The uptake exploration of 68Ga-labeled NGR in well-differentiated hepatocellular carcinoma xenografts: Indication for the new clinical translational of a tracer based on NGR
- Authors:
- Published online on: August 31, 2017 https://doi.org/10.3892/or.2017.5933
- Pages: 2859-2866
Abstract
Introduction
Hepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths worldwide (1). Ultrasound, CT and MRI have been recommended as effective examination methods for the diagnosis of HCC, but the diagnostic efficacy of these traditional imaging methods is decreased when the lesions are smaller than 1 cm (2–4). Fluorine-18-labeled fluorodeoxyglucose positron emission tomography computed tomography (18F-FDG PET/CT) is an effective technique for early detection and precise staging of a variety of malignant diseases, based on the fact that most malignant tumors are glucose-avid (5). However, 18F-FDG PET/CT does not exhibit a good efficiency in detecting HCCs, particularly when they are well-differentiated (6–7). Clinical studies have revealed that the total sensitivity of 18F-FDG PET/CT in HCC is only 39–64%, and the average false-negative rate is up to 40–50% (8–9). Hence, the current guidelines do not recommend the use of 18F-FDG PET/CT imaging for the detection of HCC (1). Novel methods, such as 18F-FDG dual phase delay imaging (10), and 18F-FDG + 11C-acetate double tracer imaging (11,12), have been proposed as alternative methods to PET/CT for the detection of HCC. However, 18F-FDG dual phase delay imaging is complicated to operate and time-consuming; the 11C isotope has a short radioactive half-life and must be produced with a cyclotron, which greatly limits the clinical application of these methods (10,11). Hence, the applicability and effectiveness of these approaches are still controversial (13,14).
The uptake of various molecular imaging tracers by the tumor is mainly determined by the differential expression of the biomarkers (15). In well-differentiated HCC, it is widely accepted that the low uptake of 18F-FDG mainly results from the overexpression of glucose-6-phosphatase (G6Pase), which leads to the release of 18F-FDG from the tumor cells by converting the 6-phosphoric acid-FDG back to the FDG prototype (12,16). However, the low expression of glucose transporter 1 (GLUT1), which serves as the primary transporter of FDG, also decreases the uptake of 18F-FDG to some degree (17). Therefore, a new PET/CT tracer that targets other molecular biomarkers rather than those related to glucose metabolism should be developed to improve the clinical applications of PET/CT for the detection of well-differentiated HCC.
CD13 is a membrane-bound exopeptidase which has been shown to be overexpressed in a variety of tumor cells and neovascular endothelial cells (18–21). Therefore, it is a potential target for cancer diagnosis and therapy (22). The high expression rate of CD13 in a variety of HCC cell lines and clinical samples has already been confirmed (23–25). The asparagine-glycine-arginine (NGR) motif has been identified as an adequate ligand for CD13, displaying high affinity and specificity (26). It has been well established that, by labelling NGR peptide with 64Cu or 68Ga isotopes, the efficiency and specificity of NGR probes for the PET/CT imaging of CD13-positive tumors can be improved (27–30). Therefore, we questioned whether the radio-labeled NGR probe could non-invasively image well-differentiated HCC tumors. To address this issue, we performed a direct quantitative comparative preclinical study between radio-labeled NGR and FDG probes in well-differentiated HCC tumors.
Herein, the uptakes of 68Ga-NGR and 18F-FDG by the malignancies were compared in mouse xenograft models, bearing SMMC-7721-derived well-differentiated HCC tumors (positive for both CD13 and G6Pase). The models bearing HT-1080 tumors (CD13-positive and G6Pase-negative) and HT-29 tumors (negative for both CD13 and G6Pase) were also analyzed for comparison.
Materials and methods
Materials
The NGR peptide with the chelator 1,4,7,10-tetraazacyclododecan-N,N′,N″,N′″-tetraacetic acid (DOTA-GGGCNGRC, Cys4 and Cys8 being conjugated by a disulfide bridge) was synthesized by GL Biochem Ltd. (Shanghai, China), with a purity >95%, as confirmed by high-performance liquid chromatography (HPLC; Agilent Technologies, Santa Clara, CA, USA). 68GaCl3 was obtained from the 68Ge-68Ga radionuclide generator (ITG Co., Munich, Germany). The cell lines SMMC-7721, HT-1080 and HT-29 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in RPMI or high glucose Dulbecco's modified Eagle's medium (DMEM) culture supplemented with 10% fetal bovine serum (FBS) (all from HyClone, Logan, UT, USA) in 37°C humidified atmosphere containing 5% CO2. All other reagents were commercially purchased and used without further purification.
68Ga-NGR synthesis
68Ga-NGR was prepared using the following protocol: 68GaCl3 (200 µl, 111–148 MBq) was added into DOTA-NGR (2 µg), which was previously dissolved in deionized water (2 µl). The pH value was adjusted to 4.0 by adding sodium acetate (10 µl, 1.25 M). The mixture obtained was subsequently incubated at 90–100°C for 10 min.
