B Academy of Molecular Imaging and Society for Molecular Imaging, 2010 Published Online: 15 July 2010

Mol Imaging Biol (2011) 13:518Y525 DOI: 10.1007/s11307-010-0378-x

RESEARCH ARTICLE

A Dual Radiologic Contrast Agent Protocol for 18F-FDG and 18F-FLT PET/CT Imaging of Mice Bearing Abdominal Tumors
Nicolas Aide,1,2,3,6 Kathryn Kinross,3,4 Jean-Mathieu Beauregard,3 Oliver Neels,3 Titaina Potdevin,4 Peter Roselt,3 Donna Dorow,3,4 Carleen Cullinane,4 Rodney J. Hicks3,4,5
1 2

Bioticla Team, EA 1772, IFR 146 ICORE, GRECAN, Franois Baclesse Cancer Centre and Caen University, Caen, France PET Centre, Caen University Hospital and Franois Baclesse Cancer Centre, Caen, France 3 Centre for Molecular Imaging, Peter MacCallum Cancer Centre, East Melbourne, Australia 4 Sir Donald and Lady Trescowthick Laboratories, Peter MacCallum Cancer Centre, East Melbourne, Australia 5 The Department of Medicine, The University of Melbourne, Parkville, Australia 6 Nuclear Medicine Department, Centre Franois Baclesse, Avenue Gnral Harris, 14076, Caen cedex 5, France

Abstract Purpose: The aim of the study was to improve abdominal tumor detection by use of a dual radiologic contrast protocol. Procedures: eXia160 (Benitio international) was mixed with 2-deoxy-2-[18F]fluoro-D-glucose or 3-[18F]fluoro-3-deoxythymidine for intravenous (IV) injections. Omnipaque 300 (GE healthcare) was used for intraperitoneal (IP) injections. Positron emission tomography/computed tomography (PET/CT) scans were acquired on a Siemens Biograph equipped with point spread function reconstruction. The optimal concentration and injection schedule of IP contrast agent was studied in 12 mice. The impact of IP contrast media on PET quantitative accuracy was investigated by phantom studies and by imaging six mice before and after IP injection of Omnipaque. The impact of a dual contrast media protocol on tumor delineation and quantitation was evaluated in 15 tumor-bearing mice using ex vivo counting as the reference. Results: The optimal sequence was a mixture of tracer plus IV contrast agent followed by 1 mL of IP contrast agent (20 mg iodine/mL) administered 10 min before PET/CT acquisition. Phantom studies showed that the use of a 20-mg iodine/mL concentration of Omnipaque led to a 4.8% overestimation of radioactivity concentration, as compared to saline. This was confirmed by animal studies that demonstrated a 4.3% overestimation. Tumor detection was excellent and correlation between PET/CT quantitative data and ex vivo counting was good (r2 =0.91, slope=0.7). Conclusions: A dual radiologic contrast protocol is useful in PET/CT scanning of mice bearing abdominal tumors. Contrast agents used in this manner lead to a small but acceptable overestimation of quantitative PET data. Key words: PET/CT, Preclinical studies, Abdominal tumors, Contrast media

A dual radiologic contrast protocol, which includes a mixture of tracer plus long-lasting intravenous contrast agent followed by intraperitoneal contrast agent, is described that provides better anatomical separation and delineation of abdominal tumors for PET/CT scanning of mice. Correspondence to: Nicolas Aide; e-mail: n.aide@baclesse.fr

