Radiopharmacological comparison of an Al18F- and 68Ga-labeled somatostatin agonist and antagonist in 2D and 3D models of BON-SSTR2 cells

Introduction:

Due to the upregulation of somatostatin receptor subtype-2 (SSTR2) in neuroendocrine tumors, radiolabeled somatostatin analogs have been successfully used so far for diagnostic investigations and peptide receptor radionuclide therapy [1]. In the present work, both the somatostatin agonist (Tyr3)octreotate (TATE) and the somatostatin antagonist JR11 were conjugated with a cyclohexanediaminetriazole (CHDT) chelator for the potential labeling with Al18F, 68Ga or 111In. The radiopharmacological behavior of Al18F and 68Ga labeled CHDT-TATE and CHDT-JR11 were studied in BON-SSTR2 cells [2] in both monolayer (ML) and spheroid (SP) culture. The binding data were determined in static cell culture to have a basis for further assays using a microphysiological system (MPS) with dynamic properties [3].
Methods:
Spheroids of BON-SSTR2 cells were created according to a protocol by the vendor (Greiner Bio-One GmbH, Germany). Freshly harvested cells were incubated with magnetic nanoparticles in RPMI medium overnight. After loading, the cells were distributed in a 24-well-plate (3 x 10-4 cells/well) and placed on a plate with corresponding 24-point magnets in order to form SPs in the incubator for up to 5 days [4].
18F-labeling was performed with Al[18F]F2+ (TR-FLEX Cyclotron, Canada) and 68Ga-labeling with [68Ga]GaCl3 (68Ge/68Ga generator, iThemba Labs, South Africa) under mild conditions (20 min at 40°C). After incubation with the radiolabeled ligands the cells were washed; for internalization purpose the cells were treated with acidic glycine buffer (pH 3.0) to remove the bound molecules from the outer cellular membrane [5]. These samples correspond to the ligand-bound fraction and are measured in a gamma counter alongside the samples with the internalized ligand molecules.
Results:
Saturation binding analysis of Al18F-labeled CHDT-TATE and CHDT-JR11 revealed very good binding affinities with similar Kd-values toward ML (Al[18F]F-CHDT-TATE 12.4 ± 2.1 nM, Al[18F]F-CHDT-JR11 11.4 ± 0.3 nM) and for SPs (Al[18F]-CHDT-TATE 30 ± 6.6 nM, Al18F-CHDT-JR11 56 ± 20 nM). In contrast, [68Ga]Ga-CHDT-TATE had better binding affinities toward ML and SPs compared to [68Ga]Ga-CHDT-JR11 ([68Ga]Ga-CHDT-TATE ML 4.3 ± 0.5 nM, SPs 19 ± 2.2 nM; [68Ga]Ga-CHDT-JR11 ML 25.3 ± 13.9 nM, SPs 118 ± 38 nM). The maximal binding capacity (Bmax) was about 25-times higher for radiolabeled CHDT-JR11 (average 5.8 ± 0.3 pmol/mg) than for CHDT-TATE (average 0.23 ± 0.08 pmol/mg) both for ML and SPs.
The internalization assays showed that radiolabeled CHDT-TATE internalized about 4-times stronger than radiolabeled CHDT-JR11 based on percent of administered dose per µg of protein both for ML and for SPs (radiolabeled CHDT-TATE 0.22 ± 0.03 %AD/µg protein, radiolabeled CHDT-JR11 0.06 ± 0.02 %AD/µg protein).
Conclusions:
Both the agonist conjugate CHDT-TATE and the antagonist conjugate CHDT-JR11 were successfully labeled with Al18F and with 68Ga and assayed with SSTR2-expressing BON-SSTR2 cells in monolayer and spheroid form. The 25-times higher Bmax values of the radiolabeled antagonist appear advantageously regarding a potential theranostic application. Despite the fact that the SSTR2 antagonist only weakly internalizes in the cells the antagonists show effective treatment in clinical studies [6]. The next step is to compare the results with assays in dynamic MPS chips.
Acknowledgements:
The authors like to express great appreciation for the financial support from the LifeScience-Stiftung (Funding code: HZDR 2021.01).
The SSTR2 expressing BON-SSTR2 cells were kindly given by Dr. Grötzinger, University Hospital Charité Berlin.
References:
[1] Eychenne et al., Molecules 2020, 25, 4012. [2] Exner et al., Front Endocrinol 2018, 9, 146. [3] Busek et al., J Sens Sens Syst 2016, 5, 228. [4] Noel et al., J Vis Exp 2017, 126, e56081. [5] Matzku et al., Cancer Res 1986, 46, 3848. [6] Borgna et al., Eur J Nucl Med Mol Imaging 2022, 49, 1113.