Preclinical Evaluation of ¹³¹I/¹⁸F-Labeled Covalent Small-Molecule Inhibitors for STING Status Imaging

The stimulator of interferon genes (STING) is a vital protein to the immune surveillance of the tumor microenvironment. In this study, we develop novel inhibitor-based radioligands and evaluate their feasibility for noninvasive visualization of STING expression in tumor-bearing mice. Analogous compounds to STING inhibitors C170 and C176 were synthesized and labeled with ¹³¹I and ¹⁸F to attain [¹³¹I]I-NFIP and [¹⁸F]F-NFEP, respectively. The radiosynthesis was achieved with high radiochemical purity (>95%) and molar activity (28.56-48.89 GBq/μmol). The affinity and specificity of tracers were assessed through cell uptake and docking experiments, demonstrating that [¹³¹I]I-NFIP exhibited high specificity for STING, with a cell-based IC₅₀ value of 7.56 nM. Small-animal PET/SPECT imaging and biodistribution studies in tumor-bearing mice models were performed to verify the tracers' pharmacokinetics and tumor-targeting capabilities (n = 3/group). SPECT imaging demonstrated that [¹³¹I]I-NFIP rapidly accumulated in the Panc02 tumor quickly at 30 min post-injection, with a tumor-to-muscle (T/M) ratio of 2.03 ± 0.30. This ratio significantly decreased in the blocking group (1.10 ± 0.14, **P < 0.01, n = 3). Furthermore, tumor uptake and the T/M ratio of [¹³¹I]I-NFIP were positively associated with STING expression. In summary, [¹³¹I]I-NFIP is the first STING-specific inhibitor-based radioligand offering the potential for visualizing STING status in tumors.