[Frontiers in Bioscience 14, 2193-2202, January 1, 2009]
Combined microPET/CT for imaging of hepatocellular carcinoma in mice
Christian von Falck1,2, Thomas Rodt1,2, Roman Halter2, Reinhard Spanel3, Michael Galanski1, Juergen Borlak2
1
TABLE OF CONTENTS
1. ABSTRACT
The EGF-transgenic mouse is a genetic model of hepatocellular carcinoma that allows for a comprehensive study of signal pathways, molecular interactions and the evaluation of novel therapeutic concepts. In this regard, non-invasive imaging tools for serial in-vivo monitoring of tumor load and growth are highly desirable. This study therefore aimed at demonstrating the feasibility of non-invasive in-vivo imaging of primary liver malignancies in mice using combined contrast-enhanced microCT and F-18 FDG microPET. In our murine disease model, microCT enabled imaging of primary liver tumors down to a lesional diameter of 0.9mm. F-18 FDG tumor-to-non-tumor ratio of HCCs was observed to be dependent on lesion size and linked to overpression of glucose transporters and hexokinase isoenzymes as determined by gene expression studies. Histopathologic analyses indicated an increased cellular dedifferentiation with increase lesion size, as well.
2. INTRODUCTION
Hepatocellular carcinoma (HCC) is the most frequent primary malignancy of the liver. It usually develops following a cascade of chronic liver injury including inflammation, regeneration, remodeling, fibrosis and, ultimately, irreversible cirrhosis. The most prominent risk factors are chronic hepatitis B and C infection (80%), Aflatoxin B1 exposure, alcohol-induced cirrhosis, hemochromatosis, fatty liver disease and alpha-1 antitrypsin deficiency. Therapeutic concepts with curative intention are mainly surgical and often limited to early stages of disease. Liver transplantation is a curative option for a subset of patients with locally advanced disease that meet the milan criteria. Different treatments applying systemic, local or combined delivery of chemotherapeutics and local ablative procedures have been described for later stages of disease, but their mid- to long-term results remain unsatisfying (1,2).
The ability to develop effective treatments requires the availability of relevant preclinical models to evaluate novel agents and approaches with respect to their activity against tumor. Different animal models have been established and allow for a comprehensive study of signal pathways, molecular interactions and the evaluation of novel therapeutic concepts. The EGF-transgenic mouse is a genetic model, that develops multiple highly malignant HCCs through targeted overexpression of a secretable form of epidermal growth factor (IgEGF) in hepatocytes (3). It mimics effectively the consequence of altered EGF signaling via the EGF receptor. There is strong evidence, that overexpression of liver mitogens like EGF or TGF-alpha may be an important mechanism in the development of HCC (4, 5). Recently, an oncogenomic study with EGF transgenic mice was reported. Essentially, genome-wide gene expression analysis at different stages of the disease enabled an improved understanding of the molecular events associated with EGF-induced HCC and an identification of novel therapeutic targets (6). In this regard, non-invasive preclinical imaging techniques are highly desirable as tools to monitor tumor growth and response to therapeutic interventions. As the use of contrast-enhanced microCT and 18F-FDG microPET has not yet been described for imaging of primary liver cancer in rodents, the rationale of this study was: 1. To demonstrate the feasibility of imaging primary liver malignancies in-vivo using contrast-enhanced micro computed tomography (microCT) and (18F)-2-fluoro-2-deoxyglucose (F-18 FDG) micro positron emission tomography (microPET) in a genetic mouse model of hepatocellular carcinoma and 2. to assess correspondence of 18F-FDG uptake with expression of glucose transporters and hexokinase isoenzymes.
3. MATERIALS AND METHODS
3.1. Tumor model and animal handling
All animal studies were approved by the institutional ethics committee and the local governmental authorities and were in accordance with national guidelines. The EGF- transgenic mouse model has been previously described in detail (3,6). Mice were maintained as hemizygotes in the Blk6 background. Transgenicity was confirmed by PCR of DNA extracted from tail biopsies. Mice were kept in cages with 1 - 4 mice per cage on sawdust on a 12-h light-dark cycle, at 50% relative humidity, in a temperature controlled (22�C) room. The animals received irradiated rodent chow and drinking water ad libitum. All imaging procedures were performed under inhalation anaesthesia with isoflurane (Isoba vet., Essex Pharma, Germany) at a concentration of 5% for anesthesia induction and 1-2% for maintenance. Animals breathed spontaneously via nose cone (Summit Anesthesia Solutions, Bend, OR, USA). The breathing rate was continuously monitored using a small pressure transducer (Biovet, m2m imaging, Newark, NJ, USA) and the mice were placed on a temperature controlled pad for warming (T/Pump, Gaymar, Orchard Park, NY, USA). Recovery time after the imaging procedure was usually less than five minutes.
