¹⁸F-5-fluoro-aminosuberic acid (FASu) as a potential tracer to gauge oxidative stress in breast cancer models

Abstract

The cystine transporter (system xC-) is an antiporter of cystine and glutamate. It has relatively low basal expression in most tissues and becomes upregulated in cells under oxidative stress (OS) as one of the genes expressed in response to the antioxidant response element (ARE) promoter. We have developed ¹⁸F-5-fluoro-aminosuberic acid (FASu), a Positron Emission Tomography (PET) tracer that targets system xC-. The goal of this study was to evaluate the suitability of ¹⁸F-FASu as a specific gauge for system xC- activity in vivo and its potential usefulness for breast cancer imaging. Methods: ¹⁸F-FASu specificity towards system xC- was studied by cell inhibition assay, cellular uptake following OS induction with diethyl maleate (DEM), with and without anti-xCT siRNA knockdown, in vitro uptake studies and in vivo uptake in a system xC- transduced xenograft model. In addition, radiotracer uptake was evaluated in three separate breast cancer models MDA-MB-231, MCF-7 and ZR-75-1. Results: Reactive oxygen species (ROS)-inducing DEM increased glutathione level and ¹⁸F-FASu uptake, while gene knocking down with anti-xCT siRNA led to decreased tracer uptake. ¹⁸F-FASu uptake was robustly inhibited by system xC- inhibitors or substrates, while the uptake was significantly higher in transduced cells and tumors expressing xCT compared to the wild type HEK293T cells and tumors (p<0.0001 for cells, P = 0.0086 for tumors). The ¹⁸F-FASu demonstrated tumor uptake in all three breast cancer cell lines studied. Among them, triple negative breast cancer MDA-MB-231 had the highest tracer uptake (P = 0.0058 when compared with MCF-7; p<0.0001 when compared with ZR-75-1), which also has the highest xCT mRNA level. Conclusion: ¹⁸F-FASu as a system xC- substrate is a specific PET tracer for functional monitoring of system xC- and OS imaging. By enabling non-invasive analysis of xC- responses in vivo, this biomarker may serve as a valuable target for the diagnosis and treatment monitoring of certain breast cancers.