The radiochemical purity (RCP) was analyzed by the mean of HPLC. The analytic HPLC experiment was performed using a RP C18 column (Zorbax; 5 µm, 4.6×250 mm). The HPLC method employed is described below: solvent A consisted of 0.05% trifluoroacetic acid (TFA) in water, and solvent B consisted of 0.05% TFA in acetonitrile. The flow rate was set to 1 ml/min; and the gradient was 0–3 min, 5–5% solvent B; 3–20 min, 5–65% solvent B. All experiments were repeated four times under the same conditions.
Western blot analysis
After the cultures (HT-1080, SMMC-7721 and HT-29) were subjected to the lysis process, the protein samples were quantified by bicinchoninic acid (BCA) assay and heated up at 95°C for 10 min, followed by the loading buffer addition. The samples, each containing 40 µg proteins, were loaded onto sodium dodecyl sulfate-polyacrylamide gel in the electrophoresis gels and transferred to polyvinylidene fluoride membrane filters (Life Technologies, Foster City, CA, USA). The membranes were blocked for 2 h in Tris-buffered saline containing 0.01% Tween-20 (TBST) and 5% bovine serum albumin. Afterwards, an overnight incubation process at 4°C was accomplished, having the appropriate primary antibodies in TBST with 2.5% bovine serum albumin as following: mouse anti-CD13 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-G6Pase (1:1,000), rabbit anti-GLUT1 (1:1,000) (both from Abcam, Cambridge, MA, USA) and anti-β-actin antibodies (loading control; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China). After 1 h of incubation with the peroxidase-conjugated secondary antibody (1:5,000; ZSGB-BIO, Beijing, China), the membranes were washed with TBST. The blots were developed with an enhanced chemiluminescence kit (Thermo Scientific), and images were acquired using a ChemiDOC XRS+ instrument (Bio-Rad, Hercules, CA, USA).
The relative expression levels of detected proteins were quantified in accordance with a previously reported method (31). The statistics were acquired based on four repeated experiments.
Cell uptake studies
For the cellular uptake studies, the incubation of SMMC-7721, HT-1080 and HT-29 cells was carried out in plastic tubes, with a volume of 450 µl containing 5×105 cells. This was followed by the addition of 50 µl (37 kBq) 68Ga-NGR or 18F-FDG into each tube. The incubation process was carried out at 37°C for 30, 60, 90 and 120 min. Afterwards, cold phosphate-buffered saline (PBS) was added to the mixture and centrifuged to discard the supernatant. For the cell blocking studies, SMMC-7721 cultures were incubated along with 68Ga-NGR (50 µl, 37 kBq) and unlabeled DOTA-NGR (2 µl, 10 µg/µl). The dosage of unlabeled DOTA-NGR was performed as previously reported (32,33). The radioactivity of the precipitate was counted using a γ-counter (with decay correction, the same below). These experiments were also repeated four times under the same conditions.
In vivo micro-PET/CT imaging studies
All the animal studies were performed according to the protocol approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University. Fifty-four male BALB/c nude mice (4–6 weeks old, with a body weight of 20–25 g) were used. The xenograft models were generated by administering a subcutaneously injection containing 2.5×106 tumor cells into the upper flanks of nude mice; the HT-1080 xenografts were constructed on the left scapular region of nude mice, the HT-29 and SMMC-7721 xenografts were both on the right side. The number of each type of xenograft model was 18. The tumors were allowed to grow for 2–3 weeks until they reached a volume of 0.5–1 cm3, as required for the micro-PET/CT imaging and biodistribution experiments. The nude mice bearing SMMC-7721 tumors were randomly divided into three groups, each group including six mice, and marked as group 1, 2 and 3; the same treatments were administered to HT-1080 and HT-29 xenografts. The mice in group 1 were injected with 7.4 MBq 18F-FDG by the tail vein, and the images were eventually acquired at 30, 60 and 90 min post-injection using a micro-PET/CT scanner (Mediso Medical Imaging Systems, Budapest, Hungary). In contrast, the mice composing group 2 were injected with 7.4 MBq 68Ga-NGR; while the mice in group 3 were administered 7.4 MBq 68Ga-NGR as well as an excessive unlabeled amount of NGR (20 mg/kg). The scanning time (30, 60 and 90 min) and blocking dosage were based on previously reported procedures (27,32); the rest of the steps involved were similar as for group 1. The regions of interest (ROIs) over tumors on the coronal images were carefully delineated, and the average signal levels in the ROIs were expressed as % ID/g (percentage of injected dose per gram of tissue).