3.2. In vivo Imaging
MicroCT and 18F-FDG microPET examinations were performed in 12 mice (age: 7.5�0.7 months, sex: m=5 / f=7). Follow-up examinations after two-weeks were performed in a subgroup of 5 mice (age: 7.5�0.5 months) to evaluate our imaging approach with regard to monitoring tumor growth.
3.3. microCT imaging
Mice were injected through the tail vein with approximately 200 - 300�l (10�l/g bodyweight) of a liver-specific iodinated contrast agent (DHOG, Fenestra LC, ART Inc., Saint-Laurent, Canada) 4 hours prior to the imaging procedure. The timing of injection and image acquisition was selected on the basis of manufacturer's guidelines, previously published studies (7) and personal experience (unpublished data). The microCT scan was obtained with a high-resolution small animal computed tomography scanner (eXplore Locus, GE Healthcare, Chalfont St. Giles, UK) immediately after the microPET scan. Misregistration artefacts were mimimized by using a multi-modality cradle that can be easily interchanged between the different scanners. Scan parameters were: tube voltage: 80kVp, tube current: 450�A, projections: 720, exposure time: 400ms, one average per frame. No respiratory gating was used. Total scan duration was 18 minutes for a single gantry rotation, covering an axial field-of-view of 33 mm. Image data was reconstructed using a cone-beam algorithm on an 8-node linux cluster. The resulting voxel size of the isotropic dataset was 45�m. Arbitrary attenuation values were converted to the Hounsfield scale using a calibration phantom with water, air and bone inserts.
3.4. microPET imaging
All animals were fasted for at least 6h prior to the imaging procedure. Anaesthesized mice received an injection of 10MBq (18F)-2-fluoro-2-deoxyglucose (18F-FDG, Department of Nuclear Medicine, Hannover Medical School, Hannover, Germany) in a total volume of 50 - 100�l sterile isotonic saline solution into the tail vein. Static images were acquired after 60 minutes using a high-resolution small animal PET camera (eXplore Vista, GE Healthcare, Chalfont St. Giles, UK). Total acquisition time was 30 minutes for a single bed position. Images were corrected for random events and scatter prior to reconstruction with a 3D-FORE/2D-OSEM iterative algorithm. No attenuation correction and no respiratory gating were used.
3.5. Image analysis and Statistics
MicroCT datasets were visualized and evaluated using the software Microview 1.2 (GE Healthcare, Chalfont St. Giles, UK). Rigid registration of PET and CT datasets based on anatomical landmarks (First galley + Professional galley + Licensed PDF was used to generate fused datasets. Image evaluation included assessment of contrast enhancement and detection and measurement of focal liver lesions. Lesion quantification was based on 2D-measurements of the largest diameter. Measurements were performed by two readers and the results were averaged. MicroPET datasets were evaluated using the software MMWKS (GE Healthcare, Chalfont St. Giles, UK). Following registration and image fusion, regions-of-interest (ROI) were manually defined in all focal liver lesions as detected by microCT and/or 18F-FDG microPET. In large, partially cystic lesions, the ROI was placed in the solid part of the tumor. The background (non-tumor) signal was determined by placing large ROI in the tumor-free liver parenchyma. The maximum count rate per volume was determined for each ROI and tumor-to-non-tumor ratios were calculated. Small lesions (<=5mm) were corrected for partial volume effects according to their size on microCT using precalculated recovery coefficients and technical specifications of the microPET scanner (8,9). Liver lesions were separated into three groups according to their size: small (<5mm), medium (5-10mm) and large (>10mm) lesions. The mean tumor-to-non-tumor ratio and the standard deviation were calculated. The students unpaired two-sided t-test was used to evaluate the statistical significance (p-value cutoff determined as 0.05).
3.6. Necropsy and histopathology
Mice were euthanized after the imaging procedure and the liver was removed for gross examination and histological analysis. Tumor tissue was fixed in 4% formaldehyde in PBS and embedded in paraffin. Paraffin blocks were sectioned into 3-5�m thick slices and stained with haematoxylin alone (H), haematoxylin and eosin (HE) and Ladewig's procedure. Single slices were additionally stained with periodic acid Schiff stain (PAS), Elastic van Giessen stain (EvG) and reticulin stain.