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Introduction
ositron emission tomography (PET) scanning of abdominal tumors [14] has potential utility for evaluating the success of engraftment of exogenous malignant cell lines or development of spontaneous tumors in transgenic mice and for monitoring tumor growth and response to therapy. It may also aid in the evaluation of new tracers. However, such imaging is challenging because of the high physiological uptake that may be observed in the gastrointestinal tract and the lack of anatomical landmarks on PET images alone. In this setting, computed tomography (CT) scans, performed either on a hybrid PET/CT device or a separate CT device, could be useful to better localize tumors. However, several studies have reported the inability of CT scans acquired without contrast media to delineate tumors from surrounding tissues due to organs and tumors having the same density [5, 6]. This drawback is particularly encountered in nude mice, which lack abdominal fat. Two long-lasting intravascular contrast media agents, eXia160 [7] and FenestraVC [8, 9], are available commercially. Also, the potential use of iohexol, an iodinebased contrast media agent, by intraperitoneal route has been reported to improve hepatic tumor delineation in CT imaging of mice [6]. The use of a CT protocol including both intravenous (IV) and intraperitoneal (IP) injections of contrast agents would therefore be very useful in PET/CT imaging but has, to our knowledge, not previously been tested. The use of both radiotracer and contrast media injections in a single imaging session in mice is challenging for two reasons. First, IV injections (contrast media and tracers) must meet the requirements of international and local ethics committees, which generally require that the total injected volume to be kept below 250 L. Second, the use of two injections increases the risk of extravasation, especially during longitudinal studies. This could hamper the calculation of quantitative indices which take into account the injected dose, specifically the percentage of injected dose (ID) per gram and the standardized uptake value (SUV). Therefore, we designed this study to determine an optimal protocol for both IV and IP contrast media injection to be used in combination with PET/CT using either 2deoxy-2-[ 18 F]fluoro- D -glucose ( 18 F-FDG) for glucose metabolism evaluation or 3-[18F]fluoro-3-deoxythymidine (18F-FLT) as a tracer of cellular proliferation. The aim of these experiments was to improve delineation of intraabdominal tumors. In particular, we evaluated the possibility of mixing eXia160 with 18F-FDG and 18F-FLT in order to minimize the number of required injections into the tail vein, as well as image quality and quantitative accuracy when IV and IP contrast media enhancement are used in mice bearing ovarian tumors.

Materials and Methods

In Vitro Studies
To test whether eXia160 could have an impact on the tracers and vice versa, high-performance liquid chromatography (HPLC) and thin layer chromatography (TLC) were performed.

Phantom Studies
First, we evaluated the optimal concentration of the iodinebased contrast agent for IP injections using phantoms designed to mimic the abdomen of a mouse. These phantoms consisted of 50 mL tubes containing a catheter and small tubes filled with air or ultrasound gel to simulate the air and feces, respectively, present in the abdomen of mice. The larger tubes of these phantoms were then filled with a solution of saline serum plus iodine-based contrast media (Omnipaque 300; GE Healthcare, Australia), the concentration of which ranged from 1 to 20 mg I/mL. Next, we evaluated the potential impact of the use of contrast media on PET quantitation. For this purpose, small tubes (volume of 2 mL, diameter of 9 mm) were filled with an 18F-FDG solution and placed at the center of a 30-mL syringe (diameter 23 mm) in order to mimic tumors surrounded by IP contrast media. The concentration of 18F-FDG was 0.75, 0.95, and 1.15 MBq/mL reflecting typical concentrations in tumor models.

In vivo Studies
Animal studies were performed under approval of the animal ethics committee of our cancer center. Mice housing was in a dedicated small animal facility adjacent to the preclinical imaging laboratory in a PC-2 facility staffed by experienced animal technologists. Imaging studies were begun when the tumor size reached a 100-mm3 volume, based on ultrasound measurements performed using a dedicated small animal ultrasound device (Vevo 770, VisualSonics, Canada). A total of 33 mice were used, comprising tumor-free nude mice, a genetically engineered murine model (Lox-stop-loxKRASG12D/+/PTENfl/fl) that develops spontaneous orthotopic ovarian cancers [10] and nude mice bearing orthotopic transplanted ovarian cancers. In groups 1 (six mice) and 2 (three mice), we determined the optimal concentration and injection schedule of IP contrast agent by varying the concentration (10 or 20 mg I/mL), volume (0.51.5 mL), and delay between injection and scan (10 or 60 min). The optimal protocol was then confirmed in three mice (group 3; Table 1). In group 4 (six mice), PET/CT acquisitions were performed to assess the impact of IP injection of contrast media on tumor detection and accuracy of quantitative data. Mice were injected with a mixture of 18F-FDG and eXia160 and euthanized after the tracer uptake period in order to stop the tracers uptake that may have occurred between the two series of PET/CT examinations. Animals were then scanned on the PET/CT over a 10-min period. Immediately thereafter, IP contrast injections