Immunofluorescence staining and immunohistostaining studies
HT-1080, SMMC-7721 and respectively HT-29 cells were planted in a 24-well plate at a density of 5×104 cells/well. After overnight incubation at 37°C in 5% CO2, the cells were washed three times with cold PBS and fixed with 4% paraformaldehyde for 15 min. The cells were then blocked with 3% bovine serum albumin at room temperature for 1 h and subsequently incubated with mouse anti-CD13 for 12–16 h at 4°C (1:200; Abcam). The cultures were washed with PBS and further incubated with a fluorescent secondary goat anti-mouse FITC-antibody (1:200; ZSGB-BIO) for 2 h at ambient temperature. This was followed by repeated washings (five times) and continued by the addition of 4,6-diamidino-2-phenylindole (DAPI) to the cells which were further incubated for 10 min to stain the nuclei. The cells were analyzed using an Olympus IX71 fluorescence microscope (Olympus, Tokyo, Japan).
The mice were sacrificed by cervical dislocation, then HT-1080, SMMC-7721 and HT-29 cell-derived tumor tissues were fixed with 4% of paraformaldehyde, embedded in paraffin and sectioned at 0.3 µm. Then, the sections were blocked with 3% serum for 30 min and underwent antigen retrieval by heat mediation in a citrate buffer; this was followed by incubation with mouse anti-CD13 antibody (1:100; Abcam) at 4°C for 16–18 h. The sections were subsequently washed three times with PBS and incubated with the peroxidase-conjugate secondary antibody (1:500; goat anti-mouse IgG for mouse anti-CD13; ZSGB-BIO) at room temperature for 1 h, washed again with PBS, stained using 0.025% 3,3′-diaminobenzidine solution in PBS, and eventually counterstained with hematoxylin. The sections were visualized by the aid of an Olympus BX51 microscope (Olympus).
Biodistribution studies
The xenograft models bearing SMMC-7721 tumors were randomly divided into two groups (n=6), which were individually injected with 7.4 MBq of either 68Ga-NGR or 18F-FDG via the tail vein. At 30 min post-injection, the mice were sacrificed by cervical dislocation and their blood, tumors and the major organs (heart, lung, liver, spleen, pancreas, stomach, intestine, kidney, muscle, bone and brain) were harvested and weighed. The radioactivity of each sample was measured by γ-counter. The data collected are expressed as % ID/g.
Statistical analysis
Quantitative data are expressed as mean ± SD. Means were compared using one-way ANOVA and Student's t-test. P values <0.05 were considered to indicate a statistically significant result.
Results
68Ga-NGR radiochemistry properties and cell uptake studies
68Ga-NGR was successfully synthesized with an RCP as high as 98.2±0.9% (Fig. 1A). The specific activity of the tracer was found to be between 55–80 MBq/nmol.
The cellular uptake studies revealed that 68Ga-NGR could bind to both SMMC-7721 and HT-1080 cells, but not to HT-29 cells and the CD13 blocked SMMC-7721 cells (Fig. 1B). However, 18F-FDG could be absorbed by HT-1080 and HT-29 cells, but not by SMMC-7721 cells (Fig. 1C). For SMMC-7721 cells, after 2 h of incubation, the uptake of 68Ga-NGR was significantly higher than that of 18F-FDG (1.23±0.11 vs. 0.515±0.14%, P=0.0024 <0.01). Furthermore, after being blocked by non-radioactive NGR (CD13 blocked), the uptake of 68Ga-NGR in SMMC-7721 (0.59±0.08%) cells was significantly lower than that in non-blocked SMMC-7721 cells (1.23±0.11%, P=0.0013 <0.01), which demonstrated that 68Ga-NGR was selectively targeting the CD13 receptor.
Western blot analysis
The western blot analysis of CD13 expression in HT-1080 and SMMC-7721 cells showed positive bands correlated to the molecular size of CD13, whereas a low signal band was observed for HT-29 cells (Fig. 2A). In addition, the western blotting analysis of G6Pase indicated that two weak bands were observed for HT-1080 and HT-29 cells, and G6Pase was expressed in the SMMC-7721 cells. The semi-quantitative analysis [the semi-quantitative analysis method based on a previously reported method (31)] results of the expression levels of CD13 (Fig. 2B), G6Pase (Fig. 2C) and GLUT1 (Fig. 2D) corresponded to our observed results. The CD13 expression levels in the HT-1080 and SMMC-7721 cells were notably higher compared to that of HT-29 (0.80±0.12 vs. 0.07±0.04, P=0.0097<0.01; and 0.54±0.10 vs. 0.07±0.04, P=0.02<0.05). The G6Pase expression level in the SMMC-7721 cells exhibited an obviously greater value in comparison with HT-1080 and HT-29 cells (0.75±0.15 vs. 0.17±0.08, P=0.0083<0.01; and 0.75±0.15 vs. 0.12±0.07, P=0.0085<0.01). In addition, the GLUT1 expression level in SMMC-7721 cells was significantly lower than that of HT-1080 and HT-29 cells (0.14±0.06 vs. 0.77±0.10, P=0.0098<0.01 and 0.14±0.06 vs. 0.79±0.06, P=0.0094<0.01).