3.7. Gene Expression Analysis
Global gene expression was analyzed in a different group of EGF transgenic (n=6) and wild-type (n=4) mice using the Affymetrix Gene Chip technology. In brief, total RNA was isolated from frozen tumor tissue. Double-stranded cDNA was synthesized and used to generated biotin-labelled cRNA. The cRNA was fragmented and hybridized onto the Murine Genome U74Av2 array. After standard washing and staining procedures the arrays were analyzed using the Agilent GeneArray scanner. The Affymetrix Micoarrays Suite 5.0 was used for data analysis. Expression levels of glucose transporters (glut-1, glut-3) and hexokinase isoenzymes (hexokinase I / II) in small (<5mm; n = 5; pooled), medium (5 - 10mm; n = 4) and large (>10mm; n = 3) liver tumors were compared to transgenic, but non-tumorous liver tissue and wild-type animals. A total of 12 analyses for large tumors (3 tumors vs. 4 controls), 16 analyses for medium sized tumors (4 tumors vs. 4 controls), 4 analyses for small tumors (5 pooled tumors vs. 4 controls) and 12 analyses for tumor-free transgenic tissue (3 livers vs. 4 controls) were performed. Fold-change (FC) values were calculated as the ratio of the average expression levels for each gene between two tissue sets.
4. RESULTS
4.1. In-vivo imaging: microCT
A total of 57 liver lesions were detected in 12 examinations with contrast-enhanced microCT with an average of 4.7 tumors (� 2.3; range: 2 - 8) per animal and a lesion size varying between 0.9mm and 23mm (long axis diameter). Mean lesion size was 2.3mm (� 1.1mm) for small (<5mm; n = 34), 6.9mm (� 1.6mm) for medium (5 - 10mm; n = 9) and 14.5mm (� 4.1mm; n = 14) for large tumors. The morphologic appearance of the tumors was primarily hypodense. Small lesions were mostly homogenous, whereas larger lesions demonstrated areas of intermediate contrast uptake, that could be differentiated from tumor-free liver parenchyma. These areas were usually located either at the tumor periphery or as linear structures in the central parts of the lesion (Figure 1, 2). Large tumors with hypodense areas in CT showed typically pseudocystic intratumorous peliosis and necrosis in histopathology (Figure 2).
4.2. In vivo imaging: microPET
To allow for a more accurate anatomical localization of focal 18F-FDG uptake, we performed registration and fusion of microCT and microPET datasets prior to quantitative analysis of PET data. The additional information from contrast-enhanced microCT enabled differentiation of physiological tracer uptake from true pathologic findings as illustrated in Figure 2. The exact anatomical correlation was especially helpful at the esophageal-gastric junction due to its close anatomical relationship to the liver and the high physiological uptake. Fused datasets demonstrated that 18F-FDG uptake was absent in large parts of the tumor with hypodense appearance in contrast-enhanced microCT. However, 18F-FDG uptake was intense in peripheral parts of the tumors with intermediate uptake of the contrast agent in microCT. In 18F-FDG microPET the average tumor-to-non-tumor ratio was 1.05 (� 0.27) for small, 1.21 (� 0.33) for medium and 1.87 (� 0.53) for large tumors (s. Figure 4b), respectively. Differences were statistically significant for large lesions as compared to small and medium sized lesions (p < 0.01). There was no statistically significant difference between medium and small lesions.
4.3. Imaging of tumor growth
Follow-up examinations were performed in a subgroup of 5 transgenic mice with microCT-proven liver lesions after an interval of 14 days. A total of 16 lesions (medium: n = 3; small: n = 13) could be identified. Rapid tumor growth was seen with all lesions with a mean increase in the axial diameter of 86.6% (� 75,1%; p<0.05) (s. Figure 3a, b). Corresponding microPET demonstrated a mean increase in tumor-to-non-tumor ratio of 23.9% (� 29.4%; p<0.05). Heterogeneity in growth kinetics was observed and contributed to the relatively high standard deviation. Individual growth kinetics of the 5 mice are depicted in figure 3c. Except for one animal with a single tumor, tumor growth is given as mean growth of all tumors per animal.
