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Aim/Introduction: A recently developed cyclohexanediaminetriazole- chelator enabling 18F-labelling using the Al[18F]F approach was conjugated via copper-catalysed azide-alkyne cycloaddition to the PSMA-binding motif glutamate-urea-lysine connected with 1-naphthyl-D-alanine[1] (ligand L). In the present study radiolabelled L will be characterized on PSMA-positive prostate cancerspheroids. Three-dimensional cell models offer structure and functional properties closer to in vivo conditions than two-dimensional cell culture models. Spheroids show altered cell-cell and cell-extracellular matrix interactions and are useful for better understanding the nature of tumours concerning drug uptake, toxicity, hypoxia, tissue growth and therapeutic effects. The aim of this study was to investigate the radiopharmacological potential of 18F- and 68Ga-labelled L using LNCaP-spheroids regarding binding properties and internalisation behaviour. The results will be compared with data obtained from LNCaP-monolayer cell culture. Materials and Methods: Spheroids of LNCaP and PC3 (PSMA-negative) cells were created according to a protocol by the vendor. Freshly harvested cells were incubated with magnetic nanoparticles overnight. After loading, the cells were distributed in a 24-well-plate (10-4 cells/well), covered with a lid equipped with 24 small magnet devices for 5 days and kept in an incubator so that the cells could form spheroids [2]. After incubation with radiolabelled ligand (RCY >95%) the spheroids were washed, exposed to a phosphor imaging plate and the data analysed. Internalisation experiments were carried out by the “acid wash” method. Results: The method of creating spheroids by magnetised prostate cancer cell lines and treating them with a magnet device was applied successfully and resulted in spheroids with a diameter size of about 500 μm. The evaluation of first saturation experiments with LNCaP spheroids provided preliminary affinity data with Kd values in the range of 30 to 40 nM. These results are comparable to that of monolayer cell homogenate. Internalisation experiments with LNCaP-spheroids are ongoing. Conclusion: The novel radiolabelled PSMA-ligand L can be radiopharmacologically characterised on prostate cancer cell homogenate but also spheroids. The establishing of three-dimensional cell aggregates by magnetised cells and, moreover, the investigation of new radiolabelled conjugates for potential PET/SPECT application or even therapeutic treatment of cancer entities using spheroids is a valuable and promising method. Next, it is planned to use the LNCaP-spheroids for evaluation of PSMA-radioligands in a proof-of-conceptstudy applying the technology of Micro-Physiological Systems (multiorgan-chips) [3]. References: [1] Sihver, Eur. J. Nucl. Med. Mol. Imaging 2019, 26 (Suppl 1), S132. [2] Turker, ACS Biomater. Sci. Eng. 2018, 4, 787. [3] Maschmeyer, Lab Chip 2015, 15, 2688.