In vivo micro-PET/CT imaging studies
The imaging efficacies of 18F-FDG and 68Ga-NGR were evaluated and compared in nude mice bearing HT-1080, SMMC-7721 and HT-29 xenografts by micro-PET/CT imaging (Fig. 3A). The results revealed that 18F-FDG uptake in SMMC-7721 tumors was significantly lower than that in HT-1080 and HT-29 tumors (0.73±0.26% ID/g vs. 3.47±0.31% ID/g, P=0.0031<0.01 and 0.73±0.26% ID/g vs. 3.17±0.29% ID/g, P=0.0197<0.05) at 30 min post-injection. Regarding the uptake of 68Ga-NGR in SMMC-7721 tumors, it was similar to that in the CD13-positive control HT-1080 tumors (2.46±0.23% ID/g vs. 2.17±0.21% ID/g, P=0.18>0.05), and was significantly higher than that in the CD13-negative control HT-29 malignancy (2.17±0.21% ID/g vs. 0.67±0.20% ID/g, P=0.0148<0.05). For SMMC-7721 tumors, the uptake of 68Ga-NGR was 2.97-fold higher compared to that of 18F-FDG (2.17±0.21% ID/g vs. 0.73±0.26% ID/g, P=0.0030<0.01). The corresponding quantitative analyses are showed in Fig. 3C. In addition, 68Ga-NGR in SMMC-7721 tumors exhibited a higher imaging efficiency than that in CD13 blocked SMMC-7721 tumors (2.17±0.21% ID/g vs. 0.55±0.12% ID/g, P=0.0078<0.01), which implies that 68Ga-NGR selectively targets CD13.
The tumor/liver (T/L) ratios of 18F-FDG and 68Ga-NGR in SMMC-7721 xenografts at 30 min post-injection were calculated and are compared in Fig. 3D, and the quantitative comparison results revealed that the T/L ratio of 68Ga-NGR was 2.05±0.16, which was significantly higher than that of 18F-FDG (1.01±0.25, P=0.0290<0.05) and 68Ga-NGR (CD13 blocked) (0.81±0.18, P=0.0294<0.05). In addition, the tumor/background (T/B) ratios of 68Ga-NGR in HT-1080 and HT-29 xenografts were 2.14±0.43 and 1.18±0.25 respectively, which indicated that 68Ga-NGR could image CD13+ tumors.
Multiple time-point (30, 60 and 90 min) micro-PET/CT scans were performed in SMMC-7721 xenograft (Fig. 3B). The tumor uptakes of 68Ga-NGR were 2.17±0.21 % ID/g, 1.66±0.17% ID/g and 1.47±0.15 % ID/g, respectively. The dynamic uptake tendencies of 68Ga-NGR in major organs (blood, liver, kidneys and muscle) are shown in Fig. 3E.
Immunofluorescence staining and immunohistostaining studies
The immunofluorescence staining study indicated that CD13 was selectively expressed at the level of the cell surface of HT-1080 and SMMC-7721 tumors, but not in HT-29 cells (Fig. 4A).
The results obtained from immunohistostaining assays further indicated that CD13 was expressed on the cell surface of HT-1080 and SMMC-7721 tumor cells as well as the neovascular endothelial cells, but not in HT-29 tumor cells, which was consistent with the results obtained by western blotting analysis and immunofluorescence staining (Fig. 4B).
Biodistribution studies
The in vivo biodistribution analysis showed that, except for the kidneys, the highest accumulation of 68Ga-NGR was detected in SMMC-7721 HCC tumors (Fig. 5). The accumulation of 68Ga-NGR in SMMC-7721 HCC tumors was 2.81-fold higher compared to that of 18F-FDG (2.70±0.65% ID/g vs. 0.96±0.19% ID/g, P=0.0298<0.05).
Discussion
In the present study, we found that the uptake of 68Ga-NGR was 2.97-fold higher than that of 18F-FDG by SMMC-7721 tumors, based on micro-PET/CT imaging studies, and 2.81-fold higher, based on biodistribution studies. However, the T/L ratio of 68Ga-NGR was 2.03-fold higher than that of 18F-FDG in the well-differentiated HCC models. These findings strongly reflected that 68Ga-NGR was potentially superior to 18F-FDG for imaging well-differentiated HCC.