4.4. Expression of glucose transporters and hexokinases
Expression of the glucose transporters glut-1 and glut-3 as well as the glycolytic enzymes hexokinase-1 and hexokinase-2 was analyzed separately for small, medium and large tumors and tumor-free transgenic livers and compared to normal liver tissue of wild-type mice (s. Figure 4a). In small and medium tumors, hexokinase-1 was moderately overexpressed (fold change (FC) = 2.4 (small) / 2.3 (medium), p<0.05) as compared with wild-type mice. No relevant change was noted for glut-1, glut-3 or hexokinase-2 (FC < 2). In large tumors, however, expression of hexokinase-1 increased further (FC = 3.7; p<0.05). Additionally, expression of glut-1 and hexokinase-2 were strongly increased (glut-1: FC = 5.7; hexokinase-2: FC = 6.3), aswell. No significant change was observed for tumor-free transgenic parenchyma when compared with wild-type animals. This data fits well with the results from the microPET studies, where a significant increase in 18F-FDG uptake was noted for large lesions, only.
4.5. Histopathology
Macroscopically, all examined mice developed multiple tumors of different size, which could be microscopically identified as HCC. Tumors ranged from highly to less differentiated. In general, small HCCs were adenoma-like tumors with well-developed hepatocytes and mainly bilayered trabeculae, whereas larger HCCs showed advanced cellular dedifferentiation, enhanced nuclear atypia and multilayered trabeculae (Figure 2, 5). Cystic degeneration which represented in fact an intratumorous pseudocystic peliosis and confluent necrosis were seen in larger tumors, only. The precursor lesions followed a regular developmental pattern in the transgenic EGF2-model: Basically, the tumor-free liver parenchyma presented large cell dysplasia with large hepatocytes and enlarged, repeatedly polymorphous nuclei. Its ubiquitous, rather diffuse appearance in the transgenic animals contrasted with the multiple consecutive nodular cell proliferations, so called dysplastic foci and nodules (according to human classification) (10).
5. DISCUSSION
Genetic disease models are invaluable for preclinical testing of novel anticancer drugs. In the past, mostly small animal models were established in immunodeficient nude mice by subcutaneous injection of tumor cells (e.g. xenotransplants). However, these models suffer from severe restrictions in terms of data translation to clinical research and the rather unphysiological tumor environment. More relevant models involve either implanting (orthotopic) tumors cells into an organ of interest or, particularly, the use of genetic animal models, where disease candidate genes and processes associated with malignant transformation can be investigated (11,12). However, the difficulty in using many of these animal models in the preclinical testing of novel anticancer drugs is the limited ability to accurately monitor development and growth of intraabdominal or visceral tumors without having to sacrifice the study animal. Here, we characterized a genetic mouse model of hepatocellular carcinoma by means of non-invasive in-vivo imaging techniques. Through combined contrast-enhanced microCT and 18F-FDG microPET we were able to visualize tumor load and growth of the primary liver malignancies. Furthermore, we were able to demonstrate correspondence of glucose transporter and hexokinase expression with tumor-size dependent uptake of 18F-FDG and histopathological findings. This mouse model together with the imaging platform may serve as a scenario for the evaluation of novel therapeutic approaches.
Micro computed tomography (microCT) is a morphologic imaging modality that depicts the anatomical features of different pathologies in great detail (13). Through serial examinations and volumetric measurements, microCT can serve as a powerful method to detect and monitor tumor burden and assess the therapeutic efficacy in small animal disease models (13-15). As in clinical CT imaging, contrast agents are mandatory for imaging of parenchymal organs due to the poor low-contrast performance of non-enhanced CT. Due to the long scan time of most commercially available microCT equipment and the rapid renal clearance of water-soluble iodinated contrast agents, liver-specific, long-lasting contrast agents are often required (7, 16, 17). Preclinical imaging of liver lesions in murine disease models with contrast-enhanced microCT has been described for metastatic disease of pheochromocytoma (18) and colon cancer (7). In both studies, microCT imaging was performed after the injection of an iodinated hepatobiliary contrast agent (1,3-bis (7-(3-amino-2,4,6-triiodophenyl) heptanoyl)-2-oleoyl-glycerol, ITG) as initially described by Weber et al. (19). Through an ApoE receptor-mediated pathway this contrast-agent is selectively taken up into hepatocytes and leads to opacification of normal liver parenchyma. Hepatic metastases are thus depicted as hypodense areas as the metastatic cells do not express the ApoE receptor (7). However, unlike for orthotopic tumor implants, the use of ITG to image primary