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Introduction

Pancreatic cancer is the 13th most common type of cancer, and the 8th leading cause of cancer-related mortality, accounting for 6.9% of all cancer-related mortalities, worldwide (1). The initial symptoms of pancreatic cancer are often nonspecific, such as nausea, fatigue, jaundice, weight loss, light-colored stools, dark urine and pain in the back or stomach area (2). Pancreatic cancer may be treated with surgery, radiotherapy or chemotherapy (3). Chemotherapy and radiation therapy are important adjuvant or neoadjuvant therapies, particularly for patients with unresectable disease (4). Pancreatic cancer has an extremely poor prognosis; the median survival time for all patients is 4–6 months, and the overall five-year survival rate is 7.2% (1). Emerging evidence suggests that the serine-protease urokinase-type plasminogen activator (uPA) and its receptor (uPAR) are significant in pancreatic cancer invasion and metastasis (5–7). Overexpression of uPAR in pancreatic cancer has been determined to be a strong and independent predictor of short overall survival (6). uPAR is recognized as a novel marker of cancer invasion and metastasis, and is a promising candidate as a molecular target for cancer therapy (8,9). The ability to visualize and quantify uPAR expression non-invasively in vivo is required for the potential clinical application of anticancer therapy based on the uPA/uPAR system (10,11).

Therefore, in the present study, a high-affinity 9-mer peptide antagonist of uPA-uPAR (AE105) was selected to develop a technetium-99m (99mTc)-labeled tracer for non-invasive single-photon emission computed tomography (SPECT) assessment of uPAR expression in pancreatic cancer. 99mTc-Hynic-PEG-AE105 was prepared, together with a non-binding version (99mTc-Hynic-PEG-AE105mut) as a control, and the quantitative association between tracer uptake and uPAR expression was investigated in pancreatic tumor tissues.

Materials and methods

Reagents

All commercially available chemical reagents were used without further purification. The peptide antagonist HYNIC-PEG-AE105 and a non-binding variant of HYNIC-PEG-AE105 (HYNIC-PEG-AE105mut) were synthesized (purity >95%) by Shanghai Apeptide Co., Ltd. (Shanghai, China). The sequences of HYNIC-PEG-AE105 and HYNIC-PEG-AE105mut were D-Cha-F-s-r-Y-L-W-S and D-Cha-F-s-r-Y-L-E-S, respectively. 99mTc-O4− was obtained from Beijing Atom HighTech Co., Ltd. (Beijing, China). SnCL2•H2O (purity >99.99%) was purchased from Gracia Chengdu Chemical Technology Co., Ltd. (Chengdu, China). Tricine (purity >99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Labeling of peptides

99mTc peptide labeling was performed at room temperature using Tricine as a co-ligand and SnCL2 as the reducing agent. Tricine and 99mTc-O4− (specific activity, 370 MBq/ml) in 100 µl SnCL2•H2O was diluted in 80 µl Hynic-PEG-AE105 dissolved in HEPES (1 mg/ml; pH 5.5) and incubated at room temperature. The labeling was optimized by changing the reaction time (0, 5, 10, 20, 30 and 60 min), dosage of SnCL2 (40, 60, 80, 100, 120 and 150 µg), dosage of Tricine (20, 40, 60, 80 and 100 mg), dosage of Hynic-PEG-AE105 (40, 80, 160, 240 and 320 µg) and dosage of 99mTc-O4− (111, 185, 370 and 555 MBq). The reaction was stopped by adding an excess of 1.0 mol/l glycine. The labeling rate of 99mTc-Hynic-PEG-AE105 was detected by thin layer chromatography, as described previously (12). Each experiment was repeated 3 times. Hynic-PEG-AE105mut was labeled under the same conditions. The optimal conditions for the 99mTc labeling of Hynic-PEG-AE105 and Hynic-PEG-AE105mut were as follows: 60 mg Tricine and 1 ml 99mTc-O4− (~10 mCi) in 80 µl SnCL2•H2O (1 mg/ml) were diluted in 160 µl Hynic-PEG-AE105 (1 mg/ml), followed by incubation at room temperature for 10 min.

Purification of 99mTc-labeled peptides

99mTc-labeled peptide was subsequently purified using Sep-Pak Light C18 cartridges (Waters Corporation, Milford, MA, USA), as described previously (13), and diluted with 8 volumes of water for injection. To determine the specific radioactivity of the labeled peptides, radioactivity was measured by a dose calibrator (CRC-25R; Capintec Inc., Ramsey, NJ, USA) following the manufacturer's instructions.