The development of radio-labeled peptide probes, such as RGD and NGR, for diagnostic and therapeutic applications, has rapidly expanded in the last decades (34–37). On the aspect of clinical practice, the safety and the applicability of these probes have been well established in lung cancer (38–40), brain metastases (41), breast cancer (42) and rheumatoid arthritis (43). Even though HCCs are worldwide malignant tumors with high incidence of morbidity, there has been little progress in their detection by radio-labeled probes. Roland et al utilized [68Ga]NODAGA-RGD to image patients with HCCs, but the probe uptake by the HCC tumors and the tumor/liver ratio were both insufficient for HCC detection (44). This may be attributed to the low receptor expression and the higher background radioactivity in the cirrhotic liver leading to poor detection. As the high expression of CD13 in well-differentiated HCC has been confirmed in previous studies (23–25), utilizing NGR-based probes for HCC detection is an attractive prospect. In the present study, the positive expression of CD13 was confirmed in SMMC-7721 and HT-1080 cell-derived tumors and neovascular endothelial cells by western blot or immunohistochemical analyses. Furthermore, the high uptake and T/L ratio of 68Ga-NGR in the well-differentiated HCC xenografts indicated the ability of 68Ga-NGR to image well-differentiated HCC. However, the high expression rate of CD13 has already been confirmed not only in a variety of HCC cell lines, but also clinical samples (23,24), which suggests that 68Ga-NGR may have high specificity and sensitivity for further clinical diagnosis of well-differentiated HCCs. Poor-differentiated HCCs have relatively low expression of G6Pase and high expression of GLUT1 (17), which may contribute to the low uptake of 18F-FDG by poor-differentiated HCC. The uptake performance of 68Ga-NGR, in poor-differentiated HCC, warrants further investigation.
Chronic hepatitis is the leading cause of cirrhosis and hepatocellular carcinoma (HCC) (45), and early discrimination of liver cirrhosis from chronic hepatitis is critical for effective treatment and optimal prognosis (46). The probe based on the RGD peptide was reported to specifically bind to integrin αvβ3 receptors in activated hepatic stellate cells (HSCs). The hepatic deposition amount of RGD probe was markedly increased in parallel with the development and progression of liver fibrosis, which indicate that we could quantitatively assess the extent of liver fibrosis with probes based on RGD (47). Since CD13 has also been reported to be a highly specific marker of hepatocyte differentiation (23,25), it is possible for 68Ga-NGR to differentiate cirrhosis from chronic hepatitis and HCCs from non-HCCs. However, how 68Ga-NGR specifically performs in cirrhosis or non-HCCs, such as cholangiocellular carcinoma (CCC) or focal nodular hyperplasia (FNH), warrants further investigation.
The additional advantages of peptide-based radiopharmaceuticals, beyond 18F-FDG, require further exploration. In the present study, we found that for the imaging of well-differentiated HCC, the 68Ga-NGR probe was more efficient, compared to 18F-FDG, in three distinct aspects, as discussed below. Firstly, the uptake of 68Ga-NGR was substantially higher compared to that of 18F-FDG in well-differentiated HCC tumors, which indicated its potential to be used as an improved alternative to 18F-FDG for the diagnosis of well-differentiated HCCs. Secondly, compared with 64Cu or 99mTc-labeled NGR, reported in previous studies (27,48), 68Ga-NGR displayed a low uptake in liver tissue; and the T/L ratio of 68Ga-NGR was 2.05±0.16, which was significantly higher than that of 18F-FDG (1.01±0.25), 64Cu-labeled NGR (0.35) (27), and 99mTc-labeled NGR (0.34) (48). Lastly, as 68Ga is a cost-effective radioisotope that could be easily obtained from a 68Ge/68Ga generator, 68Ga-NGR peptide-based radiopharmaceuticals could be effortlessly and inexpensively produced. Altogether, these 68Ga-NGR properties indicate its excellent potential for clinical translation in the future.
Generally, malignant tumors are highly phenotypically and genetically heterogeneous, which is an important contributing factor for clinical diagnosis and treatment decision (49). The molecular imaging approach provides the opportunity to non-invasively visualize the expression level of molecular markers in vivo, and hence, it is superior to traditional histopathological examination (50). Based on the present study, 68Ga-NGR could image CD13-positive tumors (SMMC-7721 and HT-1080 tumors) and 18F-FDG could image G6Pase-negative tumors (HT-1080 and HT-29 tumors), which indicates that the different uptake rates of the tracers (68Ga-NGR and 18F-FDG) are closely associated with the expression levels of the corresponding molecular markers (CD13 and G6Pase, respectively). These results imply the possibility of a non-invasive molecular imaging method to evaluate the expression of corresponding biomarkers.
Despite these promising findings, the advantages of 68Ga-NGR in imaging efficiency have only been demonstrated in in vitro and xenograft models. More HCC cell lines and orthotopic HCC models may be explored in our future studies. However, it is vital for clinical translation studies to validate the actual effect of 68Ga-NGR in order to determine the specificity and sensitivity of 68Ga-NGR for the diagnosis of well-differentiated HCCs in the clinic.
In summary, the present study demonstrated that the uptake of 68Ga-NGR was significantly higher than that of 18F-FDG in well-differentiated HCC xenografts and therefore 68Ga-NGR was more suitable for the imaging of well-differentiated HCC, which suggests its future potential for clinical translation in the PET/CT diagnosis of HCC.
