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.干扰素基因刺激因子（stimulator of interferon genes, STING）是肿瘤微环境免疫监视的关键蛋白。本研究开发了新型基于抑制剂的放射性配体，并评估其用于非侵入式可视化荷瘤小鼠体内STING表达的可行性。针对STING抑制剂C170与C176的类似化合物进行了合成，并分别以碘-131（131I）与氟-18（18F）进行标记，得到[131I]I-NFIP与[18F]F-NFEP。该放射合成产物具有较高的放射化学纯度（>95%）与摩尔活度（28.56–48.89 GBq/μmol）。通过细胞摄取与分子对接实验评估了该示踪剂的亲和力与特异性，结果显示[131I]I-NFIP对STING具有高特异性，基于细胞实验的半抑制浓度（IC50）为7.56 nM。本研究在荷瘤小鼠模型中开展了小动物PET/SPECT成像与生物分布研究，以验证该示踪剂的药代动力学与肿瘤靶向能力（每组n=3）。SPECT成像结果表明，[131I]I-NFIP在注射后30分钟即可在Panc02肿瘤中快速聚集，肿瘤/肌肉（T/M）比值为2.03 ± 0.30；阻断组的该比值显著降低至1.10 ± 0.14（**P < 0.01，n=3）。此外，[131I]I-NFIP的肿瘤摄取量与T/M比值均与STING表达呈正相关。综上，[131I]I-NFIP是首款基于STING抑制剂的特异性放射性配体，具备可视化肿瘤内STING状态的应用潜力。