Pancreatic cancer xenografts in nude mice

The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Dalian Medical University (Dalian, Liaoning, China). Sodium pentobarbital anesthesia was used to minimize animal suffering. Male nude mice (4–5 weeks old) were obtained from Dalian Medical University Animal Center, and kept under pathogen-free conditions in accordance with the guidelines of the IACUC of Dalian Medical University. For the xenograft tumor growth assay, BxPC-3 cells were obtained from the Chinese Academy of Sciences (Shanghai, China) and the cultured cells (1×106 cells) were injected subcutaneously into the right flank of the mice, which were anesthetized with 2% sodium pentobarbital (dose, 45 mg/kg weight). At 2 weeks post-inoculation, the tumor size was measured every 3–4 days until the tumors grew to a diameter of 10 mm or until the tumor burden exceeded 10% of their body weight, at which time the mice were enrolled in SPECT studies.

Biodistribution studies

In brief, the nude mice bearing BxPC-3 xenografts were injected into the tail vein with 18.5 MBq of 99mTc-Hynic-PEG-AE105 or 99mTc-Hynic-PEG-AE105mut. The mice were euthanized at 0.5, 1, 2, 4 or 8 h post-injection. Blood, tumor and major organs were collected (wet-weight) and the radioactivity was measured using a γ-counter (Perkin Elmer Inc., Waltham, MA, USA) (n=5 mice/group). Tumor/non-tumor (T/NT) ratios were calculated based on the radioscans by outlining regions of equal areas of tumor tissues and the corresponding non-tumor tissues.

SPECT imaging

Prior to being sacrificed, all the mice underwent SPECT imaging (Millennium VG; GE Healthcare, Milwaukee, WI, USA), at 2, 4 and 6 h post-injection, respectively. The mice were laid in the center of the field of view. A low-energy high-resolution parallel holes collimator was used. SPECT images were obtained with a zoom factor of 3.0 for 5 min, and were digitally stored in a 128×128 matrix and analyzed using a GE Integra workstation (GE Healthcare).

Immunohistochemistry (IHC)

IHC was performed using a standard streptavidin-biotin-peroxidase complex method. In brief, non-specific binding was blocked with 10% normal rabbit serum for 20 min. Tumor sections were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 20 min. For antigen retrieval, the sections were microwave-treated in 10 mM citrate buffer (pH 6.0) for 10 min. The sections were incubated with rabbit uPAR polyclonal antibody (1:500 dilution; Santa Cruz Biotechnology Inc., Dallas, TX, USA) overnight, then incubated with a biotinylated goat anti-rabbit immunoglobulin G antibody (1:2,000 dilution; Sigma-Aldrich, St. Louis, MO, USA) for 30 min and subsequently reacted with a streptavidin-peroxidase conjugate and 3–3′-diaminobenzidine (Sigma-Aldrich). The sections were counter-stained using Meyer's haematoxylin. Negative controls were performed by replacing the primary antibody with rabbit serum. The sections were observed under a light microscope and five fields (×400 magnification) of each section were randomly selected for analysis. The staining density was calculated based on absorbance using the Image-pro Plus 6.0 image analysis system (Media Cybernetics Inc., Rockville, MD, USA).

Statistical analysis

Statistical analysis was performed with SPSS software (version 10.0; SPSS Inc., Chicago, IL, USA). Data are presented as the mean ± standard error of the mean and were assessed by a two-tailed Student's t-test. P<0.05 was used to indicate a statistically significant difference. The correlation between tracer and uPAR expression was analyzed using Pearson's χ2 test.

Results

Radiolabeling of peptides

The efficiency of the 99mTc labeling of Hynic-PEG-AE105 and inactive Hynic-PEG-AE105 was 94.64±0.72 and 92.03±0.81%, respectively. The optimal conditions for the 99mTc labeling of Hynic-PEG-AE105 and Hynic-PEG-AE105mut were as follows: 60 mg Tricine and 1 ml 99mTc-O4− (~10 mCi) in 80 µl SnCL2•H2O (1 mg/ml) were diluted in 160 µl Hynic-PEG-AE105 (1 mg/ml), followed by incubation at room temperature for 10 min. The radiochemical purity of 99mTc-Hynic-PEG-AE105 and 99mTc-Hynic-PEG-AE105mut was 97.72±1.73 and 96.70±1.32%, respectively, following Sep-Pak purification (Fig. 1). No significant degradation of any 99mTc-labeled peptides was observed in physiological saline following incubation for 8 h (Table I).

