Development of a Promising 18F-Radiotracer for PET Imaging Legumain Activity In Vivo

Abstract

Legumain has been found overexpressed in several cancers, which serves as an important biomarker for cancer diagnosis. In this research, a novel fluorine-18 labeled radioactive tracer [18F]SF-AAN targeting legumain was designed and synthesized for positron emission tomography (PET) imaging. Nonradioactive probe [19F]SF-AAN was obtained through chemical and solid phase peptide synthesis. After a simple one-step 18F labeling, the radiotracer [18F]SF-AAN was obtained with a high radiochemical conversion rate (>85%) and radiochemical purity (99%) as well as high molar activity (12.77 ± 0.50 MBq/nmol). The targeting specificity of [18F]SF-AAN for detecting legumain activity was investigated systematically in vitro and in vivo. In vitro cellular uptake assay showed that the uptake of [18F]SF-AAN in legumain-positive MDA-MB-468 cells was twice as much as that in legumain-negative PC-3 cells at 4 h. In vivo PET imaging revealed that the tumor uptake of [18F]SF-AAN in MDA-MB-468 tumor-bearing mice was about 2.7 times of that in PC-3 tumor-bearing mice at 10 min post injection. The experimental results indicated that [18F]SF-AAN could serve as a promising PET tracer for detecting the legumain expression sensitively and specifically, which would be beneficial for the diagnosis of legumain-related diseases.

Role of LGMN in tumor development and its progression and connection with the tumor microenvironment

“…LGMN has highly expressed in neovascularization endothelial cells in the tumor microenvironment. To study the effect of LGMN on the lumen formation of human venous endothelial cells (HUVEC), it was added APEI, an inhibitor of LGMN, into the medium ( Ding et al, 2022 ; Lu et al, 2022 ). The experimental results show that the number of intact tubules formed by HUVEC cells was significantly reduced and further decreased with the increase of APEI addition, suggesting that the high expression of LGMN in vascular endothelial cells of tumor tissue may promote the formation of tumor neovascularization, improve tumor blood supply, and promote tumor growth.…”

Dual-Targeting PET Tracers Enable Enzyme-Mediated Self-Assembly for the PET Imaging of Legumain Activity

“…The method for determining the water partition coefficient (log P) was consistent with that of [ 18 F]SF-AAN. 22 2.6. Reduction and Legumain-Controlled Self-Assembly.…”

“…The method for determining the response ability to legumain and the relevant enzyme kinetics was performed with the established protocols. 22 2.7. Cell Culture.…”

“…However, there are very few legumain-targeted radiotracers to meet those requirements. 21,22 In the past decade, the condensation reaction occurring in the physical environment between 2-cyanobenzothiazole (CBT) and the aminothiol group from cysteine (Cys) has been widely used to develop small-molecule probes with the property of controlled self-assembly, 23 which unusually displays a prolonged tumor retention time. 12,22−28 We have reported a legumain-activatable radiotracer 18 F-2 based on CBT-Cys scaffold for detecting legumain activity.…”

“…29 On the basis of this scaffold, we devolved a legumain-activatable radiotracer ([ 18 F] SF-AAN) by the conjugation of legumain-mediated cleavable Ala-Ala-Asn (AAN) peptide with SF. 22 Even though [ 18 F]SF-AAN displays high uptake in legumain-overexpressed tumors, certain deficiencies of [ 18 F]SF-AAN imaging still need to be addressed. Primary, the rapid efflux of [ 18 F]SF-AAN at the target sites limits the detection time.…”