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Table 1. Study design Group Number of mice Imaging modality

N. Aide, et al.: Dual Contrast Agent Protocol for PET/CT Scanning of Mice

Aim

Tissue samples used for quantification Tumors Organs 7 15

1 2 3 4 5 6

6 3 3 6 9 6

CT CT CT PET/CT PET/CT PET/CT

Optimal delay between IP injection and CT Optimal volume for IP injections Confirmation of optimal concentration and injection schedule Impact of IP contrast agent on quantification 18 F-FDG imaging: image quality and quantification 18 F-FLT imaging: image quality and quantification

8 8 5

were performed and mice were scanned a second time. The duration of the second acquisition was increased to account for fluorine-18 decay. In groups 5 and 6, PET/CT acquisitions were performed after injection of IV and IP contrast agents to assess image quality and accuracy of quantitative data when imaging mice with 18F-FLT (group 5, nine mice) or 18F-FDG (group 6, six mice). Animals were euthanized after imaging and tumors were harvested for ex vivo scintillation counting.

Animal Studies Tumor activity was obtained from a volume of

interest (VOI) encompassing the entire lesion. The VOI was determined by means of an isocontour, which was set so that the VOI matched the apparent tumor volume on PET and CT images. When discordance was encountered between PET metabolic volume and CT volume, the VOI was drawn according to CT images, so that PET/CT images could be compared to ex vivo counting for which the entire tumor was harvested, irrespective of the presence of non-viable areas. Immediately after the PET examination had been performed, tumors and organs were harvested and ex vivo counting was performed as follows: The weight of the lesions was measured with precision scales (precision 0.01 mg). Tumor radioactivity was counted for 1 min in a cylinder-well counter (Wallac 1470 automatic gamma counter) and corrected for instrument efficiency and decay. Counts per minute were converted in becquerel and normalized by sample weight, assuming a density of 1 g/mL.

PET/CT Acquisitions
All PET imaging studies were performed on a Biograph TrueV (Siemens Healthcare). This PET/CT device is equipped with a 64slice spiral CT component. After a 3-h fasting period, mice were injected in the tail vein with a solution of 18F-FDG (1632 MBq) or 18 F-FLT (3237 MBq). The volume of the syringes was always kept below 0.2 mL in order to meet the requirements of our ethics committee. To minimize muscle and brown fat uptake in the case of 18 F-FDG imaging, animals were kept anesthetized under warming lights for a 20-min period after injection. Animals were imaged simultaneously in groups of three with the PET/CT scanner, one was placed at the center of the FOV and the two others were placed on each side of the central animal, at a 5-cm and at a 5-cm radial offset. The PET/CT acquisition began with a CT scan acquired with the following parameters: 80 mAs, 130 kV, pitch 0.8, and 640.6 mm collimation. Then an emission scan was acquired in 3D mode. PET images were reconstructed in a reduced FOV (35 cm) applying a scaling factor of 2. Images were reconstructed with an algorithm which models point spread function of the scanner and leads to a 2.2-mm spatial resolution at the center of the FOV [11]. The following parameters were used: six iterations, 16 subsets, no filtering, and a matrix size of 3362, resulting in a 1.021.021-mm voxel size. Scatter and attenuation corrections were applied.

Statistical Analysis The relationship between (1) the CT density and the concentration of contrast media in phantoms and (2) the radioactivity in animal tumors, as determined by clinical PET/CT and by gamma counter, was estimated using linear regression analysis. In addition to regression analysis, BlandAltman plots were performed.

Results
Lack of Chemical Interaction When Mixing eXia160 and 18F-FDG and 18F-FLT for IV Injection
eXia160 was mixed in a 1:1 volume ratio with the tracer. Neither tracer metabolites nor decomposition of the contrast agent was observed by HPLC and TLC directly after mixing for 10, 30, and 60 min at room temperature. The concentration of ethanol in the 18F-FLT solution (7%) had no impact on the stability of eXia160.

