Small-Molecule Probe [68Ga]Ga-SMIC-2501 Targeting the V-Domain Immunoglobulin Suppressor of T Cell Activation Protein for Positron Emission Tomography Imaging

V-domain immunoglobulin suppressor of T cell activation (VISTA) inhibits T-cell effector functions and helps tumor immune evasion, making it an important biomarker and therapeutic target in cancer immunotherapy. To facilitate VISTA-targeted strategy development and enable the accurate detection of tumor VISTA expression, a novel small-molecule probe [⁶⁸Ga]Ga-SMIC-2501 was developed for noninvasive imaging. The radiochemical purity was over 95%, with a radiochemical yield of around 90%. [⁶⁸Ga]Ga-SMIC-2501 demonstrated good in vitro stability at room temperature in physiological saline and fetal bovine serum for 2 h tested. WB results showed that VISTA expression in B16–F10 tumor cells was higher than that in CT26 and MC38 cells. [⁶⁸Ga]Ga-SMIC-2501 accumulated in tumors in both high (B16–F10) and moderate (CT26) VISTA-expression animal models, with uptake levels of 2.53 ± 0.55% and 1.89 ± 0.38% ID/g, respectively. In blocking experiments, tumor uptake was 1.56 ± 0.20% ID/g, and the inhibition rate reached 36%. Immunohistochemistry (IHC) analysis showed that VISTA expression levels were positively correlated with [⁶⁸Ga]Ga-SMIC-2501 uptake in tumors, with a correlation coefficient greater than 0.75. [⁶⁸Ga]Ga-SMIC-2501 shows high potential as a novel molecular tool for imaging VISTA-positive tumors and expression. It provides a foundation for the future development of VISTA-based cancer theranostic agents and guidance for VISTA-targeted immunotherapy.