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Basic Science Investigation |
1 Molecular Imaging Program at Stanford (MIPS), Departments of Radiology and Bioengineering, Bio-X Program, Stanford University School of Medicine, Stanford University, Stanford, California; and 2 Crump Institute for Molecular Imaging, Department of Pharmacology, David Geffen School of Medicine, UCLA, Los Angeles, California
Correspondence: For correspondence or reprints contact: Sanjiv S. Gambhir, MD, PhD, James H. Clark Center, 318 Campus Dr., East Wing, First Floor, Stanford, CA 94305-5427. E-mail: sgambhir{at}stanford.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Key Words: microPET • small-animal CT • bioluminescence imaging • metastasis • mouse • melanoma
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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The biologic mechanism of metastasis has become better understood through the study of the migration and seeding of tumoral cells, tumor–stroma interactions, vascularization of tumors, genetic mouse models, and gene expression and proteomic patterns that correlate with metastasis. These insights have highlighted the need for more realistic models of tumor xenografts that suffer from altered vascularization, have no relevant tumor–stroma interactions, and often rely on immunodeficient mice (2). Recently, our group and others have made progress in adapting whole-body imaging techniques to small animals so that tumor burden can be visualized and quantified in a single animal over time. For this purpose, we have developed reporter gene systems for PET (3) and for bioluminescence imaging (BLI) (4). We have used these systems to study the role of specific genes in cancer progression (5), to study viral vector targeting of metastasis (6), and to noninvasively monitor novel treatments for melanoma metastases (7,8). We have also developed a trimodality fusion reporter gene that allows detection of gene expression by fluorescence, BLI, and PET (9). For the latter, the reporter probe used is 9-[4-18F-fluoro-3-(hydroxymethyl)butyl]guanine (18F-FHBG), which is phosphorylated by the reporter gene product, a mutant herpes simplex virus type 1 thymidine kinase (HSV1-tk) enzyme.
Recently developed small-animal CT systems (10,11) allow imaging of the anatomy of living mice with a high spatial resolution of 50–200 µm based on differential x-ray absorption by different tissues. Tissue contrast depends mainly on differences in tissue density and on the presence of mass amounts of contrast agents, providing limited if any molecular information. Hence, most current applications of small-animal CT have focused on specific tissues, such as bone (11), that can be well depicted because of their favorable contrast properties or tissues that have required the use of specific contrast agents (12). We have previously used small-animal CT to study a localized model of prostate cancer bone metastasis in the tibia of mice (13).
The high specificity of PET tracers for their molecular targets provides few anatomic landmarks—a disadvantage in studies in which the origin of the signal is not known, such as cell and metastasis trafficking studies. To improve this weakness of PET, we investigated the use of small-animal PET and small-animal CT as a multimodality imaging method allowing visualization of molecular information within an adequate anatomic framework. We used BLI to validate our findings and to compare combined small-animal PET/CT with BLI within the same animal.
Our study was a preliminary assessment of combined PET/CT with the following specific goals: establishment of a methodology to obtain coregistered small-animal PET and small-animal CT images, validation of these fused PET/CT images in tumor xenografts, evaluation of fused PET/CT in the detection of pulmonary melanoma metastases in a mouse model with 18F-FDG and 18F-FHBG (intravenously administered), evaluation of fused PET/CT in the detection of pulmonary melanoma metastases in a mouse model with 18F-FDG and 18F-FHBG, and comparison of this result to BLI metastasis detection.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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PET Procedure
The animals were kept fasting for a minimum of 6 h before undergoing PET. They were anesthetized using 1 L/min of 2% isoflurane in 100% oxygen. After receiving the anesthesia, they were given a 7.4-MBq injection of 18F-FDG or 18F-FHBG. After 1 h had been allowed for clearance of nonspecifically bound tracer, the animals received an intraperitoneal injection of 30 µL of a mixture of ketamine and xylazine (80 and 20 mg/mL, respectively) and subsequently were fixed in the prone position on a polystyrene bed in the presence of 6 fiducial markers (Fig. 1A). These consisted of a polymerase chain reaction tube filled with 2 µL of iohexol (Omnipaque; GE Healthcare) and 74 kBq of 18F-FDG to allow detection by CT and PET and were positioned next to the mouse. A 10-min static single-frame scan was acquired on the small-animal PET camera (microPET P4; Concorde Microsystems Inc.). Images were reconstructed by filtered backprojection using a ramp filter. The pixel size was 0.845 x 0.845 x 1.260 mm, and the resolution was approximately 1.85 mm.
