Comparative Evaluation of [99mTc]Tc-HYNIC-FAPI-04 and [99mTc]Tc-HYNIC-FAPI-46 for FAP-Targeted SPECT Imaging.

Fibroblast activation protein (FAP) is highly expressed in cancer-associated fibroblasts (CAFs) and has emerged as an important target for tumor imaging and therapy. Although numerous FAP-targeted PET tracers have demonstrated promising clinical performance, their high cost and limited accessibility restrict their widespread clinical application. In contrast, SPECT imaging is a more cost-effective and widely available alternative. Among the developed FAPI derivatives, FAPI-04 and FAPI-46 are two of the most extensively studied compounds with high translational potentials. In this study, we performed a systematic preclinical comparison of [99mTc]Tc-HYNIC-FAPI-04 and [99mTc]Tc-HYNIC-FAPI-46 to evaluate their imaging performance and provide guidance for optimal tracer selection in clinical SPECT. Bioinformatic analyses were performed to evaluate FAP expression and its association with prognosis and CAF-related markers. [99mTc]Tc-HYNIC-FAPI-04 and [99mTc]Tc-HYNIC-FAPI-46 were synthesized and assessed for their radiochemical purity and in vitro stability. FAP expression in FAP-HT1080 and HT1080 cells was validated using western blotting and immunofluorescence. Cellular uptake and saturation binding assays were conducted to evaluate the tracer affinity, and blocking experiments were conducted to confirm specificity. Small-animal SPECT/CT imaging and biodistribution studies were performed using FAP-HT1080 xenograft mouse models to assess in vivo tumor targeting. Immunohistochemical staining was used to further validate FAP expression in the tumor tissue. [99mTc]Tc-HYNIC-FAPI-04 and [99mTc]Tc-HYNIC-FAPI-46 exhibited high radiochemical purity and stability. FAP overexpression in FAP-HT1080 cells was confirmed by western blotting, immunofluorescence, and immunohistochemistry, and both tracers showed a high affinity for FAP. The cellular uptake of FAPI-04 was slightly higher than that of FAPI-46, and blocking experiments confirmed specific binding. SPECT/CT imaging demonstrated strong tumor accumulation with prolonged retention for both tracers, enabling clear visualization even 24 h post-injection. Quantitative analysis revealed no significant differences in tumor uptake or tumor-to-normal tissue ratios, despite the slightly higher tumor uptake of FAPI-04 observed in biodistribution studies. Consistently, comparable T/M, T/B, T/K, and T/L ratios were observed, indicating similar overall imaging performance. Bioinformatic analyses further revealed that FAP was upregulated in multiple tumors and correlated with CAF markers (COL1A1 and ACTA2). Both [99mTc]Tc-HYNIC-FAPI-04 and -46 showed high tumor uptake and clear visualization. Despite differences in pharmacokinetics, a similar tumor-to-normal tissue contrast was observed, supporting the suitability of both tracers for clinical SPECT imaging of FAP-expressing tumors.