Acknowledgements
The present study was supported by the National Natural Science Foundation of China (grant nos. 81230033, 81401442, 81227901 and 81601521), the National Key Research and Development Program of China (grant no. 2016YFC0103804), the Xijing Hospital Subject Promoting Plan (grant nos. XJZT15G01, XJZT15M07), the Shaanxi Science & Technology Co-ordination & Innovation Project (grant no. S2016TQSF0021) and the Shaanxi Science & Technology Co-ordination & Innovation Project (grant no. 2016KTCQ03-09). We would like to thank Professor Fan Wang from the Medical Isotopes Research Center of Peking University (Beijing, China) for her generous support, and Guiyu Li, Mingru Zhang and Shu Zong for their technical assistance in conducting the present study.
References
|
Jemal A, Bray F, Center MM, Ferlay J, Ward E and Forman D: Global cancer statistics. CA Cancer J Clin. 61:69–90. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Benson AB III, Abrams TA, Ben-Josef E, Bloomston PM, Botha JF, Clary BM, Covey A, Curley SA, D'Angelica MI, Davila R, et al: NCCN clinical practice guidelines in oncology: Hepatobiliary cancers. J Natl Compr Canc Netw. 7:350–391. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Kwon HJ, Byun JH, Kim JY, Hong GS, Won HJ, Shin YM and Kim PN: Differentiation of small (≤2 cm) hepatocellular carcinomas from small benign nodules in cirrhotic liver on gadoxetic acid-enhanced and diffusion-weighted magnetic resonance images. Abdom Imaging. 40:64–75. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Yu NC, Chaudhari V, Raman SS, Lassman C, Tong MJ, Busuttil RW and Lu DS: CT and MRI improve detection of hepatocellular carcinoma, compared with ultrasound alone, in patients with cirrhosis. Clin Gastroenterol Hepatol. 9:161–167. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Zhan HW, Xu W, Ye XJ, Zhao CL, Zhang H, Li J, Yao Q and Zhang LJ: Application of FDG-PET for detection of malignant lesions in patients with elevated blood tumor markers but without a history of malignancy. Mol Med Rep. 2:837–842. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Delbeke D, Martin WH, Sandler MP, Chapman WC, Wright JK Jr and Pinson CW: Evaluation of benign vs malignant hepatic lesions with positron emission tomography. Arch Surg. 133:510–515. 1998. View Article : Google Scholar : PubMed/NCBI | |
|
Iwata Y, Shiomi S, Sasaki N, Jomura H, Nishiguchi S, Seki S, Kawabe J and Ochi H: Clinical usefulness of positron emission tomography with fluorine-18-fluorodeoxyglucose in the diagnosis of liver tumors. Ann Nucl Med. 14:121–126. 2000. View Article : Google Scholar : PubMed/NCBI | |
|
Hayakawa N, Nakamoto Y, Nakatani K, Hatano E, Seo S, Higashi T, Saga T, Uemoto S and Togashi K: Clinical utility and limitations of FDG PET in detecting recurrent hepatocellular carcinoma in postoperative patients. Int J Clin Oncol. 19:1020–1028. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Chen YK, Hsieh DS, Liao CS, Bai CH, Su CT, Shen YY, Hsieh JF, Liao AC and Kao CH: Utility of FDG-PET for investigating unexplained serum AFP elevation in patients with suspected hepatocellular carcinoma recurrence. Anticancer Res. 25:4719–4725. 2005.PubMed/NCBI | |
|
Cheng G, Torigian DA, Zhuang H and Alavi A: When should we recommend use of dual time-point and delayed time-point imaging techniques in FDG PET? Eur J Nucl Med Mol Imaging. 40:779–787. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Park JW, Kim JH, Kim SK, Kang KW, Park KW, Choi JI, Lee WJ, Kim CM and Nam BH: A prospective evaluation of 18F-FDG and 11C-acetate PET/CT for detection of primary and metastatic hepatocellular carcinoma. J Nucl Med. 49:1912–1921. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Ho CL, Yu SC and Yeung DW: 11C-acetate PET imaging in hepatocellular carcinoma and other liver masses. J Nucl Med. 44:213–221. 2003.PubMed/NCBI | |
|