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50 x 50 mm), and a precision-motion translation stage. The detector and x-ray source rotate about a fixed bed, allowing the mouse to be kept in the same horizontal position in the CT scanner as in the PET scanner. Directly after the PET scan, the animals were transported to and positioned in the CT scanner while still fixed to their polystyrene bed containing the fiducial markers. After administering a booster dose of 15 µL of ketamine/xylazine intraperitoneally, we scanned 2 bed positions (separated by 40.6 mm along the longitudinal axis) for 15 min each (190 projection images) to acquire the entire body of the mouse (total axial field of view, 90.6 mm). The total procedure took about 40 min, including 2 intervals of 5 min for positioning and system initialization. The images were acquired in step-and-shoot mode with the x-ray source set at 50 kVp and 450 µA. Two-dimensional slices of each bed position were reconstructed using a fanbeam resorting algorithm with a standard ramp filter. Images were reconstructed on a 256 x 256 pixel grid with a pixel size of 70 x 70 µm. For reconstruction in Hounsfield units, the system was calibrated using a 50-mL polypropylene tube filled with water. The resolution of the reconstructed images was 200 µm. We then merged the images of both bed positions into a single whole-body image dataset by discarding the 18 outer slices of each bed position and by computing a progressive weighted average of the overlapping 18 slices. No x-ray contrast medium was used.
Image Fusion and Quantitative Analysis
Both the PET and the CT datasets were imported using the nonproprietary AMIDE software (14), version 0.8.2 (http://amide.sourceforge.net). In the datasets of both modalities, "fiducial marker points" were placed at the center of each image of the fiducial markers. Subsequently, the fiducial marker points, and their respective datasets, were aligned using the alignment wizard and the Procrustes rigid-body alignment algorithm without scaling. The CT images were displayed in Hounsfield units. The PET images were converted to percentage injected dose per gram (%ID/g) by means of a calibration constant obtained from scanning a cylindric phantom in the small-animal PET scanner, assuming a tissue density of 1 g/mL, and dividing by the injected dose, decay-corrected to the time of scanning. Three-dimensional ellipsoidal regions of interest were drawn over the tumors and metastases. In the metastatic models, control regions of interest were drawn contralaterally or on nonpathologic tissue. The mean and maximum pixel values were obtained for both datasets. Tumor-to-background contrast was calculated using the %ID/g values and the formula (tumor – background)/(tumor + background).
BLI Procedure and Image Analysis
Each mouse received 30 µL of ketamine/xylazine by intraperitoneal injection and was placed supine. To generate the bioluminescence reaction catalyzed by Renilla luciferase, we injected its substrate, coelenterazine (20 µg in 100 µL of a 10% methanol/90% phosphate-buffered saline [pH 7.4] solution), in the lateral tail vein. Immediately afterward, we placed the animal supine in an IVIS system (Xenogen Inc.), which consists of a cooled charge-coupled-device camera (–105°C) mounted on a lighttight chamber. A photograph and a bioluminescent image consisting of a 1-min photon collection were acquired 3 times successively. The photograph and bioluminescent image were superimposed using Living Image software (version 2.20; Xenogen Inc.) and IGOR Pro image analysis software (version 4.06A; Wavemetrics). A region of interest was manually drawn over the body of the mouse, and the average radiance was measured in photons·s–1·cm–1·sr–1 (steradians). The highest value of the 3 scans is reported.
Radiotracers and Chemicals
18F-FDG was synthesized using the method described by Hamacher et al. (15) at a specific activity of approximately 185 TBq/mmol. 18F-FHBG was synthesized as previously described (16) at a specific activity of approximately 37 TBq/mmol. Coelenterazine was purchased from Biotium, Inc.
Cell Lines and Transfection Procedures
N2a murine neuroblastoma cells were obtained from Dr. Vincent Mauro (Scripps Research Institute). B16F0 and A375M human melanoma cells were provided by Dr. Michael Kolodney (UCLA). These cell lines were cultured in high-glucose Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution (respectively, 100 and 292 µg/mL). C6 rat glioma cells were provided by Dr. Margaret Black (Washington State University) and cultured in high-glucose deficient Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% L-glutamine (100 nM).
The A375M cell line was transfected with Firefly luciferase (A375M-Fluc (7)) or transduced with a lentiviral vector expressing the triple-fusion gene hRL-mRFP-tTK (humanized Renilla luciferase, monomeric red fluorescent protein, and truncated herpes simplex virus type 1 thymidine kinase) (A375M-3F (9)).
