Activatable Near-Infrared Fluorescent Probe for In Vivo Imaging of Fibroblast Activation Protein-alpha

Abstract

Fibroblast activation protein-alpha (FAPα) is a cell surface glycoprotein which is selectively expressed by tumor-associated fibroblasts in malignant tumors but rarely on normal tissues. FAPα has also been reported to promote tumor growth and invasion and therefore has been of increasing interest as a promising target for designing tumor-targeted drugs and imaging agents. Although medicinal study on FAPα inhibitors has led to the discovery of many FAPα-targeting inhibitors including a drug candidate in a phase II clinical trial, the development of imaging probes to monitor the expression and activity of FAPα in vivo has largely lagged behind. Herein we report an activatable near infrared (NIR) fluorescent probe (ANPFAP) for in vivo optical imaging of FAPα. The ANPFAP consists of a NIR dye (Cy5.5) and a quencher dye (QSY21) which are linked together by a short peptide sequence (KGPGPNQC) specific for FAPα cleavage. Because of the efficient fluorescence resonance energy transfer (FRET) between Cy5.5 and QSY21 in ANPFAP, high contrast on the NIR fluorescence signal can be achieved after the cleavage of the peptide sequence by FAPα both in vitro and in vivo. In vitro assay on ANPFAP indicated the specificity of the probe to FAPα. The in vivo optical imaging using ANPFAP showed fast tumor uptake as well as high tumor to background contrast on U87MG tumor models with FAPα expression, while much lower signal and tumor contrast were observed in the C6 tumor without FAPα expression, demonstrating the in vivo targeting specificity of the ANPFAP. Ex vivo imaging also demonstrated ANPFAP had high tumor uptake at 4 h post injection. Collectively, these results indicated that ANPFAP could serve as a useful NIR optical probe for early detection of FAPα expressing tumors.

Molecular imaging with engineered physiology

“…Targets included fibroblast activation protein (FAP), a cell surface-bound collagenase and cancer biomarker20, which removes the dipeptide AP from the N terminus of polypeptides; TEV protease and enterokinase, both well-studied endopeptidases that are absent in the central nervous system and could, therefore, be used as bio-orthogonal markers for transgenesis or cell tracking2122; and caspase-3 (CASP3), another endopeptidease and cytosolic apoptosis actuator that can be released following apoptosis and secondary necrosis23 and serve as a prognostic indicator during cancer treatments2425. FAP and CASP3 have been targeted by previous optical262728 or MRI2930 probes, but improvements in sensitivity for deep tissue detection would be valuable. CGRP constructs targeted towards these proteases and incorporating AP repeats, green fluorescent protein (GFP), or biotin as blocking domains were produced in Escherichia coli or by solid phase peptide synthesis.…”

Molecular Imaging of the Tumor Microenvironment for Precision Medicine and Theranostics

“…FAPα-targeted imaging probes could be used for early detection of cancer, stratification of cancer patients for FAPα-targeted therapy, and monitoring treatment response. A new probe that consists of a NIR dye (Cy5.5) and a quencher dye (QSY21) which are linked together by a short peptide sequence (KGPGPNQC) specific for FAPα cleavage was recently developed to detect CAFs (Li, Chen, et al, 2012). NIR fluorescence signal was observed after cleavage of the peptide sequence by FAPα both in vitro and in vivo.…”

“…NIR fluorescence signal was observed after cleavage of the peptide sequence by FAPα both in vitro and in vivo. In vivo imaging demonstrated that the probe was rapidly activated in FAPα-positive U87MG tumors but not in negative C6 tumors, providing a novel FAPα-specific probe to quantify the presence of CAFs (Li, Chen, et al, 2012). …”

Advances in fluorescent-image guided surgery

“…These fluorescent probes become activated upon degradation of the micelle after its uptake into HER2-positive tumors, validating their potential use in visualizing and classifying tumors (40). Another probe design that relies on the unique surface environment of cancer cells takes advantage of the cell surface glycoprotein fibroblast activation protein-alpha to activate a fluorescence signal (41). Novel activatable probes targeting additional physiologic characteristics of cancer cells, such as thiol concentration, decreased pH, surface lectins, and antibody binding, are also currently in development (8,(42)(43)(44)(45)(46).…”