Synthesis and Evaluation of a ^99m Tc-Labeled Fibroblast Activation Protein (FAP)-Specific Radiotracer for SPECT Imaging

Fibroblast activation protein (FAP), which is overexpressed in malignant epithelial tumors, represents a key therapeutic and diagnostic target. This study reports the development and evaluation of a novel FAP-targeted SPECT radiotracer, [^99mTc]<b>4</b>. The precursor was synthesized and labeled with ^99mTc using a one-pot labeling procedure, yielding a radiochemical purity exceeding 95%. The tracer exhibited high hydrophilicity (log <i>P</i> = -1.366 ± 0.004) and demonstrated excellent stability in PBS and serum over 7 h. <i>In vitro</i>, [^99mTc]<b>4</b> showed specific uptake and rapid internalization in FAP-positive cells, which could be effectively blocked by competitive FAP inhibitors. <i>In vivo</i> SPECT/CT imaging and biodistribution studies in U87MG tumor-bearing mice revealed modest and specific tumor uptake, which was significantly reduced by co-injection with FAPI-04, confirming FAP-mediated targeting. These findings indicate that [^99mTc]<b>4</b> is a highly promising candidate for noninvasive SPECT imaging of FAP expression in tumors, providing a valuable tool for tumor diagnosis and therapeutic monitoring.