Herpes Simplex Virus 1 Thymidine Kinase Activity Assay
Cell or tumor extracts were incubated for 20 min with 8-3H-penciclovir to determine the formation of phosphorylated 8-3H-penciclovir. This phosphorylated 8-3H-penciclovir was trapped on a DE-81 filter (Whatman) while the parent compound was washed away. The remaining activity was counted in an LS-9000 liquid scintillation counter (Beckman) with BioSafe II scintillation fluid (Beckman). The results are reported as the percentage conversion of 8-3H-penciclovir ([disintegrations per minute per microgram of cell protein or tumor extract protein]/[disintegrations per minute of control sample] x 100).
Renilla Luciferase Assay
The Renilla luciferase assay was performed using a dual-luciferase reporter assay system (Promega). Each of the luciferase reactions was measured in a TD 20/20 luminometer (Turner Designs) for 10 s and normalized for protein content.
Statistical Analysis
All data were processed using Excel 2000 software (Microsoft). Results are expressed as mean ± SD of the mean. Linear regression analysis was performed to obtain the Pearson correlation coefficient. Comparisons of consecutive scans within the same animal and with control animals were performed using a paired and unpaired 2-sided Student t test, respectively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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18F-FDG PET/CT for Monitoring Growth of Tumor Xenografts
Small-animal CT measurements using an ellipsoidal ROI correlated well with caliper measurements for the 13 scans of the xenograft models, with tumor size ranging from 15.6 to 1,720 mm3 (r2 = 0.92; P < 0.0001) (Fig. 1B). The small-animal PET images showed a hypermetabolic signal in all tumors that could be detected by external palpation. The PET data showed increasing uptake of 18F-FDG in the N2a xenografts from day 8 to day 20 after inoculation, with a maximum final value of 4.52 ± 0.69 %ID/g. In the C6 xenograft model, 18F-FDG uptake did not significantly differ from day 12 to day 17 after implantation: the mean 18F-FDG uptake evolved from 4.54 ± 0.94 to 4.70 ± 1.67 %ID/g, and the maximum evolved from 7.07 ± 1.86 to 8.28 ± 0.78 %ID/g. Necrotic areas were visualized on the PET images for all 4 C6 tumors but were not detected on the corresponding CT images, regardless of the settings of the Hounsfield thresholding windows (Fig. 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Our method was based on the use of fiducial markers for fusion of the PET images with the CT images. Because the alignment error was about 5 times lower than the resolution of the small-animal PET scanner, we consider this method sufficiently accurate. We did not perform any partial-volume or attenuation correction, because this was beyond the scope of the study. The reported values are therefore underestimations for lesions smaller than about 4 mm in diameter.
The tumor xenografts could be visualized with 18F-FDG PET and CT. The N2a xenografts showed increasing uptake with time, whereas the C6 xenografts showed no differences in mean or maximum uptake over time. This finding was due to a partial-volume effect in the N2a xenografts, because the volume in the early scans was around 45 mm3 (diameter, 4.5 mm). In the C6 xenografts, the increase in mean uptake was less than the increase in the maximum pixel value because of necrosis in the tumor periphery. The fact that this necrosis was not seen on the CT images in any of the tissue windows we examined was due to the poor soft-tissue contrast of small-animal CT. This finding highlights the need for contrast agents to characterize tumors in small-animal CT. These agents can be of the classic iodine-based type, can be tissue-specific (12), or can come from another imaging modality, as in our approach using 18F-FDG PET.
To study metastasis, we first used an established model of lung metastasis (7) to take advantage of the contrast between tumoral soft tissue and the air in the lungs. We were successful in detecting pulmonary metastases with CT and 18F-FDG PET in 4 of 5 SCID mice 45 d after intravenous injection of A375M-Fluc melanoma cells. These mice did represent an advanced stage of disease, with lesions ranging in size from 7.5 to 58 mm3. High background uptake of 18F-FDG in heart, muscle, and brown fat did interfere with lesion delineation in 2 mice, and because pulmonary metastases of B16-F0 melanoma cells did not show increased 18F-FDG uptake, we further focused on reporter gene–based metastasis detection.
