genic signaling pathways. An example is avastin, a monoclonal antibody, which neutralizes vascular endothelial growth factor-A (VEGF-A), the main inducer of angiogenesis. 10 Tyrosine kinase inhibitors with specificity towards angiogenic receptors have also been developed and are entering clinical trials now. 11–13 Preclinical testing of inhibitors of VEGF signaling resulted in potent antitumor effects in a variety of animal models of can- cer. 13,14 However, in the clinical setting, results of monotherapy with angiogenesis inhibitors have not met the initial expectations. Yet, combination of avastin with chemotherapy in patients with advanced colorectal cancer has resulted in prolongation of median survival of 4 months as compared to chemotherapy alone. 15 It has been hypothesized that the beneficial effect of combination ther- apy is based on avastin-induced normalization of tumor vascula- ture, resulting in reduced interstitial pressure and, consequently, improved distribution of cytotoxic compounds to tumor cells. 16,17 Such vessel normalization also occurred in a mouse model of cere- bral Mel57 melanoma metastases in response to vandetanib (ZD6474, ZACTIMA TM ), 18 a tyrosine kinase inhibitor with speci- ficity towards VEGFR2 and epidermal growth factor receptor (EGFR). 13 Although vandetanib treatment effectively inhibited angiogenesis, it did not induce tumor regression or even dor- mancy: tumors progressed via cooption of preexistent vascula- ture. 18,19 Importantly, vessel normalization resulted in restoration of the BBB, and consequently the invisibility of progressing tumors in Gd-DTPA-enhanced MRI scans. 18 We also previously reported that Mel57 melanoma xenografts may grow in brain parenchyma in an angiogenesis-independent fashion by vessel cooption. 20 Although these infiltrative lesions could not be detected in Gd-DTPA contrast enhanced MRI (CE- MRI), the relatively low vascular volume in the tumors, as com- pared to the surrounding tissue, could be exploited to detect these lesions using blood pool contrast agents such as ultrasmall par- ticles of iron oxide (USPIO). 21 Because antiangiogenic treatment may result in conversion of an angiogenic tumor phenotype into a coopting one, USPIO imaging may therefore be an attractive com- plementary tool to detect glioma and evaluate response to antian- giogenic therapy. USPIO (Sinerem 1 , Guerbet, France) consists of iron oxide crystals, coated by a dextrane layer. 22 Because of its physicochem- ical characteristics [mean particle size 29.5 nm 6 23.1 (volume weighted)], it remains in the blood circulation for a long time (plasma half-life: 25–30 hr) and can therefore be considered to be a blood pool contrast agent early after intravenous injection. 23,24 USPIOs have a low toxicity profile and have been tested in clinical trials for detection of lymph node metastases in patients with blad- der and prostate carcinoma. 23,25 Abbreviations: BBB, blood brain barrier; EGFR, epidermal growth fac- tor receptor; Gd-DTPA, gadolinium diethylenetriaminepenta-acetic acid; MRI, magnetic resonance imaging; RET, rearranged during tranfection; USPIO, ultrasmall particles of iron oxide; VEGF, vascular endothelial growth factor. Grant sponsor: Hersenstichting Nederland; Grant number: 12F04.(02)2. Grant sponsor: Dutch Cancer Society; Grant number: 2003-2975. *Correspondence to: Department of Pathology, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Nether- lands. Fax: 131-243668750. E-mail: w.leenders@pathol.umcn.nl Received 10 August 2007; Accepted after revision 23 October 2007 DOI 10.1002/ijc.23306 Published online 14 December 2007 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 122, 1981–1986 (2008) ' 2007 Wiley-Liss, Inc. Publication of the International Union Against Cancer