Figure 1. Radiochemical purity of technetium-99m (99mTc)-labled peptides. The peptides were purified by Sep-Pak and then analyzed by thin layer chromatography (TLC). (A) TLC pattern of 99mTc-Hynic-PEG-AE105. (B) TLC pattern of 99mTc-Hynic-PEG-AE105mut. The peaks indicate labeled peptides.

Table I. Radioactivity of 99mTc-labeled peptides following incubation in saline.

Table I.

Radioactivity of 99mTc-labeled peptides following incubation in saline.

Incubation time	Hynic-PEG-AE105, %	Hynic-PEG-AE105mut, %
2 h	96.14±1.26	96.38±1.15
4 h	95.22±0.91	95.27±1.43
6 h	94.93±1.12	94.26±0.96
8 h	94.15±1.44	93.66±0.83

[i] ^99mTc, technetium-99m.

Biodistribution and specificity of 99mTc-Hynic-PEG-AE105

Next, the study investigated the in vivo pharmacokinetics of 99mTc-Hynic-PEG-AE105 and 99mTc-Hynic-PEG-AE105 in pancreatic cancer BxPC-3 cell-bearing animals. A fast clearance rate of radiolabeled peptides from the blood and all organs investigated following resection was found (Tables II and III). The two radiolabeled peptides were distributed to the various organs of the body and cleared rapidly from the blood, primarily via the hepatic-intestinal route and kidneys. The tumor uptake of 99mTc-Hynic-PEG-AE105 was significantly higher than the normal pancreatic tissue uptake at 4 h and 6 h post-injection (P<0.01), whereas the uptake in the blood was 2.87±0.13 (4 h)/2.73±0.35 (6 h), and the uptake in the muscle was 0.53±0.21 (4 h)/0.49±0.08 (4 h), thus generating a tumor-to-blood and tumor-to-muscle ratio of 1.09±0.12 (4 h)/1.11±0.20 (6 h) and 6.29±1.59 (4 h)/6.26±1.20 (6 h), respectively. The tumor uptake of the control peptide, 99mTc-Hynic-PEG-AE105mut, at 4 or 6 h was significantly reduced to 1.65±0.53 (4 h) and 1.41±0.38 (6 h) (P<0.01), respectively, indicating the specificity of 99mTc-Hynic-PEG-AE105 to human uPAR.

Table II. Biodistribution and specificity of 99mTc-Hynic-PEG-AE105.

Table II.

Biodistribution and specificity of 99mTc-Hynic-PEG-AE105.

	Radioactivity (%ID/g)
Location	0.5 h	1 h	2 h	4 h	6 h	8 h
Blood	5.38±0.25	3.74±0.43	2.31±0.53	2.87±0.13	2.73±0.35	2.44±0.22
Tumor	4.65±0.41	3.96±0.26	2.72±0.45	3.12±0.27	2.98±0.15	2.15±0.29
Heart	3.43±0.51	1.87±0.53	1.37±0.20	1.56±0.44	1.71±0.48	1.03±0.13
Liver	4.86±0.30	3.86±0.61	3.19±0.29	2.99±0.65	3.31±0.93	2.09±0.20
Spleen	4.09±1.07	1.41±0.70	0.95±0.10	1.61±0.74	1.53±0.45	0.93±0.06
Pancreas	3.74±0.47	1.30±0.20	1.12±0.72	1.45±0.73	1.30±0.41	0.52±0.09
Lung	4.63±0.18	3.46±1.70	1.78±0.36	2.06±0.23	1.97±0.38	1.39±0.36
Kidney	5.72±0.65	4.35±0.28	2.52±0.17	3.21±0.21	3.32±0.21	2.50±0.37
Stomach	2.83±0.27	1.46±0.42	0.65±0.12	1.23±0.42	1.00±0.35	0.54±0.02
Intestine	2.67±1.19	1.18±0.65	0.89±0.33	1.28±0.88	0.79±0.30	0.46±0.03
Bone	2.32±0.36	1.60±1.10	0.56±0.12	1.12±0.36	1.34±0.47	0.67±0.07
Muscle	1.60±0.34	0.55±0.12	0.40±0.07	0.53±0.21	0.49±0.08	0.30±0.01
Brain	0.35±0.07	0.35±0.31	0.11±0.01	0.14±0.05	0.13±0.04	0.08±0.01

[i] ^99mTc, technetium-99m; ID, injected dose.

Table III. Biodistribution and specificity of 99mTc-Hynic-PEG-AE105mut.

Table III.

Biodistribution and specificity of 99mTc-Hynic-PEG-AE105mut.