We used melanoma cells expressing the trimodality fusion reporter gene hRL-mRFP-tTK after lentiviral transduction (A375M-3F). We showed that reporter gene activity was still present after 12 cell culture generations, indicating that there is no significant silencing of reporter gene expression that might influence cell detection. Ten metastatic sites in total were detected by 18F-FHBG PET, with a mean uptake of 3.3 ± 1.3 %ID/g. The CT images showed changes in the lung and liver but not in the bone lesions. Earlier reports showed the presence of osteolytic lesions caused by A375 cells (17,18), but this is not the case for the derived cell line A375M, which was selected for its capacity to form lung metastases (19,20). Despite a higher uptake of 18F-FDG in the A375M-3F cells, 18F-FHBG was superior to 18F-FDG because of its much lower background uptake in the thorax and the head. Tumor-to-background contrast showed a significant increase of at least 0.27 for 18F-FHBG in the most relevant thoracic organs. This better performance of 18F-FHBG than of 18F-FDG in the thorax was anticipated because 18F-FHBG is specifically designed to be retained only in cells expressing HSV1-tk, a viral enzyme that in these mice is present only in the tumor cells. Furthermore, 18F-FHBG has a favorable pharmacokinetic profile, with rapid washout from normal tissues. In contrast, 18F-FDG uptake is related to glucose metabolism and is determined primarily by the number of active glucose transporters on the cell membrane and by hexokinase activity, and these are not restricted to tumor cells but also occur in brain, heart, brown fat, and liver. In intestines, the nonspecific physiologic hepatobiliary clearance of 18F-FHBG causes higher activity levels that are responsible for the limited detection of metastatic lesions in the peritoneal cavity, the lumbar spine, and pelvic bones. Novel molecules with better imaging characteristics, such as 2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil (21), are currently under investigation in our laboratory, and they might improve the detection of metastasis because they show a higher absolute uptake and lower gastrointestinal activity than does 18F-FHBG. Using a rabbit model of VX2-carcinoma pulmonary metastasis, Kondo et al. (22) showed that the performance of 18F-FDG PET was significantly greater for lesions larger than 4 mm than for smaller ones. Recently, oncologic imaging of mice has been reported in clinical combined PET/CT systems (23), but the low resolution of the system (6 mm) interferes with accurate quantification of metastatic lesions.
The BLI signal intensity is depth-dependent, resulting in a higher signal for superficial lesions than for deeper lesions that showed a comparable 18F-FHBG uptake, confirming our previous observations (9). BLI has the advantage of a relatively low cost and high throughput capability, making the depth dependence of the signal the major disadvantage in small animals. The other major limitation of BLI is that the planar nature of the images impedes accurate 3-dimensional localization of the signal. Recent studies have used BLI for evaluating metastases noninvasively (24), for evaluating lymphoma therapy (25), for studying the influence of differential gene expression on the pattern of metastasis formation (26), and for studying the importance of cellular receptors for metastasis development (27). The development of tomographic optical systems is progressing (28). These systems are using novel hardware that allows the acquisition of bioluminescence images from multiple angles and the use of reconstruction algorithms that apply models of photon transport in tissue to localize and quantify photon sources, but tomographic optical systems still need substantial validation regarding localization and quantification of the luminescent signal. The low cost, high sensitivity, and high throughput of planar BLI also make it suited as a routine monitoring tool that, when metastases are detected, can be complemented by 18F-FHBG small-animal PET/CT for anatomic localization and quantitation. A slightly different strategy was used by Kang et al. (29). They used BLI to detect MDA-MB-231 breast cancer bone metastases followed by PET reporter gene imaging of HSV1-tk expression under the control of a TGF-ß response element. Images produced by the latter represent signal transduction through the TGF-ß/Smad pathway and can show in vivo that these cells are engaged in Smad-dependent transcription while growing as bone metastases. Thus, PET and BLI can be used to detect 2 different molecular signals from the same metastasis.
We have demonstrated that multimodality imaging with PET, CT, and BLI is a powerful tool in the study of metastatic mouse models. Because of the low contrast of 18F-FDG, even in a cell line that has a high 18F-FDG uptake, PET reporter gene strategies are superior to 18F-FDG imaging except for abdominal metastases. On the basis of our results, we suggest the following imaging strategy: frequent BLI scanning should be used initially to monitor the animals while the metastases are below the detection thresholds of PET and CT. When BLI shows lesions that have reached the detection thresholds of PET or CT, combined PET/CT scans can than be used for detection (PET and CT), quantitation (PET), anatomic localization (CT), or density and volume measurement (CT).
A major limitation of the current study is the lack of a histologic gold standard for assessing tumor metastases. For identifying lesions as metastases, this lack was partially compensated for by the double detection of lesions on PET and BLI and by the in vitro analysis of tissue extracts. However, there might have been lesions that did not reach the threshold of detection by either imaging method, resulting in false-negative findings. This study, however, was designed to show the feasibility of using multimodality PET/CT and BLI to detect metastases. Future work will have to address the issue of sensitivity and determine the detection thresholds for the different modalities. For instance, scan times longer than 10 min for PET might increase sensitivity. Histologic standards will be needed, and the monomeric red fluorescent protein moiety of the multimodality reporter gene will be useful for detecting and identifying fluorescent metastatic cells in tissue specimens. The monomeric red fluorescent protein moiety is also important for fluorescence-activated cell sorting, rather than for in vivo fluorescent imaging, in which the high background and the weak signal generate a signal-to-noise ratio that is much lower than for BLI (30).
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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References |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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