Noninvasive Evaluation of Metabolic Tumor Volume in Lewis Lung Carcinoma Tumor-Bearing C57BL/6 Mice with Micro-PET and the Radiotracers 18F-Alfatide and 18F-FDG: A Comparative Analysis

Purpose To explore the value of a new simple lyophilized kit for labeling PRGD2 peptide (18F-ALF-NOTA-PRGD2, denoted as 18F-alfatide) in the determination of metabolic tumor volume (MTV) with micro-PET in lewis lung carcinoma (LLC) tumor-bearing C57BL/6 mice verified by pathologic examination and compared with those using 18F-fluorodeoxyglucose (FDG) PET. Methods All LLC tumor-bearing C57BL/6 mice underwent two attenuation-corrected whole-body micro-PET scans with the radiotracers 18F-alfatide and 18F-FDG within two days. 18F-alfatide metabolic tumor volume (VRGD) and 18F-FDG metabolic tumor volume (VFDG) were manually delineated slice by slice on PET images. Pathologic tumor volume (VPath) was measured in vitro after the xenografts were removed. Results A total of 37 mice with NSCLC xenografts were enrolled and 33 of them underwent 18F-alfatide PET, and 35 of them underwent 18F-FDG PET and all underwent pathological examination. The mean ± standard deviation of VPath, VRGD, and VFDG were 0.59±0.32 cm3 (range,0.13~1.64 cm3), 0.61±0.37 cm3 (range,0.15~1.86 cm3), and 1.24±0.53 cm3 (range,0.17~2.20 cm3), respectively. VPath vs. VRGD, VPath vs. VFDG, and VRGD vs. VFDG comparisons were t = -0.145, P = 0.885, t = -6.239, P<0.001, and t = -5.661, P<0.001, respectively. No significant difference was found between VPath and VRGD. VFDG was much larger than VRGD and VPath. VRGD seemed more approximate to the pathologic gross tumor volume. Furthermore, VPath was more strongly correlated with VRGD (R = 0.964,P<0.001) than with VFDG (R = 0.584,P<0.001). Conclusions 18F-alfatide PET provided a better estimation of gross tumor volume than 18F-FDG PET in LLC tumor-bearing C57BL/6 mice.


Results
A total of 37 mice with NSCLC xenografts were enrolled and 33 of them underwent 18 F-alfatide PET, and 35 of them underwent 18 F-FDG PET and all underwent pathological examination. The mean ± standard deviation of V Path , V RGD, and V FDG were 0.59±0.32 cm 3 (range,0.13~1.64 cm 3 ), 0.61±0.37 cm 3 (range,0.15~1.86 cm 3 ), and 1.24±0.53 cm 3 (range,0.17~2.20 cm 3 ), respectively. V Path vs. V RGD , V Path vs. V FDG , and V RGD vs. V FDG comparisons were t = -0.145, P = 0.885, t = -6.239, P<0.001, and t = -5.661, P<0.001, respectively. No significant difference was found between V Path and V RGD . V FDG was much Introduction Improving the accuracy of target volume estimation will help avoid unnecessary radiation of normal tissues and help avoid geographic tumor misses in patients with non-small cell lung cancer (NSCLC). Accurate metabolic tumor volume (MTV) assessment is a promising method for defining volumes in radiotherapy because of the development of functional imaging tools and image-guided radiotherapy.
PET with 18 F-FDG has been widely used to define target volumes in radiotherapy with increased metabolism. Studies have shown that FDG PET/CT can reduce inter-and intraobserver variability of target volume delineation to improve radiotherapy treatment planning [1,2]. In recent years, the possibly substantial impact of 18 F-FDG PET on the size and form of target volumes in lung cancer was demonstrated [3]. Most methods currently used in clinical practice are based on the use of some form of binary threshold, either fixed [4] or adaptive, that use tumor-to-background ratios [5]. Unfortunately, sometimes 18 F-FGD PET fail to provide satisfactory delineation of tumors characterized by heterogeneous activity distributions and fail to provide reproducible results for small tumors with low contrast because of intense cardiac uptake and high lung background [6]. Realistically, it is preferable that the radiotherapist uses the 18 F-FDG PET images as a reference only [7]. The radiation oncologist are expecting more accurate tool to contour target volume precisely.
Arginine-glycine-aspartic acid peptide (Arg-Gly-Asp, RGD) can specifically bind with integrin αvβ3, which is highly expressed in angiogenic tumors, to detect angiogenesis in noninvasive PET imaging. Angiogenesis plays an important role in the regulation of tumor growth, local invasiveness, and metastatic potential [8]. Chen et al invented a new simple lyophilized kit for labeling PRGD 2 peptide ( 18 F-ALF-NOTA-PRGD 2 , denoted as 18 F-alfatide) [9]. PET scanning with RGD allows specific imaging of integrin αvβ3 expression with minimal nonspecific activity accumulation in normal lung and heart tissue. Therefore, RGD PET may render high-quality orthotopic lung cancer images, enabling clear demarcation of both the primary tumor at the upper lobe of the left lung, as well as metastases in the mediastinum, contralateral lung, and diaphragm.
This study is designed to explore the value of 18 F-alfatide in the determination of MTV with micro-PET in lewis lung carcinoma (LLC) tumor-bearing C57BL/6 mice verified by pathologic examination and compared with those using 18 F-FDG PET.

