Preparation and Bioevaluation of 99mTc-Labeled FAP Inhibitors as Tumor Radiotracers to Target the Fibroblast Activation Protein

Fibroblast activation protein (FAP) is overexpressed in cancer-associated fibroblasts (CAFs) in a majority of human epithelial cancers. With low expression in normal organs, FAP has become a promising molecular target for tumor theranostics. To develop a lower cost and more widely available alternative to positron emission tomography (PET), two isocyanide-containing FAP inhibitors (CN-C-5-FAPI and CN-PEG(4)-FAPI) were synthesized and radiolabeled with Tc-99m to obtain [Tc-99m][Tc-(CN-C-5-FAPI)(6)](+) and [Tc-99m][Tc-(CN-PEG(4)-FAPI)(6)](+) in high yields (>95%). They showed good stability in saline and mouse serum. The partition coefficient (log P) values of [Tc-99m][Tc-(CN-C-5-FAPI)(6)](+) and [Tc-99m][Tc-(CN-PEG(4)-FAPI)(6)](+) were -0.86 +/- 0.03 and -2.38 +/- 0.07, respectively, indicating that they were good hydrophilic complexes. The low nanomolar IC50 values of CN-C-5-FAPI and CN-PEG(4)-FAPI indicated that they had specificity to FAP. In vitro cellular uptake and blocking experiments implied a FAP-targeted uptake mechanism. The nanomolar K-d values from the saturation binding assay indicated that they had significantly high target affinity to FAP. The biodistribution and blocking study in BALB/c nude mice bearing U87MG tumors showed that both exhibited specific tumor uptake. [Tc-99m][Tc-(CN-PEG(4)-FAPI)(6)](+) showed a higher tumor uptake and a higher tumor/nontarget ratio than [Tc-99m][Tc-(CN-C-5-FAPI)(6)](+). The results of micro-single-photon emission computed tomography (SPECT) imaging studies of [Tc-99m][Tc-(CN-C-5-FAPI)(6)](+) and [Tc-99m][Tc-(CN-PEG(4)-FAPI)(6)](+) were in accordance with the biodistribution results, suggesting that [Tc-99m][Tc-(CN-PEG(4)-FAPI)(6)](+) is a promising tumor imaging agent for targeting FAP.

Automated summary

Fibroblast activation protein (FAP) is highly expressed in cancer-associated fibroblasts (CAFs) and has become a promising target for tumor therapy. Two isocyanide-containing FAP inhibitors were synthesized, radiolabeled, and showed good stability and specificity to FAP. These inhibitors demonstrated high target affinity and promising tumor imaging potential in preclinical studies.