Nerve damage is the major morbidity of many surgeries, resulting in chronic pain, loss of function, or both. The sparing of nerves during surgical procedures is a vexing problem because surrounding tissue often obscures them. To date, systemically administered nerve-highlighting contrast agents that can be used for nerve-sparing image-guided surgery have not been reported. In the current study, physicochemical and optical properties of 4,4’-[(2-methoxy-1,4-phenylene)di-(1E)-2,1-ethenediyl]bis-benzenamine (BMB) and a newly synthesized, red-shifted derivative 4-[(1E)-2-[4-[(1E)-2-[4-aminophenyl]ethenyl]-3-methoxyphenyl]ethenyl]-benzonitrile (GE3082) were characterized in vitro and in vivo. Both agents crossed the blood-nerve barrier and blood-brain barrier, and rendered myelinated nerves fluorescent after a single systemic injection. Although both BMB and GE3082 also exhibited significant uptake in white adipose tissue, GE3082 underwent a hypsochromic shift in adipose tissue that provided a means to eliminate the unwanted signal using hyperspectral deconvolution. Dose and kinetic studies were performed in mice to determine the optimal dose and drug-imaging interval. Results were confirmed in rat and pig, with the latter used to demonstrate, for the first time, simultaneous fluorescence imaging of blood vessels and nerves during surgery using the FLARE™ (Fluorescence-Assisted Resection and Exploration) imaging system. These results lay the foundation for the development of ideal nerve-highlighting fluorophores for image-guided surgery.

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 18F-FASu as a specific gauge for system xC− activity in vivo and its potential 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 levels and 18F-FASu uptake, while gene knockdown 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). 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. Conclusions 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.

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