General
64CuCl2 was obtained from the department of Nuclear Medicine, Peking University of Cancer Hospital. All chemicals, reagents and solvents were purchased commercially without further purification. Sep-Pak C18-Light cartridges were purchased from Waters. The product was analyzed by reversed-phase high performance liquid chromatography (RP-HPLC; Eclipse Plus C18, 4.5×250 mm, 5μm; Agilent) performed using a linear A-B gradient (15%-60% of B in 15 min) with a flow of 1 mL/min. Solvents were 0.1% aqueous TFA (A) and 0.1% TFA in acetonitrile (B). The HPLC system was equipped with UV and γ detectors. UV absorbance was measured at 220 nm. Micro-PET was performed on Super Argus PET (Sedecal, Spain). PET/CT scans were obtained on a Biograph mCT Flow 64 scanner (Siemens, Erlangen, Germany) with unenhanced low-dose CT. PSMA (+) 22Rv1 and PSMA (-) PC-3 cell lines were obtained from China Cell Line Resource.
Cell culture and animal models
Human prostate cancer cell lines 22Rv1 and PC-3 were cultured and the tumor models were established as previously reported [18]. All animal experiments were conducted in accordance with the guidelines approved by Peking University Cancer Hospital Animal Care and Use Committee.
Radiochemistry and quality control
64CuCl2 was obtained in solution of 0.01 M HCl (3.7 GBq/mL) [4]. 5 μL of PSMA-BCH (2 mM), 200 μL of NaAc (0.1 M) and 50 μL of 64CuCl2 (187 MBq) were added in a tube and reacted at 95 ℃ for 10 min. After cooling to room temperature, 64Cu-PSMA-BCH was purified by C18 Light Cartridge and obtained as shown in supporting information. 64Cu-PSMA-BCH was diluted with saline for further studies and analyzed by radio-HPLC for radiochemical purity, checked for pH value and sterility tested.
Partition coefficient
The partition coefficient of 64Cu-PSMA-BCH was studied in the PBS (0.1 M, pH 7.4)-octanol system (supporting information) and the value was presented as log P±SD, P was calculated as below:
P= (average of CPM in octanol / average of CPM in PBS).
In vitro stability
The in vitro stability of 64Cu-PSMA-BCH (3.7 MBq) was tested in solution of saline and 5% HSA at 37 ℃ till 36 h and analyzed by radio-HPLC (see supporting information).
Pharmacokinetics in blood
200 μL of 64Cu-PSMA-BCH (3.7 MBq) was intravenously injected to BALB/c male mice (n=5). The blood was collected from ophthalmic artery at 1, 2, 5, 10, 15, 30, 45, 60, 90, 120, 180 and 540 min p.i.. Then, the blood was weighted and measured for the radioactivity by γ-counter. The results were expressed as the percent of injected dose per gram (%ID/g).
In vitro cell uptake assay
PSMA (+) 22Rv1 and PSMA (-) PC-3 cell lines were used and the cell uptake study was performed as shown in supporting information. For blocking, 0.5 μg of ZJ-43 ((S)-2-(3-((S)-1-carboxy-3-methylbutyl)ureido), pentanedioic acid), a PSMA inhibitor, was added.
Biodistribution
Biodistribution of 64Cu-PSMA-BCH in normal BALB/c male mice was performed (see supporting information) and results were expressed as the percent of injected dose per gram (ID%/g).
Micro-PET imaging, biopsy and histology study in tumor model mouse
18.7 MBq of 64Cu-PSMA-BCH were injected into mice bearing 22Rv1 and PC-3 vial a tail vein. At 3, 12 and 20 h p.i., the mice were anaesthetized and performed micro-PET imaging on Super Argus PET (Sedecal, Spain) acquired with 80 mm diameter Transaxial FOV, OSEM 3D reconstruction algorithms with attenuation and random corrections. Finally, the images were displayed by MMWKS Super Argus. The milicounts/sec values of ROI (regions of interest) over tumor, kidneys and liver were collected.
After imaging at 20 h p.i., the mouse was anaesthetized with 3% (v/v) and sacrificed. Then puncture in tumor lesions was performed. The samples were immobilized in 10% neutral formaldehyde fixative and then performed radioautography to further investigate the possibility of methodology.
Radioautography and immunohistochemistry
The samples were stored on slides with 10% neutral formaldehyde fixative and exposed on a phosphorus plate (Perkin-Elmer, USA) for 12 h. The plate was scanned using a phosphor imaging system (Cyclone, Packard) to obtain the images. The slides were prepared and analyzed for PSMA expression by immunochemistry as described previously [7].
PET/CT imaging and analysis
With the approval of Ethics Committee of Beijing Cancer Hospital (No. 2017KT97), four patients (age 77.25±5.36, range 68-81, PSA 35.40±31.05 ng/mL, range 8.5-86.15; Gleason score 8.25±0.43, range 8-9) with suspected prostate cancer, who were clinically appropriate for biopsies, were included in this study (Table 1). Three patients without metastasis were performed whole-body PET/CT scans at 1 and 24 h p.i. and pelvic cavity scans at 4h p.i.. One patient with multiple bone and lymph nodes metastases performed whole-body PET/CT scans at 1, 4 and 24 h p.i.. Imaging was performed and all images were read by 2 experienced nuclear medicine specialists, the SUVmean, radioactivity concentration (Bq/mm3) and volume (mm3) of each organ and SUVmax of tumor lesions were obtained as literature.as previously reported [18].
Absorbed dosimetry
The radioactivity concentration (Bq/mm3) and volume (mm3) were used for calculating ID% of each organ. And the data was used for estimating human organ radiation dosimetry. OLINDA/EXM 2.0 software (Hermes Medical Solution, Sweden) was used with adult male model without special kinetics.
PET-ultrasound guided targeted biopsy
After imaging at 24 h p.i., the images were reconstructed and analyzed, then the patients were performed classic ultrasound-guided biopsies with visual fusion of PET images according to PROMISE criteria (version 1.0) as reported [19, 3].