Investigating Temporal Fluctuations in Tumor Vasculature With Combined Carbogen and Ultrasmall Superparamagnetic Iron Oxide Particle (CUSPIO) Imaging Jake S. Burrell, 1 * Simon Walker-Samuel, 1,2 Lauren C. J. Baker, 1 Jessica K. R. Boult, 1 Anderson J. Ryan, 3y John C. Waterton, 3 Jane Halliday, 3z and Simon P. Robinson 1 A combined carbogen ultrasmall superparamagnetic iron ox- ide (USPIO) imaging protocol was developed and applied in vivo in two murine colorectal tumor xenograft models, HCT116 and SW1222, with established disparate vascular morphology, to investigate whether additional information could be extracted from the combination of two susceptibility MRI biomarkers. Tumors were imaged before and during carbogen breathing and subsequently following intravenous administration of USPIO particles. A novel segmentation method was applied to the image data, from which six cate- gories of R 2 * response were identified, and compared with his- tological analysis of the vasculature. In particular, a strong association between a negative DR 2 * carbogen followed by posi- tive DR 2 * USPIO with the uptake of the perfusion marker Hoechst 33342 was determined. Regions of tumor tissue where there was a significant DR 2 * carbogen but no significant DR 2 * USPIO were also identified, suggesting these regions became temporally isolated from the vascular supply during the experimental timecourse. These areas correlated with regions of tumor tis- sue where there was CD31 staining but no Hoechst 33342 uptake. Significantly, different combined carbogen USPIO responses were determined between the two tumor models. Combining DR 2 * carbogen and DR 2 * USPIO with a novel segmenta- tion scheme can facilitate the interpretation of susceptibility contrast MRI data and enable a deeper interrogation of tumor vascular function and architecture. Magn Reson Med 66:227–234, 2011. V C 2011 Wiley-Liss, Inc. Key words: tumor; hypoxia; vasculature; USPIO It is well established that most solid tumors require a func- tional vascular network to grow beyond a few millimetres in diameter. Tumor neovasculature differs considerably from healthy blood vessel networks, being characterized by chaotically ordered vessels with leaky discontinuous vessel walls, composed of highly proliferating endothelial cells (1). Vascular phenomena associated with tumor neovascu- lature include arteriovenous shunts, fluctuating red blood cell concentration, an inability to innervate, and regions with no erythrocyte perfusion leaving only ‘‘plasma chan- nels’’ (2). The characteristically irregular vascular network is associated with many key tumor hallmarks, including hypoxia, and raised interstitial fluid pressure, which are linked to poor clinical treatment outcome and prognosis (3). Hypoxic tumor regions have been shown to respond poorly to radiotherapy and chemotherapy and to be less ac- cessible to therapeutic molecules (4). In addition, the high interstitial fluid pressure in the centre of tumors can hinder drug delivery (5). Furthermore, transient or acute hypoxia resulting from intermittent shutdown of blood vessels is another different yet equally important characteristic that impacts on drug delivery and tumor oxygenation (6). Clinical development of novel therapeutic agents is being facilitated by evaluation and qualification of biomarkers of antitumor response and/or mechanism of action. This includes the use of functional imaging biomarkers afforded by techniques such as magnetic resonance imaging (MRI), positron emission tomography, and computed tomography. The clinical development of therapeutics targeting tumor vasculature requires imaging biomarkers of tumor vascular function and morphology, and in this regard two suscepti- bility contrast MRI approaches have been developed. Intrinsic susceptibility contrast MRI works on the principle that deoxyhemoglobin is paramagnetic and compartmental- ized, and therefore causes local susceptibility gradients that extend radially around blood vessels, increasing both the homogeneous (R 2 ) and inhomogeneous (R 2 0 ) contributions to the effective transverse relaxation rate, R 2 *, of water protons in and around the blood vessels. Breathing a high oxygen content gas such as carbogen (95% O 2 and 5% CO 2 ) can lower the concentration of deoxyhemoglobin in the blood stream and thereby reduce the susceptibility gradients, resulting in a slower R 2 * rate. However, under certain condi- tions carbogen breathing can paradoxically increase R 2 *. The reasons for this are not fully understood but may include vascular steal (7). This change in relaxation rate, DR 2 * (sec 1 ), can provide a biomarker for hemodynamic functional vasculature (8). 1 Cancer Research UK and EPSRC Cancer Imaging Centre, The Institute of Cancer Research, Belmont, Sutton, Surrey, United Kingdom. 2 Centre for Advanced Biomedical Imaging, University College London, London, United Kingdom. 3 AstraZeneca, Alderley Park, Macclesfield, United Kingdom. yPresent address: Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Headington, Oxford, United Kingdom zThe authors would like to acknowledge the contribution made by our colleague and friend, Jane Halliday, now sadly deceased. This article was published online on 8 Feb 2011. Errors were subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 21 June 2011. Grant sponsor: BBSRC/AstraZeneca Industrial Partnership Studentship; Grant number: BB/E528979/1; Grant sponsor: Medical Research Council; Grant number: G0700017; Grant sponsor: Cancer Research UK; Grant number: C1060/A10334. *Correspondence to: Jake S. Burrell, B.Sc., The Institute of Cancer Research, 15 Cotswold Rd, Belmont, Sutton, Surrey SM2 5NG, United Kingdom. E-mail: Jake.Burrell@icr.ac.uk Received 12 August 2010; revised 27 October 2010; accepted 24 November 2010. DOI 10.1002/mrm.22779 Published online 8 February 2011 in Wiley Online Library (wileyonlinelibrary. com). Magnetic Resonance in Medicine 66:227–234 (2011) V C 2011 Wiley-Liss, Inc. 227