	Radioactivity (%ID/g)
Location	0.5 h	1 h	2 h	4 h	6 h	8 h
Blood	4.16±0.79	2.82±0.68	3.20±0.20	1.56±0.47	1.53±0.23	1.33±0.22
Tumor	3.53±0.42	2.53±0.87	3.21±0.29	1.65±0.53	1.41±0.38	1.21±0.20
Heart	2.54±0.28	2.11±0.67	2.46±0.31	1.25±0.46	1.21±0.28	1.01±0.28
Liver	3.92±0.33	2.92±1.31	3.52±0.63	3.26±1.33	1.90±1.81	1.65±0.87
Spleen	2.71±0.05	2.04±0.90	2.96±0.80	2.15±1.29	0.96±0.69	0.82±0.20
Pancreas	1.74±0.09	1.50±0.38	1.55±0.18	0.75±0.08	0.88±0.49	0.73±0.26
Lung	1.82±0.12	1.62±0.11	1.57±0.21	1.37±0.51	1.12±0.17	1.00±0.18
Kidney	4.41±0.59	3.07±0.66	3.47±0.33	2.41±0.41	2.28±0.26	2.03±0.40
Stomach	1.57±0.26	1.37±0.19	1.82±0.55	0.70±0.19	0.47±0.18	0.42±0.13
Intestine	2.45±0.27	2.36±0.31	2.60±1.04	0.56±0.10	0.66±0.27	0.61±0.20
Bone	1.79±0.34	1.59±0.45	1.70±0.33	0.69±0.19	0.56±0.05	0.56±0.21
Muscle	0.89±0.05	0.72±0.20	0.80±0.19	0.54±0.21	0.36±0.10	0.31±0.10
Brain	0.34±0.05	0.18±0.15	0.24±0.03	0.08±0.03	0.08±0.02	0.07±0.01

[i] ^99mTc, technetium-99m; ID, injected dose.

SPECT study

The BxPC-3 tumor-bearing mice were SPECT-scanned at 2, 4 and 6 h post-intravenous injection of 99mTc-Hynic-PEG-AE or 99mTc-Hynic-PEG-AE105mut. The representative images for each group of mice at 2, 4 and 6 h post-injection are shown in Fig. 2. The tumor was clearly visible as early as 2 h post-injection of 99mTc-Hynic-PEG-AE105, and the uptake kept increasing and reached a plateau at 6 h post-injection. By contrast, in the mice injected with 99mTc-Hynic-PEG-AE105mut, the tumor was not clear at 2, 4 and 6 h post-injection. Using quantitative region of interest analysis, a significantly higher radioactive uptake ratio (T/NT) was found for 99mTc-Hynic-PEG-AE105 than for control peptide 99mTc-Hynic-PEG-AE105mut at 4 h (3.37±0.11 vs. 1.36±0.18; P<0.001) and 6 h (3.64±0.25 vs. 1.28±0.20; P<0.001).

Figure 2. In vivo imaging of xenografted nude mice. (A) Coronal single-photon emission computed tomography (SPECT) images of mice bearing BxPC-3 tumors at 2, 4 and 6 h post-injection of 99mTc-Hynic-PEG-AE105. (B) Coronal SPECT images of mice bearing BxPC-3 tumors at 2, 4 and 6 h post-injection of 99mTc-Hynic-PEG-AE105mut. The arrows indicate xenografted tumors. 99mTc, technetium-99m.

uPAR expression is correlated with the tumor uptake of 99mTc-Hynic-PEG-AE105

IHC showed that uPAR was mainly stained in the cytoplasm and on the membrane surface of the BxPC-3 cells (Fig. 3). Semi-quantification of uPAR staining showed that uPAR expression was not significantly different between the experimental and control groups (0.481±0.024 vs. 0.574±0.021; P=0.173). By association analysis, a significant correlation was found between the tumor uptake of 99mTc-Hynic-PEG-AE105 and uPAR expression at 4 to 6 h post-injection (r=0.791, P=0.006; Fig. 4).

Figure 3. Immunohisotchemical staining of urokinase-type plasminogen activator receptor (uPAR) in xenografts. uPAR was mainly stained in the cytoplasm and on the membrane surface of BxPC-3 cells (brown).

Figure 4. uPAR expression is significantly correlated with the uptake of 99mTc-Hynic-PEG-AE105 in tumors at 4 to 6 h (n=10; r=0.791, P=0.006). Intergrated optical density indicated the relative uPAR protein level based on immunohistochemistry analysis. uPAR, urokinase-type plasminogen activator receptor; 99mTc, technetium-99m; ID, injected dose.

