PET Imaging of Click-engineered PSMA-targeting Immune Cells in Normal Mice

This study aimed to increase the targeting ability against PSMA in cell therapy using metabolic glycoengineering and biorthogonal chemistry and to visualize cell trafficking using PET imaging. Cellular membranes of THP-1 cells were decorated with azide(-N₃) using Ac₄ManNAz by metabolic glycoengineering. Engineered THP-1 cells were conjugated with DBCO-bearing fluorophore (ADIBO-Cy5.5) for 1 h at different concentrations and analyzed by confocal fluorescence microscopy and flow cytometry. For PSAM ligand conjugation to THP-1 cells, Ac₄ManNAz treated THP-1 cells were incubated with DBCO-PSMA ligand (ADIBO-GUL) at a final concentration with 100 µM for 1 h. To evaluate the effect on cell recognition, PSMA ligand conjugated THP-1 cells(as effectors) were co-cultured with PSMA positive 22RV1 (as target cells) at 3 : 1 a effector-to-target cell (E/T) ratio. The interaction between THP-1 and 22RV1 was monitored by confocal fluorescence microscopy. For preparing the radiolabeled THP-1, the cells were treated at the activity of ~ 740 kBq of [⁸⁹Zr]Zr(oxinate)₄/5 × 10⁶ cells. Radiolabeled cells were analyzed for determination of cell-associated radioactivity by gamma counting and viability using MTS assay. In the cytotoxicity assay, THP-1 cells did not have any cytotoxicity even when the Ac₄ManNAz concentration was 100 µM. In confocal microscopy and flow cytometry, THP-1 cells were efficiently labeled ADIBO-Cy5.5 in a dose-dependent manner, and the dose of 100 µM was the optimal concentration for the following experiments. The clusters of PSMA ligand-conjugated THP-1 cells and 22RV1 cells were identified, indicating cell-cell recognition over the cell surface between two types of cells. Cell radiolabeling efficiency was 54.5 ± 17.8%. THP-1 labeled with 0.09 ± 0.03 Bq/cell showed no significant cytotoxicity compared to unlabeled THP-1 up to 7 days. We successfully demonstrated that Ac₄ManNAz treated cells were efficiently conjugated with ADIBO-GUL for preparing the PSMA-targeting cells, and [⁸⁹Zr]Zr(oxinate)₄ could be used to label cells without toxicity. It suggested that PSMA-ligand conjugated cell therapy could be improved cell targeting and be monitored by PET imaging.