Data Analysis
Phantom Studies A 0.13-cm2 region of interest (ROI) was drawn in a transverse slice of each mouse-sized phantom, in a zone close to the structures mimicking the gastrointestinal tract. The same ROI was applied in the liver and spleen of five nude mice having being imaged in a previous experiment. A 0.13-cm2 ROI was drawn in a transverse slice of each 2 mL tube filled with 18F-FDG and surrounded by various concentrations of Omnipaque 300 or saline serum.

Determination of the Optimal Concentration and Injection Schedule of Contrast Agent for IP Injection
Phantoms studies (Fig. 1) showed a strong correlation between the concentration of the contrast media and CT

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Fig. 1. Optimal concentration for IP contrast media. a Transverse slices of phantoms mimicking the small intestine and the colon of mice were filled with a solution of saline serum plus iodine-based contrast media at different concentrations. b Representative CT coronal slice of mice injected with 0.1 mL of eXia160. Liver and spleen enhancement is visualized. c Plot of CT densities against concentration of the iodine-based contrast agent solution. Dotted lines represent the range of CT densities observed in the liver and spleen in five nude mice injected with eXia160 and scanned from 60 to 90 min post-injection.

densities, which ranged from 29 to 293 HU. The CT densities of liver and spleen in five nude mice injected with eXia160 (data from a previous experiment) ranged from 110 to 194 HU. CT acquisitions in mice showed that (1) a 20-mgI/mL concentration was required to obtain a peritoneal enhancement well above that obtained in liver and spleen, (2) a volume of 1 mL was appropriate to well visualize the pelvic organs, and (3) the IP injections had to be done 10 min before the CT acquisition began, as an absorption occurred in animals imaged 60 min post-IP injection and led to lower densities in the peritoneal fluid. Fig. 2 displays typical CT acquisitions in nude mice imaged prior to and following IP contrast media.

delineation of tumors was particularly useful in the case of tumors harboring heterogeneous tracer uptake and/or which had moved outside the site of expected location, as they could be mistaken for physiological uptake. The kidneys were also well visualized because a peritoneal absorption occurs, even 10 min post-IP injection, leading to the presence of contrast media in the renal collecting systems. The injection of eXia160 was also useful in providing additional anatomical landmarks by providing liver and spleen enhancement. In two cases, the transplanted ovarian tumor had invaded the spleen. In these cases, splenic enhancement by eXia160 was particularly useful, as the tumor was clearly visible as a tissular density surrounded by the higher density related to splenic enhancement, while the spleen was surrounded by the higher density of the IP fluid.

Impact of IV and IP Contrast Media Injections on Tumor Detection

The use of IP contrast media leads to a good delineation of ovarian tumors from the surrounding organs, i.e., small bowel loop, colon, and kidneys (Figs. 3 and 4).This better

Impact of High CT Density on PET Quantification

The phantom studies showed that while the ratio of measured activity on images and true activity from ex vivo

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Fig. 2. Illustration of intraperitoneal (IP) contrast enhancement on computed tomography scans. Three tumor-free nude mice were scanned prior to and after injection of 1 mL of iodinated contrast material at a concentration of 20 mgI/mL. Maximum intensity projection views and transaxial sections are displayed before and after contrast media injection. Note the poor contrast between abdominal organs before contrast media injection. IP contrast media provides better anatomical separation and delineation of anatomical structures.

counting was almost equal to 1 when the 18F-FDG source was surrounded by water, it increased as the concentration of the contrast media increased (Fig. 5a). At a contrast agent concentration representing a density of approximately 300 HU, a drop of the curve was observed. This phenomenon has been previously described [12] and is due to the fact that 300 HU is typically used as a threshold for differentiating soft tissue and bone on segmented attenuation map of the Biograph PET/CT device. The mean overestimation of tracer radioactivity ranged from 2.6% to 5.6%, depending on contrast media concentration (Fig. 5b). For the 20-mgI/mL concentration that was shown to be optimal in the present study, the mean overestimation was 4.8%. Subsequent PET/CT acquisitions in mice without and then with IP injection of contrast agent showed an average overestimation of 4.3% (range from 6.2% to 15.5%).

rhagic ascites, the IP contrast media did not distribute well within the peritoneal space, and in three other animals with malignant ascites, the amount of liquid inside the abdomen prior to the IP contrast media injection led to a dilution of the contrast agent and the CT densities were lower than expected.

