Fibroblast activation protein peptide-targeted NIR-I/II fluorescence imaging for stable and functional detection of hepatocellular carcinoma

Purpose: Cancer-associated fibroblasts (CAFs) are the primary stromal component of the tumor microenvironment in hepatocellular carcinoma (HCC), affecting tumor progression and post-resection recurrence. Fibroblast activation protein (FAP) is a key biomarker of CAFs. However, there is limited evidence on using FAP as a target in near-infrared (NIR) fluorescence imaging for HCC. Thus, this study aims to develop a novel NIR fluorescent imaging strategy targeting FAP⁺ CAFs in HCC.

Methods: The ICG-FAP-TATA probe was synthesized by conjugating a novel cyclization anti-FAP peptide with an indocyanine green derivative (ICG-NH₂) as fluorophore, capable for NIR window I (NIR-I, 700-900 nm) and II (NIR-II, 1000-1700 nm) imaging. Its efficacy in lesion localization and other potential applications was evaluated.

Results: In vivo imaging of subcutaneous HCC models revealed that ICG-FAP-TATA specifically targeted FAP⁺ CAFs in the stroma and detected differences in CAFs loading within lesions. The fluorescence intensity/tumor-to-background ratio (TBR) positively correlated with FAP expression (R² > 0.8, p < 0.05). Ex vivo incubation of tumor tissues with ICG-FAP-TATA provided stable fluorescence imaging of tumors in subcutaneous and orthotopic HCC models, including different cell line co-culture systems (LM3-luc, MHCC97H-luc, HepG2-luc + LX2), and various liver backgrounds (healthy/fibrotic) (n = 5 per group). TBR of the tumor mice models was higher for NIR-II than NIR-I imaging (3.89 ± 1.27 vs. 2.64 ± 0.64, p < 0.05). Moreover, NIR-I/II imaging of fresh tissues from seven patients with HCC undergoing surgery incubated with ICG-FAP-TATA visually provided the spatial distribution heterogeneity of CAFs. The targeted fluorescence was relatively enriched more in the blood flow direction and at the tumor edge, both of which were associated with tumor metastasis (all p < 0.05).

Conclusion: This study presents a rapid and effective method for detecting HCC lesions, locating FAP⁺ CAFs, and visualizing high-risk areas for tumor metastasis at the macroscopic level. It offers a new promising approach with translational potential for imaging HCC.