Lam MG, Kwee TC, Basu S and Alavi A: Underestimated role of 18F-FDG PET for HCC evaluation and promise of 18F-FDG PET/MR imaging in this setting. J Nucl Med. 54:1510–1511. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Cheung TT, Ho CL, Chen S, Chan SC, Poon RT, Fan ST and Lo CM: Reply: Underestimated role of 18F-FDG PET for HCC evaluation and promise of 18F-FDG PET/MR imaging in this setting. J Nucl Med. 54:1511–1512. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Tichauer KM, Wang Y, Pogue BW and Liu JT: Quantitative in vivo cell-surface receptor imaging in oncology: Kinetic modeling and paired-agent principles from nuclear medicine and optical imaging. Phys Med Biol. 60:R239–R269. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Sharma B, Martin A and Zerizer I: Positron emission tomography-computed tomography in liver imaging. Semin Ultrasound CT MR:. 34:66–80. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Izuishi K, Yamamoto Y, Mori H, Kameyama R, Fujihara S, Masaki T and Suzuki Y: Molecular mechanisms of [18F]fluorodeoxyglucose accumulation in liver cancer. Oncol Rep. 31:701–706. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Zhang Q, Wang J, Zhang H, Zhao D, Zhang Z and Zhang S: Expression and clinical significance of aminopeptidase N/CD13 in non-small cell lung cancer. J Cancer Res Ther. 11:223–228. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Wickström MI, Larsson R, Nygren P and Gullbo J: Aminopeptidase N (CD13) as a target for cancer chemotherapy. Cancer Sci. 102:501–508. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Ikeda N, Nakajima Y, Tokuhara T, Hattori N, Sho M, Kanehiro H and Miyake M: Clinical significance of aminopeptidase N/CD13 expression in human pancreatic carcinoma. Clin Cancer Res. 9:1503–1508. 2003.PubMed/NCBI | |
|
Fukasawa K, Fujii H, Saitoh Y, Koizumi K, Aozuka Y, Sekine K, Yamada M, Saiki I and Nishikawa K: Aminopeptidase N (APN/CD13) is selectively expressed in vascular endothelial cells and plays multiple roles in angiogenesis. Cancer Lett. 243:135–143. 2006. View Article : Google Scholar : PubMed/NCBI | |
|
Su L, Cao J, Jia Y, Zhang X, Fang H and Xu W: Development of synthetic aminopeptidase N/CD13 inhibitors to overcome cancer metastasis and angiogenesis. ACS Med Chem Lett. 3:959–964. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Rocken C, Licht J, Roessner A and Carl-McGrath S: Canalicular immunostaining of aminopeptidase N (CD13) as a diagnostic marker for hepatocellular carcinoma. J Clin Pathol. 58:1069–1075. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Nagano H, Ishii H, Marubashi S, Haraguchi N, Eguchi H, Doki Y and Mori M: Novel therapeutic target for cancer stem cells in hepatocellular carcinoma. J Hepatobiliary Pancreat Sci. 19:600–605. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Röcken C, Carl-McGrath S, Gräntzdörffer I, Mantke R, Roessner A and Lendeckel U: Ectopeptidases are differentially expressed in hepatocellular carcinomas. Int J Oncol. 24:487–495. 2004.PubMed/NCBI | |
|
Corti A, Curnis F, Arap W and Pasqualini R: The neovasculature homing motif NGR: More than meets the eye. Blood. 112:2628–2635. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Chen K, Ma W, Li G, Wang J, Yang W, Yap LP, Hughes LD, Park R and Conti PS: Synthesis and evaluation of 64Cu-labeled monomeric and dimeric NGR peptides for MicroPET imaging of CD13 receptor expression. Mol Pharm. 10:417–427. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Zhang J, Lu X, Wan N, Hua Z, Wang Z, Huang H, Yang M and Wang F: 68Ga-DOTA-NGR as a novel molecular probe for APN-positive tumor imaging using MicroPET. Nucl Med Biol. 41:268–275. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Mate G, Kertesz I, Enyedi KN, Mező G, Angyal J, Vasas N, Kis A, Szabó É, Emri M, Bíró T, et al: In vivo imaging of Aminopeptidase N (CD13) receptors in experimental renal tumors using the novel radiotracer 68Ga-NOTA-c(NGR). Eur J Pharm Sci. 69:61–71. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Shao Y, Liang W, Kang F, Yang W, Ma X, Li G, Zong S, Chen K and Wang J: A direct comparison of tumor angiogenesis with 68Ga-labeled NGR and RGD peptides in HT-1080 tumor xenografts using microPET imaging. Amino Acids. 46:2355–2364. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Kang F, Ma W, Ma X, Shao Y, Yang W, Chen X, Li L and Wang J: Propranolol inhibits glucose metabolism and 18F-FDG uptake of breast cancer through posttranscriptional downregulation of hexokinase-2. J Nucl Med. 55:439–445. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Zhao M, Yang W, Zhang M, Li G, Wang S, Wang Z, Ma X, Kang F and Wang J: Evaluation of 68Ga-labeled iNGR peptide with tumor-penetrating motif for microPET imaging of CD13-positive tumor xenografts. Tumour Biol. 37:12123–12131. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Alberici L, Roth L, Sugahara KN, Agemy L, Kotamraju VR, Teesalu T, Bordignon C, Traversari C, Rizzardi GP and Ruoslahti E: De novo design of a tumor-penetrating peptide. Cancer Res. 73:804–812. