The effects of tumor-derived platelet-derived growth factor on vascular morphology and function in vivo revealed by susceptibility MRI Simon P. Robinson 1,2 * , Christian Ludwig 3 , Janna Paulsson 4 and Arne € Ostman 4 1 Division of Basic Medical Sciences, St. George’s, University of London, Cranmer Terrace, London, United Kingdom 2 Cancer Research UK Clinical Magnetic Resonance Research Group, The Institute of Cancer Research, Cotswold Road, Sutton, Surrey, United Kingdom 3 The Henry Wellcome Building for Biomolecular NMR Spectroscopy, Cancer Research UK Institute for Cancer Studies, University of Birmingham, Vincent Drive, Edgbaston, Birmingham, United Kingdom 4 Department of Pathology-Oncology, Cancer Center Karolinska, Karolinska Institutet, Stockholm, Sweden Platelet-derived growth factors (PDGF) play a major role in peri- cyte recruitment in tumor capillaries. Pericytes are required for proper vessel development, and contribute to tumor angiogenesis by promoting stabilization and maturation of newly formed ves- sels. To investigate the effects of pericyte coverage on tumor vessel morphology and function in vivo, tumors derived from B16 mela- noma cells transfected with either control plasmid (B16/ctr) or plasmid encoding full-length PDGF-BB (B16/PDGF), the latter previously shown to have enhanced blood vessel pericyte coverage and an increased tumor growth rate, were assessed using histo- pathological methods, Hoechst 33342-based perfusion analyses, and two noninvasive susceptibility magnetic resonance imaging (MRI) methods. Susceptibility-contrast MRI, incorporating the use of ultrasmall superparamagnetic iron oxide particles, revealed a significant (p < 0.05) reduction in vessel size index (R v ) of B16/ PDGF tumors, and which was validated histologically by the pres- ence of significantly smaller (p < 0.001), more punctate blood ves- sels identified by fluorescence microscopy of the perfusion marker Hoechst 33342. Intrinsic-susceptibility MRI was used to measure the transverse MRI relaxation rate R 2 *, sensitive to changes in en- dogenous paramagnetic [deoxyhaemoglobin], and used to probe for vascular maturation and function. Hypercapnia (5% CO 2 / 95% air) induced a negligible DR 2 * response in the B16/ctr and B16/PDGF tumors. In contrast, hyperoxia (5% CO 2 /95% O 2 ) induced a significantly greater R 2 * reduction in the B16/PDGF tumors (p < 0.02). Together the susceptibility MRI-derived bio- markers reveal novel pericyte-dependent changes in the morphol- ogy and function of the perfused tumor vasculature in vivo. ' 2007 Wiley-Liss, Inc. Key words: pericyte; PDGF; MRI; biomarker Angiogenesis, the development of new blood vessels to provide a nutritive blood supply, is a prerequisite for tumor growth and survival. 1–3 Angiogenesis is stimulated by the release of specific growth factors from tumor cells, endothelial cells or associated macrophages, and production of these factors is upregulated by physiological conditions associated with the classical hallmarks of the tumor microenvironment. 4 Pericytes are vascular mural cells which are embedded in the basement membrane of microvessels. These cells are required for proper vessel development, and contribute to angiogenesis by pro- moting stabilization and maturation of newly formed vessels. 5,6 Pericytes are also present in tumor capillaries. 7 However, in gen- eral, pericyte coverage of tumor capillaries is sparser than in nor- mal vessels, and the extent of pericyte coverage of tumor vessels shows large variability. 8 Also, pericytes of tumor vessels grow in a less organized manner, and are more loosely attached to the vessels. 7,9 The platelet-derived growth factor (PDGF) family of growth factors consists of 4 PDGF polypeptide chains and 5 dimeric iso- forms, PDGF-AA, -AB, -BB, -CC and -DD, and play a major role in pericyte recruitment in both normal and tumor capillaries. 10 These effects are predominantly mediated by PDGFb-receptors expressed on pericytes, and which are activated by PDGF-BB and PDGF-DD only. 11,12 A series of studies have demonstrated that stimulation of PDGF b-receptors stimulates pericyte recruitment to tumor capillaries. 11,13,14 Furthermore, inhibition of PDGFb- receptors on pericytes has been shown to increase the antiangio- genic effects of VEGF inhibitors. 15,16 However, the effects of pericyte coverage on tumor vessel structure and function in vivo remain poorly characterized. Imaging methods for assessing the tumor vascular status are continually being developed and applied. 17,18 Magnetic resonance imaging (MRI), with its high temporal and spatial resolution, presents itself as an ideal, clinically-translatable, noninvasive assay of tumor angiogenesis in vivo. 19,20 The ultimate aim is the identification, development and validation of quantitative clinical MRI biomarkers associated with tumor blood vasculature to assist in the planning of individual patient treatment protocols. These biomarkers are also proving importance in clinical trials of anti- cancer therapies directed at tumor blood vessels. 21 Two complementary magnetic susceptibility-based MRI approaches are being exploited for the investigation of tumor angiogenesis. First, tumor blood volume has been assessed using susceptibility contrast MRI, in which the tumor uptake of intravas- cular ultrasmall superparamagnetic iron oxide (USPIO) particles is measured. Estimates of fractional tumor blood volume, derived from measurements of the absolute changes in the MRI transverse relaxation rates R 2 and R 2 * following administration of USPIO particles, have been shown to correlate with the tumor blood vol- ume determined by uptake of the perfusion marker Hoechst 33342 determined by fluorescence microscopy. 22–24 In addition, the ratio of the change in relaxivities, DR 2 */DR 2 , can be used to derive maps of capillary diameter (vessel size index, R v ), 22,25–27 and which has shown good agreement with histologically determined vessel size. 24,27 Second, intrinsic susceptibility MRI offers a sensi- tive method of monitoring tumor vascular maturation and function in vivo. 23,28–31 Deoxyhaemoglobin, which is paramagnetic, creates susceptibility variations in the magnetic field, increasing the MRI transverse relaxation rate R 2 * of water in blood and in the tissue surrounding blood vessels. MRI methods such as the gradient recalled echo (GRE) sequence are sensitive to R 2 * and thus to blood deoxyhaemoglobin levels. Deoxyhaemoglobin therefore acts as an intrinsic, blood oxygenation level dependent (BOLD) contrast agent. 29 Paracrine stimulation of PDGFb-receptors on pericytes through overexpression of PDGF in B16 melanoma cells has previously been shown to enhance tumor growth. 13 The PDGF-producing B16 melanomas had similar vessel density as control tumors, but displayed higher pericyte coverage. 13 The aim of this study was to Grant sponsor: The Royal Society and Cancer Research UK. *Correspondence to: Cancer Research UK Clinical Magnetic Reso- nance Research Group, The Institute of Cancer Research, Cotswold Road, Sutton, Surrey, SM2 5PT, UK. Fax: 144-208-661-0846. E-mail: Simon.Robinson@icr.ac.uk Received 25 April 2007; Accepted after revision 5 October 2007 DOI 10.1002/ijc.23279 Published online 21 November 2007 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 122, 1548–1556 (2008) ' 2007 Wiley-Liss, Inc. Publication of the International Union Against Cancer