64Cu-PSMA-617: A novel PSMA-targeted radio-tracer for PET imaging in gastric adenocarcinoma xenografted mice model

Here, we report that it’s feasible for imaging gastric adenocarcinoma mice model with prostate-specific membrane antigen (PSMA) targeting imaging agents, which could potentially provide an alternate and readily translational tool for managing gastric adenocarcinoma. DKFZ-PSMA-617, a PSMA targeting ligand reported recently, was chosen to be radio-labeled with nuclide 64Cu. 64Cu-PSMA-617 was radio-synthesized in high radio-chemical yield and specific activity up to 19.3 GBq/µmol. It showed good stability in vitro. The specificity of 64Cu-PSMA-617 was confirmed by cell uptake experiments in PSMA (+) LNCaP cell and PSMA (-) PC-3 and gastric adenocarcinoma BGC-823 cells. Micro-PET imaging in BGC-823 and PC-3 xenografts nude mice was evaluated (n = 4). And the tumors were visualized and better tumor-to-background achieved till 24 h. Co-administration of N- [[[(1S)-1-Carboxy-3-methylbutyl]amino]-carbonyl]-L-glutamic acid (ZJ-43) can substantially block the uptake in those tumors. Dissected tumor tissues were analyzed by auto-radiography and immunohistochemistry, and these results confirmed the PSMA expression in neo-vasculature which explained the target molecular imaging of 64Cu-PSMA-617. All those results suggested 64Cu-PSMA-617 may serve as a novel radio-tracer for tumor imaging more than prostate cancer.


In vitro stability study in saline
The in vitro stability of 64 Cu-PSMA-617 was evaluated in saline (pH 7.4) at 0 min and 28 h post preparation ( Supplementary Figure 1). At least three sets of experiments were performed for each time point. HPLC analysis was applied. The column used for HPLC analysis was YMC-Pack ODS reversed-phase column (5 μm, 250 mm × 4.6 mm). The HPLC condition used was 15% to 60% acetonitrile in water with 0.1%TFA within 10 min. In vitro stability of 64 Cu-PSMA-617 in saline was also evaluated (pH= 7.4) at 28 h post preparation analyzed by ITLC (n = 3). Both methods demonstrated the stability of 64 Cu-PSMA-617 in saline.

Electrophoresis analysis of radio-tracer
Using Whatman3 filter paper (length = 20 cm) as carrier, 0.05 mol/L phosphate buffer (pH = 7.4) as solution, 64 Cu-PSMA-617 was point in the middle of the paper. Then electrophoresis at 200 V for 4 h to see the final position of the radioactivity.

In vitro cell binding assay
Confluent cells were detached with 0.25% trypsin-0.53 mM EDTA solution, washed twice and resuspended at the concentration of 2 × 10 6 cells per mL. 24-48 h prior to the binding assay, aliquots of the cell suspension (0.5 mL) were added to each well in the 24well plate. 37 kBq/mL (1 μCi/mL) of 64 Cu-PSMA-617 was added to each well. To determine the specific uptake, the selected wells were treated with the inhibitor ZJ-43 for blocking. After incubation at 37°C for 5, 30, 60 and 120 min, cells were washed twice with 1 mL ice-cold PBS to terminate the cellular uptake, trypsinized with 0.3 nM NaOH buffer. Then, the NaOH solutions were collected and counted in a γ-counter. All radioactivity values were converted into percentage of incubated dose per million cells (%ID/10 6 cells). Experiments were performed in triplicate (Supplementary Figure 3).

Biodistribution
Normal BALB/c mice, BGC-823 and PC-3 tumor bearing mice (n = 4) were injected 0.74-1.11 MBq 64 Cu-PSMA-617 via the tail vein. After 1 h, 4 h, 24 h and 48 h the blood was immediately collected from normal BALB/c mice, and 24h from BGC-823 and PC-3 tumor bearing mice. Then, the animals were sacrificed. The heart, liver, spleen, kidney, muscle, brain and tumor were collected, weighted and the radioactivity counted as %ID/g by comparison with a 1:100 diluted standard dose. All animal experiments were carried out following the guidelines of the institutional animal ethics committee. The results were shown in Supplementary Figure 5

Mass spectrometry analysis of the decayed product of 64 Cu-PSMA-617
The radio-tracer 64 Cu-PSMA-617 was characterized by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) after ten half lives of decay at 4 o C. The samples were diluted to 1.0 μg/ml by 0.1 % trifluoroacetic acid (TFA) in water. Sinapinic acid was dissolved in acetonitrile/water/ TFA(50/50/0.1) solution to a concentration of 10 mg/ml, and this solution was used for MALDI. 1 μL solution, containing 1:1 mixture sample and matrix, was used for mass analysis. The spectrum was acquired in a positive linear mode and analyzed using the FlexAnalysis v3.