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Chen H, Niu G, Wu H and Chen X: Clinical application of radiolabeled RGD peptides for PET imaging of integrin αvβ3. Theranostics. 6:78–92. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Ma W, Shao Y, Yang W, Li G, Zhang Y, Zhang M, Zuo C, Chen K and Wang J: Evaluation of 188Re-labeled NGR-VEGI protein for radioimaging and radiotherapy in mice bearing human fibrosarcoma HT-1080 xenografts. Tumour Biol. 37:9121–9129. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Tillmanns J, Schneider M, Fraccarollo D, Schmitto JD, Länger F, Richter D, Bauersachs J and Samnick S: PET imaging of cardiac wound healing using a novel [68Ga]-labeled NGR probe in rat myocardial infarction. Mol Imaging Biol. 17:76–86. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Hendrikx G, De Saint-Hubert M, Dijkgraaf I, Bauwens M, Douma K, Wierts R, Pooters I, Van den Akker NM, Hackeng TM, Post MJ, et al: Molecular imaging of angiogenesis after myocardial infarction by 111In-DTPA-cNGR and 99mTc-sestamibi dual-isotope myocardial SPECT. EJNMMI Res. 5:22015. View Article : Google Scholar : PubMed/NCBI | |
|
Zheng K, Liang N, Zhang J, Lang L, Zhang W, Li S, Zhao J, Niu G, Li F, Zhu Z, et al: 68Ga-NOTA-PRGD2 PET/CT for integrin imaging in patients with lung cancer. J Nucl Med. 56:1823–1827. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Wan W, Guo N, Pan D, Yu C, Weng Y, Luo S, Ding H, Xu Y, Wang L, Lang L, et al: First experience of 18F-alfatide in lung cancer patients using a new lyophilized kit for rapid radiofluorination. J Nucl Med. 54:691–698. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Kang F, Wang S, Tian F, Zhao M, Zhang M, Wang Z, Li G, Liu C, Yang W, Li X, et al: Comparing the diagnostic potential of 68Ga-Alfatide II and 18F-FDG in differentiating between non-small cell lung cancer and tuberculosis. J Nucl Med. 57:672–677. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Yu C, Pan D, Mi B, Xu Y, Lang L, Niu G, Yang M, Wan W and Chen X: 18F-Alfatide II PET/CT in healthy human volunteers and patients with brain metastases. Eur J Nucl Med Mol Imaging. 42:2021–2028. 2015. View Article : Google Scholar : PubMed/NCBI | |
|
Iagaru A, Mosci C, Shen B, Chin FT, Mittra E, Telli ML and Gambhir SS: 18F-FPPRGD2 PET/CT: Pilot phase evaluation of breast cancer patients. Radiology. 273:549–559. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Zhu Z, Yin Y, Zheng K, Li F, Chen X, Zhang F and Zhang X: Evaluation of synovial angiogenesis in patients with rheumatoid arthritis using 68Ga-PRGD2 PET/CT: A prospective proof-of-concept cohort study. Ann Rheum Dis. 73:1269–1272. 2014. View Article : Google Scholar : PubMed/NCBI | |
|
Haubner R, Finkenstedt A, Stegmayr A, Rangger C, Decristoforo C, Zoller H and Virgolini IJ: [68Ga]NODAGA-RGD - Metabolic stability, biodistribution, and dosimetry data from patients with hepatocellular carcinoma and liver cirrhosis. Eur J Nucl Med Mol Imaging. 43:2005–2013. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Sherigar JM, Gayam V, Khan A, Mukhtar O, Arefiev Y, Khalid M, Siddiqui I, Rangaraju AM, Budhathoki N, Mansour M, et al: Clinical efficacy and tolerability of direct-acting antivirals in elderly patients with chronic hepatitis C. Eur J Gastroenterol Hepatol. 29:767–776. 2017. View Article : Google Scholar : PubMed/NCBI | |
|
He X, Hong Y, Wang X, Zhang X, Long J, Li H, Zhang B, Chen S, Liu Q, Li H, et al: Identification and clinical significance of an elevated level of serum aminoacylase-1 autoantibody in patients with hepatitis B virus-related liver cirrhosis. Mol Med Rep. 14:4255–4262. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Li F, Yan H, Wang J, Li C, Wu J, Wu S, Rao S, Gao X and Jin Q: Non-invasively differentiating extent of liver fibrosis by visualizing hepatic integrin αvβ3 expression with an MRI modality in mice. Biomaterials. 102:162–174. 2016. View Article : Google Scholar : PubMed/NCBI | |
|
Ma W, Kang F, Wang Z, Yang W, Li G, Ma X, Li G, Chen K, Zhang Y and Wang J: 99mTc-labeled monomeric and dimeric NGR peptides for SPECT imaging of CD13 receptor in tumor-bearing mice. Amino Acids. 44:1337–1345. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Berman JJ: Tumor classification: Molecular analysis meets Aristotle. BMC Cancer. 4:102004. View Article : Google Scholar : PubMed/NCBI | |
|
Pantel AR and Mankoff DA: Molecular imaging to guide systemic cancer therapy: Illustrative examples of PET imaging cancer biomarkers. Cancer Lett. 387:25–31. 2017. View Article : Google Scholar : PubMed/NCBI |
