Evaluation of 99mTc-HYNIC-βAla-Bombesin(7-14) as an agent for pancreas tumor detection in mice

Pancreatic adenocarcinoma is important in oncology because of its high mortality rate. Deaths may be avoided if an early diagnosis could be achieved. Several types of tumors overexpress gastrin-releasing peptide receptors (GRPr), including pancreatic cancer cells. Thus, a radiolabeled peptide derivative of gastrin-releasing peptide (GRP) may be useful as a specific imaging probe. The purpose of the present study was to evaluate the feasibility of using99mTc-HYNIC-βAla-Bombesin(7-14)as an imaging probe for Capan-1 pancreatic adenocarcinoma. Xenographic pancreatic tumor was developed in nude mice and characterized by histopathological analysis. Biodistribution studies and scintigraphic images were carried out in tumor-bearing nude mice. The two methods showed higher uptake by pancreatic tumor when compared to muscle (used as control), and the tumor-to-muscle ratio indicated that99mTc-HYNIC-βAla-Bombesin(7-14)uptake was four-fold higher in tumor cells than in other tissues. Scintigraphic images also showed a clear signal at the tumor site. The present data indicate that99mTc-HYNIC-βAla-Bombesin(7-14)may be useful for the detection of pancreatic adenocarcinoma.


Introduction
Cancer is one of the main causes of death worldwide. In 2012, about 14.1 million new cases of the disease were registered, resulting in approximately 8.2 million deaths. Pancreatic adenocarcinoma has a high mortality rate. Global data showed 337,872 new cases of this disease in 2012, followed by 330,372 deaths, which represent approximately 98% of the cases (1). Deaths could be delayed if an early diagnosis could be achieved.
Cancer cells are characterized by the pathological upregulation of several physiological processes, including the overexpression of a variety of peptide receptors in cancer cell membranes (2). This upregulation enables the use of radiolabeled peptides for the differentiation between tumor and normal tissues by molecular imaging. For example, pancreas, prostate, lung, colon and breast cancer cells present an increased expression of gastrin-releasing peptide receptors (GRPr). Thus, a radiolabeled peptide derivative of gastrin-releasing peptide (GRP) may be used as a specific imaging probe for these types of tumors (3)(4)(5).
The Capan-1 cell line was isolated from a liver metastasis of a human pancreatic adenocarcinoma. This tumor cell line is of ductal origin and exhibits characteristics of a well-differentiated adenocarcinoma. In addition, it produces a well-defined tumor nodule after subcutaneous inoculation in nude mice, mimicking the tumor in humans (15). It has been reported that this ductal cell line expresses functional GRPr on its membrane surface (16)(17)(18). Therefore, a GRP analog such as a Bombesin derivative may be used to identify Capan-1 tumor tissues.

Material
The peptide HYNIC-bAla-Bombesin (7)(8)(9)(10)(11)(12)(13)(14) was purchased from GL Biochem Ltd. (China). 99m Tc was obtained from a 99 Mo/ 99m Tc generator supplied by Instituto de Pesquisas Energéticas e Nucleares (IPEN; Brazil). Other reagents and solvents were acquired from Sigma-Aldrich (Brazil). The Capan-1 human cell line, Iscove's Modified Dulbecco's Medium (IMDM) and fetal bovine serum were purchased from American Type Culture Collection (ATCC; USA). Trypsin was acquired from Invitrogen (Brazil). Nude male BALB/c mice (18-20 g) were supplied by Fundac¸ão de Apoio e Fomento à Inovac¸ão Tecnológica, à Pesquisa e ao Ensino of IPEN (Brazil). Animals were kept under specific pathogen-free conditions, with ad libitum access to chow and water. The housing was temperature-controlled with filtered air and a light-dark cycle (12/12 h). The experiments were conducted according to animal-use principles approved by the local Ethics Committee on Animal Use of the Universidade Federal de Minas Gerais (CEUA/UFMG).

Radiolabeling yield
Radiolabeling yields were determined by thin-layer chromatography (TLC) on silica gel strips (Merck s , Germany). Methyl ethyl ketone was used to determine the free technetium ( 99m TcO 4 -) and a solution of acetonitrile:water (1:1) was used to quantify hydrolyzed technetium ( 99m TcO 2 ). Radioactivity was measured using an automatic gamma counter (Wizard, Finland).

Capan-1 cell culture
Capan-1 cells were maintained at 37°C in a humidified atmosphere containing 5% CO 2 and were continuously grown in IMDM supplemented with 10% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich, USA), 100 IU/mL penicillin (Sigma-Aldrich), and 10 mg/mL streptomycin (Sigma-Aldrich). The cells were grown to confluence and then harvested by trypsinization. After centrifugation (5 min at 241 g), cells were re-suspended in IMDM for development of the pancreas tumor in an animal model.