Discussion
In abdominal tumor models, a moderate-to-high physiological uptake in the gut is observed with many tracers including 18F-FDG and 18F-FLT and may hamper tumor detection. In this setting, correlative CT acquisition could be useful to improve the localization of tracer uptake. However, it is known that CT scan without contrast media injection is unable to delineate tumors from surrounding organs because they have similar CT attenuation characteristics [5, 6]. To the best of our knowledge, the present study is the first to address the methodological issues related to the use of combined IV and IP contrast enhancement. Specifically, we assessed (1) whether PET tracers and the IV contrast agent can be safely mixed to minimize the required number of injections, which is especially important for longitudinal studies; (2) the optimal volume and concentration of IP contrast media, keeping in mind that low administered volumes are less likely to have an adverse impact on respiration or to induce iodine toxicity; (3) the optimal delay between IP contrast agent injection and imaging; and finally, (4) whether the optimal protocol chosen as a result of these experiments improve tumor delineation from surrounding

Accuracy of PET Quantitative Data

A strong correlation between PET/CT quantitative data and ex vivo counting was found (r2 =0.93, slope=0.98). Bland Altman analysis demonstrated that the mean ratio between ex vivo quantitation and clinical PET quantitation was 0.96 and that the 95% limits of agreement were narrow (Fig. 6).

Impact of Ascites on Image Quality

In the case of clinically important ascites, a reduction in the injected volume to 0.5 mL was required, in order not to impact the animals breathing. In one animal with hemor-

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Fig. 3. 18F-FDG dual contrast-enhanced PET/CT acquisition in two mice bearing a transplanted left ovary tumor. Coronal PET slices at the level of the tumors (a) and fused PET/CT slices (b) are shown. In mouse 1, a heterogeneous tracer uptake is seen. Thanks to clear delineation of tumors on correlative contrast-enhanced CT slices, 18F-FDG foci were well located within tumors and not mistaken for physiological uptake in gut, especially in the case of mouse 1, in which tumor has migrated from the pelvis.

tissues without compromising accurate quantitative values, with focus on the potential impact of high CT densities on the accuracy of PET quantitative data. We showed that eXia160 can be safely mixed with both 18 F-FDG and 18F-FLT, two tracers widely used in preclinical cancer research. This is of importance because a single injection minimizes the risk of extravasation and its consequences on the calculation of quantitative indexes that take into account the injected activity (percentage of ID per gram or SUV). Also, a single injection minimizes the stress and pain induced when injections are done on awake animals. The results we have obtained could be extended to other tracers that contain low concentrations of ethanol [13].