Cell Culture and Animal Tumor Model Preparation
Murine LLC cells, recently used in a number of high-profile preclinical studies [10,11], was purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Murine LLC cells were grown in RPMI 1640, supplemented with 10% fetal bovine serum and 1% penicillin streptomycin antibiotic mixture in a humidified incubator (Heraeus, Hanau, Germany) at 37°C with 5% CO 2 atmosphere. LLC was injected (2.5×10 5 cells/100 μl/ mouse) into the right hind leg muscle of C57BL/6 mice.
Thirty-seven C57BL/6 inbred male mice were housed in a limited access animal facility. Animal room temperature and relative humidity were set at 22±2°C and 55±10%, respectively. Artificial lighting provided a 24 h cycle of 12 h light/12 h dark (7 a.m. to 7 p.m.). All animal procedures were in accordance with the Shandong Cancer Hospital & Institute Ethical Committee Guide for the care and use of Laboratory Animals. The Shandong Cancer Hospital & Institute Ethical Committee specifically approved this study. 18

F-alfatide PET and 18 F-FDG PET image acquisition
All mice underwent PET scans (Siemens Medical Solutions) using 18 F-alfatide and 18 F-FDG PET respectively within 2 days when tumor diameter reached approximately 10 mm. With the assistance of the Inveon system's positioning laser, the LLC tumor-bearing mouse was placed with its tumor located at the center of the field of view (FOV), where the highest imaging sensitivity can be achieved. 18 F-alfatide PET scans were performed 60 minutes after tail-vein injection of 2.4-3.5 MBp of 18 F-Alfatide under isoflurane anesthesia. During the acquisition period, a thermostat-controlled thermal heater maintained the body temperature of the mice. 18 F-FDG PET scans were performed for alll mice after 4 hours fasting and 60 minutes after injection of 18 F-FDG at a dosage of 2.6-3.6 MBp. The mice rested quietly in a warm box under isoflurane anesthesia for approximately 1 hour. Subsequently, the mice underwent the scanning, using the same parameters that had been employed for the 18 The images were reconstructed and analyzed using Inveon Acquisition Workplace v1.4.3 SP1 software. In the bio-distribution analysis of static images, regions of interest (ROIs) were placed over the tumor. The attenuation-corrected PET images were reconstructed and reviewed in axial, coronal, and sagittal planes; the same procedure was performed with a cine display of maximum-intensity projections of the PET data.
The 18 F-alfatide metabolic tumor volume (V RGD ) and 18 F-FDG metabolic tumor volume (V FDG ) were manually delineated slice by slice on the 18 F-alfatide and 18 F-FDG PET images (Fig 1). As a first step, an experienced physician (Z.F) used the ROI standard evaluation tool provided by the manufacture of the micro-PET system and a global logarithmic scaling to generate a "visual" PET GTV, comprising the tissue considered visually as part of the malignant primary tumor. ROI was positioned around the tumors slice by slice and obtained a set of data such as max (ROImax), mean and so on. The results were expressed as the standardized uptake value (SUVmax), which was calculated according to the following formula: ROImax×9500(CF value)×body weight [g]/injected activity [Bq]). The MTV was delineated on the PET images in transaction slice by slice with the 40% of the SUVmax, a threshold that has been used for the delineation tumor volume in previously study [12].

Pathologic volume measurement
All mice underwent tumor resection after the end of imaging (Fig 2). Specimens that were submitted fresh from the operating laboratory had three dimensional gross measurements taken from the tumor before being placed into 10% formalin. The pathologic tumor volume (V Path ) was estimated from the volume of an ellipsoid: where D long and D short were the longest and shortest diameters on the transverse plane before formalin fixation. D long and D short were the two orthogonal diameters obtained from the resected tumor specimen.

Statistical analysis
Statistical analysis was performed using SPSS software (version 17.0; SPSS, Inc). Student's unpaired t-test was used to detect differences between two sample means. The relationships between V Path and V RGD and between V Path and V FDG were tested by the Linear Regression Equation.
All analyses were 2-sided, and a P value of less than 0.05 was considered statistically significant.