liver malignancies has not been reported, yet. Depending on the degree of differentiation, these primary tumors have the potential to accomplish hepatocyte-specific functions. It is known from gadolinium-based liver-specific contrast media for magnetic resonance imaging that highly differentiated HCCs tend to show an uptake of liver-specific contrast agents (20, 21). This observation for liver specific contrast agents may help to explain the microCT appearance of the liver lesions in our genetic murine disease model. Through correlation of microCT with 18F-FDG microPET we were able to demonstrate that the solid parts of large tumors show an intermediate contrast-enhancement and can be differentiated from hypodense areas that correspond to necrotic or cystic parts of the tumor as well as from regular liver parenchyma with hyperdense appearance. Histopathology demonstrated necrotic or pseudocystic peliosis in these hypodense areas (Fig.2). Furthermore, we were able to demonstrate that serial contrast enhanced microCT examinations can be used to visualize changes in tumor size in our disease model through morphometric measurements. Side-by-side reading allows for comparison of microCT datasets acquired at different time points and enables lesion-by-lesion follow-up for RECIST- or WHO-like evaluation of therapeutic efficacy. Semi-automatic 3D volumetry tools are available and may allow a more precise measurement of tumor load and growth. However, in this work we relied on 2D measurements to allow comparability with our previous work (6)
(18F)-2-fluoro-2-deoxyglucose positron emission tomography (18F-FDG PET) is a metabolic imaging method that depicts the glucose metabolism of tissues and organs. It is well known, that most malignant tumors show an increased uptake of 18F-FDG due to enhanced glucose utilization (22). In general, 18F-FDG uptake in malignant tissue largely depends on the presence of glucose transporters and the glycolytic enzyme hexokinase (22). It is known, that HCCs show a heterogeneous biological and clinical behavior that is reflected in the variable uptake patterns of HCC in 18F-FDG PET. Increased uptake of 18F-FDG is only seen in about half of hepatocellular carcinoma patients (23-25). However, it has been demonstrated that 18F-FDG uptake is closely linked to the pathologic grading in HCC and that clinical outcome is poorer in HCC patients with a high tumor-to-non-tumor 18F-FDG ratio (26). In patients with cirrhosis and HCC scheduled for liver transplantation, 18F-FDG PET is helpful for estimating the post-transplantation risk of tumor recurrence, reflecting its ability to differentiate between different grades of biological aggressiveness (27). Comparable to these results from clinical PET, 18F-FDG uptake in HCC lesions in our murine disease model was not uniform, but dependent on lesion size and the degree of differentiation (26). We found for small liver lesions a glucose metabolism comparable to regular liver parenchyma. A significant increase in 18F-FDG uptake is seen particularly in large tumors (>10mm). These results were in concordance with gene expression data, where a significant overexpression of glut-1, hexokinase-1 and hexokinase-2 was observed in large tumors, only. As previously described, it is well known from clinical studies, that the uptake of 18F-FDG in HCC is closely linked to the degree of differentiation (25, 26). Our histopathologic evaluation demonstrates increasing cellular dedifferentiation of the hepatocellular carcinomas with expanding lesion size (Fig 2, 6), as recently reported (6). As our tumor model follows a well-defined genetic program based on EGF mitotic signalling, the link between lesion size and degree of dedifferentiation can be regarded as rather uniform.Indeed, contrast-enhanced microCT allowed for visualization and monitoring of tumor growth in our transgenic disease model; the practical application of serial microCT studies, especially in combination with microPET may, however, be limited due to the biological effects of the ionizing radiation (28, 29). The influence of cumulative radiation exposure through repeated studies on tumor development and therapeutic response has not been evaluated for our combined imaging approach, as yet. Further studies are therefore on the way to evaluate the usability of our imaging approach in preclinical therapy studies. In conclusion, combined contrast-enhanced microCT and 18F-FDG microPET imaging allows the in-vivo visualization of tumor burden and growth in a genetic mouse model of hepatocellular carcinoma. 18F-FDG tumor-to-non-tumor ratio is dependent on tumor size and in concordance with expression levels of glucose transporters and hexokinases.
6. ACKNOWLEDGMENTS
We gratefully acknowledge the support by GE Healthcare.
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Key Words microPET, microCT, Hepatocellular Carcinoma, Mouse
Send correspondence to: Christian von Falck, Department of Radiology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany, Tel: 49-511-532-3687, Fax: 49-511-532-3797, E-mail:c.v.falck@gmx.de