Cys-ZHER2:342 and Cys-MZHER2 were purchased from Apeptide Co., Ltd. (Shanghai, China). MAL-DFO was purchased from Macrocyclics (Dallas, TX). [89Zr]-oxalate solution was supplied by Cyclotron VU(Netherlands). Human ovarian cancer cell lines SKOV-3 and breast cancer cell lines MCF-7 were obtained from Cell Bank of Shanghai Institutes for Biological Sciences. Female Balb/c nude mice were pruchased from SLAC Laboratory Animal Co., Ltd., China. Analytic and preparative high-performance liquid chromatography (HPLC) were carried out according to the literatures. [32, 33] Radio thin-layer chromatography (TLC) was operated on silica gel impregnated glass fiber sheets and analyzed by a BioScan. Sodium citrate solutions (0.1M) was used as solvent systems. Mass spectra was acquired from a Waters LC–MS system (Waters, Milford, MA).
Affibody Conjugation
MAL-DFO (0.35mg, 0.50μmol) was dissolved in 2 ml ammonium acetate solution (2M,pH=7) and reacted with Cys-MZHER2 (3mg, 0.40μmol) at room temperature overnight. (Figure 1) The product was purified with preparative HPLC, followed by lyophilization, as previously described.[32] Mass spectrometry (MS) measured m/z 8077.5 for [MH]+ (C351H560N104O111S2, , calculated molecular weight 8076.8).
Preparing of 89Zr-DFO-MAL-Cys-MZHER2
DFO conjugated affibody, DFO-MAL-Cys-MZHER2,(200μg,25nmol) was dissolved in 30µL deionized water and incubated with 185MBq [89Zr]-oxalate in 1ml 2M Na2CO3 solution (pH=4) for 20 minutes at room temperature. (Figure 2) After diluted with 10mL deionized water, the complex was purified by a Varian BOND ELUT C18 column. After washing with 10 mL deionized water again, the product was eluted with 0.3mL10 mM HCl in ethanol. The solution was diluted with 10 mL saline and passed through a 0.22-µm Millipore filter into a sterile vial. Radio HPLC and TLC were used for quality control.
In vitro stability
Aliquots of 89Zr-DFO-MAL-Cys-MZHER2 solutions were incubated with 1ml human serum or PBS for 48 hours at 37°C. At the preselected time points, the radiopurity was analyzed by TLC.
Cell lines
Cells were cultured using RPMI-1640 medium supplemented with 10% (v/v) heat inactivated fetal bovine serum and grown as a monolayer at 37°C in a humidified atmosphere containing 5% CO2.
Cell uptake studies
Uptake studies of 89Zr-DFO-MAL-Cys-MZHER2 in SKOV-3 cells were performed according to the method described. [32]Cells (1 × 106 / well) were incubated at 37 °C for various times with 37KBq labeled affibody in a 0.5 mL serum-free DMEM medium. The nonspecific binding of the tracer was determined by co-incubation with 5 μM Cys-ZHER2:342. After washed with chilled PBS, the cell pellets in the tube were obtained by centrifugation and measured using a γ-counter (PerkinElmer). The cell uptake was expressed as the percentage of the added activity (%AD/106cells) after decay correction.
Animal Model
All animal experiments were performed according to the national guidelines and approved by the Ethics Committee of Jiangsu Institute of Nuclear Medicine. Tumor models were established by subcutaneously implanted 5×106 SKOV-3 or MCF-7 tumor cells suspended in 0.2 mL PBS into the shoulder region of mice. When tumor sizes reached 100–300 mm3, the mice were used for the following experiments.
MicroPET Imaging
PET imaging was performed on a microPET scanner (Siemens Inc.). After anesthetized using isoflurane, the mice bearing tumors were placed in the center of the scanner and injected intravenously with 3.7 MBq 89Zr-DFO-MAL-Cys-MZHER2 in the presence or absence of excessive Cys-MZHER2:342 (10mg/kg body weight) via the lateral tail vein. Static PET images of 10 minutes were performed at selected times after tracer injection. Quantitative analysis was operated using the reported methods. [32, 33]
Biodistribution
Mice were injected with 0.74 MBq of the tracer via the tail vein and sacrificed at 1, 4, 8, 18, 24, 48, and 72 hours after administration, respectively. For the blocking study, four mice were coinjected with an excess of Cys-ZHER2:342 (10mg/kg body weight) and killed at 1 hour after administration. Tumor and normal tissues of interest were harvested and weighed. The radioactivity uptake in tissues was measured in the γ-counter and expressed as a percentage of the injected radioactivity per gram of tissue (% IA/g).
Autoradiography and Histology
After microPET imaging, the tumors were harvested and sectioned into slices with 5μm thickness at -80℃. Ex vivo autoradiography was conducted using the previous method[34]. To determine the intratumoral distribution of the tracer, the slices were placed on a phosphorimaging plate for 1 hour. Phosphorimaging plates were read with a plate reader. Quantitative analysis was carried out using OptiQuant software.
After radioactive decay, the slices were used for routine HE staining and HER2 analysis by immunohistochemistry. The procedures were processed following the published literature[33]. An epifluorescence microscope (Olympus, X81, Japan) was used to acquire the corresponding images.
Statistical Analysis
Statistical analyses were performed using GraphPad Prism. Data were analyzed using the unpaired, 2-tailed Student t-test. Differences at the 95% confidence level
(p< 0.05) were considered to be statistically significant.