Results
A total of 37 mice with NSCLC xenografts underwent pathologic examination after completing the PET imaging. Each mouse underwent 18 F-alfatide and 18 F-FDG PET imaging. In the 18 Falfatide PET imaging process, 4 mice were not scanned because of intravenous administration failure. For the same reason, 2 mice did not undergo 18 F-FDG PET scanning. Tumor volume was measured by various methods ( Table 1). The MTV was delineated in PET imaging performance of a novel αVβ3 integrin radiotracer, denoted as 18 Table 2 shows the comparison of V Path (n = 37) with V RGD and V FDG . V Path was considered the gold standard for MTV as it provides the closest estimation of real tumor volume. No difference was found between V Path and V RGD (0.59±0.32 vs. 0.61±0.37, t = -0.145, P = 0.885). V Path was significantly smaller than V FDG (0.59±0.32 vs. 1.24±0.53 cm 3 , t = -6.24, P<0.001).
The relationship between MTV and V Path (Fig 2) was also analyzed. V Path was strongly correlated with V RGD (n = 33, R = 0.964, P<0.001). V Path also had a moderately positive correlation with V FDG (n = 35, R = 0.584, P<0.001). The change in MTV of various specimens measured by 18 F-alfatide and 18 F-FDG PET imaging was consistent with the actual tumor volume. However, 18 F-alfatide PET more accurately reflected the actual tumor size than 18 F-FDG PET.

Discussion
The present study showed that larger MTVs were obtained with 18 F-FDG PET imaging than 18 F-alfatide PET imaging, and V RGD was similar to V Path in a mice model with NSCLC.
A study showed similar results in which the average tumor volume shown by 18 F-FDG PET imaging at 6 hours (0.37±0.08 cm 3 ), 24 hours (0.29±0.07cm 3 ), and 48 hours (0.18±0.03 cm 3 ) was larger than that estimated by histology (0.16±0.05cm 3 ), (0.17±0.06 cm 3 ), (0.06±0.02 cm 3 ) [13]. In 18 F-FDG PET imaging, many normal tissues display high uptake of 18 F-FDG [14], and this process is modulated by disease states such as diabetes [15] or physical exertion before and during scanning. These factors complicate the interpretation of 18 F-FDG PET imaging and help motivate the development of novel imaging tracers, such as 18 F-alfatide in this present study. In contrast to V FDG , we did not find any prior studies evaluating the measurement of MTV by 18 F-alfatide PET. 18 F-FDG uptake is significantly correlated with glucose metabolism, but it is phosphorylated and trapped within cells rather than metabolized and is rapidly cleared from the bloodstream [16]. Tumors are metabolically active but many normal tissues also display high uptake of 18 F-FDG. Para-tumor inflammation and infection may lead to high 18 F-FDG uptake, which potentially confounds cancer imaging. Kyoichi Kaira [17] et al showed that in tumor tissues, the amount of FDG uptake is not only associated with molecules relevant to glucose metabolism but also has a close relationship with hypoxia, angiogenesis and the mTOR signaling pathway. Research has reported that the amount of FDG uptake is determined by the expression of glucose transporter-1 (GLUT1), hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and microvessel density (MVD) [18,19]. These factors may explain why the MTV of 18 F-FDG PET is larger than V Path . Integrin αVβ3 is a member of the integrins family, and it can bind to a variety of plasma and extracellular matrix proteins containing the conserved amino acid sequence RGD [20]. The integrin αVβ3 receptor was expressed preferentially on various tumor cells and endothelial cells but was low on mature endothelial and epithelial cells [21,22]. Our findings indicate that RGD has high affinity for NSCLC in this study. However, the mechanism underlying why the MTV of 18 F-alfatide PET is closer to actual tumor volume is still unclear. Additionally, 18 F-alfatide PET is still not a perfect tool for tumor contouring due to the heterogeneity of the malignant lesions and partial volume effect. The criteria used to define MTV are subjective, and defining MTV depends on the interpretation of appropriate parameter settings in the evaluation of the image and resulting target volume contours [23]. The tumors had to be imaged with two tracers sequentially within two days as opposed to simultaneously, and therefore, variations in growth patterns could have partially influenced the results. Another limitation is that the LLC tumor model involves implantation of a tumor into a known location, and therefore, observers were aware of the tumor location. Furthermore, clinical situations are far more complicated than the animal models. Whether 18 F-alfatide PET has additional value than 18 F-FDG PET on tumor volume contouring is still unknown in patients with NSCLC and it deserves further study.

Conclusions
18 F-alfatide PET provided a better estimation of gross tumor volume than 18   Evaluation of Metabolic Tumor Volume with 18 F-Alfatide and 18 F-FDG PET