Characterization of Dimeric Fibroblast Activation Protein Inhibitor (FAPI) Probes for Cancer Molecular Imaging in Diverse Tumor Models

Cancer-associated fibroblasts are critical components of the tumor microenvironment (TME) and play pivotal roles in tumorigenesis, invasion, and metastasis. Fibroblast activation protein (FAP), a type II serine protease selectively expressed by activated fibroblasts, has emerged as a key biomarker in various solid tumors. Targeting FAP with specific molecular probes, such as fibroblast activation protein inhibitors (FAPI), provides a powerful approach to visualizing TME remodeling through optical and PET imaging. FAP overexpression, observed in many cancer types, is associated with tumor progression, metastasis, and immune evasion, while normal tissues exhibit minimal FAP expression. To enhance tumor detectability, multimeric FAP molecular imaging probes have been developed in recent years, demonstrating tremendous potential to improve cancer diagnostics and precise staging. This study aimed to characterize novel dimeric FAPI (FAPI-D) probes and validate their enhanced tumor-targeting affinity and PET imaging capabilities using 68Ga-labeled FAPI-D in diverse xenograft tumor models. Methods: FAPI-D was synthesized based on a monomeric FAPI-47 precursor (FAPI-M) by incorporating multiple FAPI targeting moieties into a single molecule. Derivatives of FAPI-D, including a Cy5.5-labeled FAPI-D and a 68Ga-labeled FAPI-D conjugate (68Ga-DOTA-FAPI-D), were characterized via in vitro FAP enzymatic inhibition and cell-binding assays using transgenic HeLa cell lines with and without FAP expression. FAPI-M derivatives labeled with Cy5.5 and 68Ga were used as controls for comparative studies. Xenograft tumor models, including HeLa, MDA-MB-231, and HEK-293T cells with varying levels of FAP expression, were established in NSG mice to evaluate the in vivo performance of 68Ga-DOTA-FAPI-D via PET imaging. Results: FAPI-D exhibited a 14-fold increase in FAP inhibition compared to FAPI-M. In vitro cell-binding assays confirmed the enhanced binding efficacy and specificity of FAPI-D in FAP-positive HeLa cells compared to FAP-negative HeLa cells. The IC₅₀ values for FAPI-M and FAPI-D were 130 nM and 8 nM, respectively. Imaging results were consistent with the in vitro findings, demonstrating the superior tumor-binding capacity of FAPI-D probes and low non-specific radioactive distribution in both optical and PET imaging of cancer xenografts with varying levels of FAP expression. In vivo images further showed that FAPI-D was more sensitive in detecting tumors with lower FAP expression levels compared to FAPI-M. SUV values for FAP-expressing HEK-293T tumors (3.95 ± 1.24 and 4.48 ± 0.67) on PET images of 68Ga-DOTA-FAPI-D acquired at 1 hour and 2 hours post-injection were significantly higher (P < 0.05) compared to blocked HEK-293T tumors (1.07 ± 0.14 and 2.81 ± 0.62) and FAPI-M analogs (0.38 ± 0.02 and 0.57 ± 0.19), respectively. Conclusion: This study demonstrates the potential of multimeric FAP targeting to advance cancer molecular imaging. FAPI-D significantly improves FAP binding specificity and affinity, exhibits favorable biodistribution, and enhances tumor detectability in PET imaging. These findings highlight its promise for translational applications in cancer theranostics.