We found that the optimal protocol first was to inject a mixture of tracer plus IV contrast agent and then IP contrast agent 10 min before the PET/CT examination. This 10-min delay could be lowered to 5 min since relatively rapid peritoneal absorption occurs, as demonstrated by the presence of high CT densities in the renal collecting system and in the bladder in some mice. In all but one case, the use of IP contrast agent was particularly useful to detect intra-abdominal tumors, which were surrounded by high-density IP liquid. These tumors were clearly distinguishable from surrounding organs. It is noteworthy that ovary tumors can move within the abdomen, some of them being located midline and anterior rather than laterally in the pelvis. The use of eXia160 provided additional landmarks and was also useful when extension of the transplanted tumor into adjacent organs occurs. For example, involvement of the spleen could be detected. The only case in which tumor localization was difficult was a mouse harboring hemorrhagic ascites. In this case, the IP contrast agent did not mix well with the ascitic fluid or was constrained by malignant peritoneal deposits or adhesions. When hemorrhagic ascites is suspected, the IP injection should be done as close as possible to the expected area of tumor involvement, together with a longer period of abdominal massage. Also, the volume of IP contrast agent should be restricted to 0.5 mL in animals with a large volume ascites, in order not to compromise respiration. Phantom studies showed that the use of IP contrast agent led to a mean overestimation of radioactivity concentration ranging from 2.6% to 5.6%, as compared to saline. This was confirmed by animal studies that demonstrated an overestimation of 4.3%. This overestimation is below the values reported in human studies when high-density oral contrast agents are given to patients [12, 14, 15] and is acceptable, given the high diagnostic value associated with the use of IP contrast agent, particularly with respect to more accurate delineation of tumor volumes of interest used in quantitative analysis. It has been recently shown that current clinical PET/CT systems, using advanced reconstruction algorithms that model the point spread function of detector elements, can provide good quality images and accurate quantification of the radioactivity within the imaged lesions of mice bearing tumors [16, 17]. Furthermore, they offer the capacity of simultaneous imaging of multiple mice in static or dynamic mode. Therefore, clinical PET/CT devices could be a practical option in experiments requiring high scanning throughput and where dedicated small animal PET is not available. Although this study confirms the potential use of new generation PET/CT devices in cancer research, one could question to what extent our results can be extrapolated to PET/CT protocols using dedicated small animal imaging devices. First, the optimal sequence would be similar, as these parameters depend only on the kinetics of contrast agents in mice. Also, the volume of IV contrast agent is driven by the recommendation of the manufacturer and the

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Fig. 4. 18F-FLT dual contrast-enhanced PET/CT acquisition in a mouse bearing a transplanted left ovary tumor. Coronal PET slice at the level of the tumor (a), correlative CT slice (b; with zoomed image centered on the tumor), and correlative necropsy (c) are shown.

ethics committee requirements and should be kept identical. Regarding the volume and concentration of the IP contrast media, only the concentration might need to be adapted since the CT attenuation obtained on CT images will depend on the tube settings, which are significantly different in small animal CT devices. Choquet et al. [5] reported on the use of a 80 mgI/mL when using a 10-min acquisition at

80 kV and 0.45 mA, while Graham et al. [6] used a 100-mg I/mL concentration for a 3-min acquisition at 80 kV and 50 mA on the same small animal CT device. This concentration should be adapted to the microCT protocol, with the goal of obtaining a peritoneal enhancement well above that obtained in liver and spleen but without causing streak artifacts.

Fig. 5. Impact of high-density CT values on PET quantification: phantom studies. a Ratios of measured to true activity plotted against contrast media concentration in phantoms mimicking a tumor surrounded by various concentrations of contrast media; b percentage of overestimation of the PET values (mean SD), as compared to control (18F-FDG source surrounded by saline), in the same phantoms.

Fig. 6. Relationship between PET/CT quantitative data for 15 mice and ex vivo counting. Linear regression analysis (a) and Bland and Altman analysis (b) between quantitative PET/ CT data and ex vivo counting are shown.

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Conclusion
This study demonstrated that a dual radiologic contrast agent protocol is useful in PET/CT scanning of mice bearing abdominal tumors. The high densities induced by IP injection of contrast agent lead to a small but acceptable overestimation of quantitative PET data.
Acknowledgments. This work was supported and by a Fellowship from the Fondation de France, and by a grant from the French Ligue contre le cancer, Comit du Calvados. The authors thank Emily Hong, Jason Callahan, David Binns and Mark Scalzo for their help during animals PET acquisitions and ex vivo counting and the animal technologists and research assistants (Kerry Ardley, Rachel Walker, Susan Jackson, Jeannette Valentan, Ekaterina Bogatyreva and Laura Kirby) from the Centre for Molecular Imaging for animal care and tracer injections. Dr. Aide is indebted to Dr. Lerouge for her continuous support during this work. Conflict of Interest. The authors declare that they have no conflict of interest.

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