Discussion

In the present study, 99mTc-labeled Hynic-PEG-AE105 was introduced as a SPECT tracer for imaging of uPAR expression for the first time. 99mTc-Hynic-PEG-AE105 exhibited high affinity and specificity to uPAR in vivo, and uPAR expression was significantly correlated with the uptake of 99mTc-Hynic-PEG-AE105 in the pancreatic cancer xenograft mouse model.

Despite its relatively low incidence, pancreatic cancer ranks fourth in the number of cancer mortalities each year (14). Overall, <5% of individuals will survive 5 years beyond their diagnosis (15). Therefore, novel and improved therapy options are required. Recent studies demonstrated that RNAi-mediated uPAR-knockdown was able to retard the invasive ability and angiogenic potential of cancer cells in vitro and in vivo (5,8,9). These results suggest that the targeting of uPAR has significant therapeutic potential for the treatment of pancreatic cancer. For the potential clinical application of anticancer therapy based on the uPA/uPAR system, we sought to develop a non-invasive imaging method for the detection of pancreatic cancer based on uPAR expression.

Previous studies investigated the use of a high-affinity 9-mer peptide antagonist of the uPA-uPAR (AE105) for positron emission tomography (PET) imaging of uPAR expression, and showed that copper-64 (64Cu)-labeled DOTA-AE105 exhibited specific and high-affinity binding to uPAR in vitro and in vivo (16–18). However, the clinical application of this protocol is restricted due to the limited availability of 64Cu and the high cost of PET imaging. 99mTc is a nuclear isomer of 99Tc that is detectable within the body using medical equipment such as γ-ray cameras, which emit readily detectable CXL keV γ rays (the same wavelength as emitted by conventional X-ray equipment), and the half-life for γ emission is only 6 h (19). Safe and fast scanning procedures are a result of the relatively short physical half-life of 99Tc and its biological half-life of 1 day in terms of human activity and metabolism (20). Therefore, 99mTc-labeled peptides have been used for in vivo targeting of tumors, including pancreatic cancer (21–23).

In the present study, 99mTc-labeled peptide was employed for SPECT imaging of uPAR in pancreatic cancer. First, the conditions for the 99mTc labeling of Hynic-PEG-AE105 and Hynic-PEG-AE105 were optimized. It was found that under the conditions optimized, the radiochemical purity of 99mTc-Hynic-PEG-AE105 was 97.72±1.73% following Sep-Pak purification. Similarly, the radiochemical purity of 99mTc-Hynic-PEG-AE105mut was 96.70±1.32%.

uPAR is widely expressed in pancreatic cancer cells such as Panc-1, MIA PaCa-2 and BxPC-3 (24). Preliminary experiments in the present study showed that among these cell lines, the expression level of uPAR is the highest in the BxPC-3 cell line (data not shown), thus BxPC-3 cells were chosen for further analysis. To analyze the biodistribution and specificity of 99mTc-Hynic-PEG-AE105 in vivo, a nude mouse xenografted with BxPC-3 cells was used as the animal model. It was found that the distribution of radioactivity in the tumor tissue was significantly higher than that in the normal tissue. Taken together, these data demonstrate the specificity of 99mTc-Hynic-PEG-AE105 for pancreatic cancer cells that highly express uPAR. SPECT imaging of nude mice further confirms the sensitivity and specificity of 99mTc-Hynic-PEG-AE105. The tumor xenograft was clearly visible as early as 2 h post-injection of 99mTc-Hynic-PEG-AE105, and the uptake kept increasing and reached a plateau at 6 h post-injection. In addition, a significant correlation was found between the tumor uptake of 99mTc-Hynic-PEG-AE105 and uPAR expression in the xenografted tumors, thus providing a strong argument for the specificity of 99mTc-Hynic-PEG-AE105.

However, the blood clearance rate of 99mTc-AE105 is not fast enough, leading to continued retention of the tracer in the blood, liver and kidneys, and interference in detecting the tumor lesion. Therefore, further studies are required to speed up the rate of the blood clearance of 99mTc-AE105 and improve the imaging of the target/background ratio.

In summary, the present study reported the radiosynthesis of 99mTc-Hynic-PEG-AE105 and achieved a high yield of 99mTc labeling of Hynic-PEG-AE105. Significantly, it was demonstrated that the distribution of 99mTc-Hynic-PEG-AE105 in the xenografted tumor tissue was correlated with the level of uPAR expression in pancreatic cancer. 99mTc-Hynic-PEG-AE105 is a promising agent for the non-invasive determination of uPAR expression in pancreatic cancer.

Acknowledgements

This study was funded by the National Natural Science Foundation of China (grant no. 81071173).

References