¹⁸F-5-Fluoroaminosuberic Acid 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 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.

Synthesis, radiosynthesis, and positron emission tomography neuroimaging using 5‐[18F]fluoro‐L‐amino suberate

“…The synthesis of FASu 1 is shown in Scheme . The syntheses were carried out using reported methods with some modifications. In brief, commercially available, functional group protected L ‐ glutamate 2 was used as a starting material.…”

Addressing Chirality in the Structure and Synthesis of [18F]5‐Fluoroaminosuberic Acid ([18F]FASu)

“…[12] Cells were maintained in 10 cm tissue culture dishes in ah umidified incubator at 37 8Cw ith 5% CO 2 and routinely subcultured at around 85 %c onfluency.F or uptake studies, cells were subcultured into 24 well plates such that they achieved confluency the next day.E ach plate was washed two times with cold HEPES buffer and uptake initiated by the addition of 0.148 MBq [ 18 F]FASu in 400 mLH EPES buffer.O ne well from each cell line was used to assess the cell number using aM oxi cell counter.S ubsequently the samples were incubated rocking at 37 8Cf or the indicated duration. HEK:xCT cell lines were generated using reported method.…”

“…[11] Previously,w eh ave demonstrated 18 FASu is as pecific tracer towards the cystine transporter and cellularo xidative stress.I th as been used for PET imaging of lymphoma, ovarian-, and breast cancer xenograft-bearing mice. [12] Most aminoa cid tracers are fluorinated by nucleophilic displacement, assisted by phase-transfer catalysts (PTC) with heating;r eaction conditions that may cause racemization of the inherentc hiral centers within the compound. [13] The chirality of the radiotracers, especiallyt hose modelled on amino acids,i s likely to be importantt ot he biological properties of the compound, and changes in opticalc onfiguration have long been knownt oa ffect pharmacokinetic behaviour.I na ddition, for radiopharmaceutical administration,t he isomer with the best imaging properties should be used to minimize patient dose due to extraneous uptake of the non-specific isomer in nontargett issue.…”

Metabolic Imaging of Glutamine in Cancer

“…A similar tracer targeting the Xc transporter system, 5-18 F-fluoro-aminosuberic acid, also demonstrated potential usefulness as a tracer for monitoring of upregulation of system Xc transporter and oxidative stress (30). Currently reported 18 F-labeled amino acid-based PET imaging agents are designed to take advantage of the increase of amino acid transporters on the membrane of tumor tissue.…”