Pancreas tumor animal model
An aliquot (100 mL) containing 5 Â 10 6 Capan-1 cells in IMDM was injected subcutaneously in the right upper flank of each nude mouse. Tumors were allowed to grow in vivo for 25 days after the inoculations, with a tumor diameter of no more than 10 mm being obtained at this time. Capan-1 tumor-bearing mice were then used for ex vivo biodistribution studies and scintigraphic images.

Ex vivo biodistribution studies
Aliquots containing 99m Tc-HYNIC-bAla-Bombesin (7-14) (7.4 MBq) were administered into the tail vein of Capan-1 tumor-bearing mice (n=5). After 4 h of radiolabeled peptide administration, the mice were anesthetized with a solution of 80 mg/kg ketamine and 15 mg/kg xylazine, and then euthanized. Organs and tissues of interest, such as spleen, heart, stomach, liver, small intestine, contralateral muscle, kidneys, blood, and tumor were removed and weighed. The associated radioactivity was determined with an automatic gamma counter (Wizard, Finland). Results are reported as percent of the injected dose per gram of tissue (% ID/g).

Histopathological analysis
After performing ex vivo biodistribution studies, Capan-1 tumor tissues were fixed in formalin (10% w/v in phosphate-buffered saline, pH 7.4), and sections (4 mm) were prepared for light microscopy studies. All staining procedures were performed on paraffinembedded sections mounted on glass slides. Histological section images were captured by a digital camera (Spot Insight Color, USA) adapted to an Olympus microscope (BX-40; Japan). SPOT www.bjournal.com.br mice. At 1 and 4 h after injection, the mice were anesthetized with 80 mg/kg ketamine and 15 mg/kg xylazine, and then placed in a prone position under a gamma camera (Mediso, Hungary) with a low-energy high-resolution collimator. Images were acquired using a 256 Â 256 Â 16 matrix size with a 20% energy window set at 140 keV for 10 min. We chose an early time (1 h) and a late time (4 h) in order to demonstrate the clearance of the radiolabeled peptide. Regions of interest (ROIs) were analyzed by scintigraphic images outlining the tumor (target). The ROIs were automatically copied to the contralateral muscle (non-target). The target/non-target ratios were calculated using the total ROI counts.

Statistical analysis
Data are reported as means ± SD. The means of the 2 groups of ROIs (target and non-target) were compared using Student's t-test. P values of less than 0.05 were considered to be significant. Data were analyzed using the Prism software (version 5.00, USA).

Histopathological analysis
A xenographic pancreatic adenocarcinoma was developed by the subcutaneous inoculation of the human ductal Capan-1 cell line in the right upper flank of nude male BALB/c mice. In order to obtain a suitable tumor focus, cells were allowed to grow for about 25 days after their inoculation into the animals. Histopathological examination (Figure 1) revealed that tumor tissue presented a delicate conjunctive stroma and more necrosis in the central area. Histopathological analysis also showed evident mitotic activity and the presence of papillary projections and granular cells.

Scintigraphic images and ex vivo biodistribution studies
Ex vivo biodistribution ( Figure 2) showed that 99m Tc-HYNIC-bAla-Bombesin (7)(8)(9)(10)(11)(12)(13)(14) presented high kidney uptake. Scintigraphic images (Figure 3) corroborate the biodistribution results, showing high radioactivity accumulation in the abdominal region, which was mainly due to 99m Tc-HYNIC-bAla-Bombesin (7)(8)(9)(10)(11)(12)(13)(14) depuration, showing high levels of radioactivity in kidneys and bladder. These results indicate radiopeptide elimination by the urinary tract, which is consistent with the hydrophilic nature of the molecule (20)(21)(22). In addition, low levels of radioactivity were observed in stomach, liver, and spleen, corresponding to the low quantities of radiochemical impurities, such as 99m TcO 4 and 99m TcO 2 . The xenographic pancreas tumor was successfully developed and identified 30 days after the subcutaneous inoculation of the Capan-1 human cell line into nude male BALB/c mice, as can be clearly seen by the scintigraphic images obtained 1 and 4 h after radiopeptide administration . This increase may be explained by a higher clearance of 99m Tc-HYNIC-bAla-Bombesin (7)(8)(9)(10)(11)(12)(13)(14) from blood and contralateral muscle compared to its elimination from the tumor focus. The visual analysis of scintigraphic images corroborated the quantitative data, since the tumor focus was more evident at 4 h than at 1 h after radiopeptide injection into Capan-1 tumor-bearing mice. This finding was due to an increase in the target/nontarget ratio due to clearance of the radiopharmaceutical in the non-target organ.