18F-5-Fluoroaminosuberic Acid as a Potential Tracer to Gauge Oxidative Stress in Breast Cancer Models

Abstract

The cystine transporter (system x_C^-) 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 promoter. We have developed ¹⁸F-5-fluoroaminosuberic acid (FASu), a PET tracer that targets system x_C^- The goal of this study was to evaluate ¹⁸F-FASu as a specific gauge for system x_C^- activity in vivo and its potential for breast cancer imaging. Methods:¹⁸F-FASu specificity toward system x_C^- was studied by cell inhibition assay, cellular uptake after OS induction with diethyl maleate, with and without anti-xCT small interfering RNA knockdown, in vitro uptake studies, and in vivo uptake in a system x_C^--transduced xenograft model. In addition, radiotracer uptake was evaluated in 3 breast cancer models: MDA-MB-231, MCF-7, and ZR-75-1. Results: Reactive oxygen species-inducing diethyl maleate increased glutathione levels and ¹⁸F-FASu uptake, whereas gene knockdown with anti-xCT small interfering RNA led to decreased tracer uptake. ¹⁸F-FASu uptake was robustly inhibited by system x_C^- inhibitors or substrates, whereas uptake was significantly higher in transduced cells and tumors expressing xCT than in wild-type HEK293T cells and tumors (P < 0.0001 for cells, P = 0.0086 for tumors). ¹⁸F-FASu demonstrated tumor uptake in all 3 breast cancer cell lines studied. Among them, triple-negative breast cancer MDA-MB-231, which has the highest xCT messenger RNA level, had the highest tracer uptake (P = 0.0058 when compared with MCF-7; P < 0.0001 when compared with ZR-75-1). Conclusion:¹⁸F-FASu as a system x_C^- substrate is a specific PET tracer for functional monitoring of system x_C^- and OS imaging. By enabling noninvasive analysis of x_C^- responses in vivo, this biomarker may serve as a valuable target for the diagnosis and treatment monitoring of certain breast cancers.

Keywords: F-18; PET; oxidative stress; system xC−; tumor imaging.

FIGURE 1.

Radiosynthesis of ¹⁸F-FASu. DMSO = dimethyl sulfoxide; TFA = trifluoroacetic acid.

FIGURE 2.

¹⁸F-FASu uptake specificity study. (A) ¹⁸F-FASu uptake in MDA-MB-231 cells at 60 min with addition of DEM at 0, 2, 5, 10, 20, 50, and 100 μM to induce OS. (B) Glutathione concentration in MDA-MB-231 cells with addition of DEM at 0, 2, 5, 10, 20, 50, and 100 μM. (C) Uptake in MDA-MB-231 cells after addition of xCT siRNA, control siRNA, no siRNA, or 500 μM sulfasalazine. (D) Uptake in HEK wild-type and HEK::xCT cells at 60 min (P < 0.0001). *P = 0.0017. **P = 0.0010. SSZ = sulfasalazine.

FIGURE 3.

(A) Biodistribution with (n = 5) and without (n = 5) coinjection of 100 mg/kg dose of aminosuberic acid in mice bearing both HEK::xCT and HEK wild-type tumors. (B) PET maximum-intensity projection at 45–60 min after injection. Kidneys and bladder are visible on both images, whereas pancreas and spleen are visible only in unblocked condition. WT = HEK wild-type tumor; xCT = HEK::xCT tumor.

FIGURE 4.

(A) ¹⁸F-FASu uptake in MDA-MB-231, MCF-7, and ZR-75-1 cell lines at 20, 40, and 60 min with and without system x_C⁻ inhibitor sulfasalazine (0.5 mM). (B) xCT messenger RNA expression levels in ZR-75-1, MCF-7, and MDA-MB-231 cells. *P < 0.0001. **P < 0.0001. SSZ = sulfasalazine.

FIGURE 5.

¹⁸F-FASu PET image and biodistribution in breast cancer tumor–bearing mice. (A) Tumor-to-blood and tumor-to-muscle uptake ratio at various time points in MDA-MB-231 tumor–bearing mice. (B) PET imaging of MDA-MB-231 tumor–bearing mouse at 2 h after injection (fused PET/CT axial, coronal, and sagittal slices). (C) Biodistribution in MDA-MB-231 tumor–bearing mice at 2 h after injection. (D) Comparison of tumor uptake in mice bearing MDA-MB-231, MCF-7, and ZR-75-1 tumors at 2 h after injection. *P = 0.0058. **P < 0.0001. T = tumor; T/B = tumor-to-blood ratio; T/M = tumor-to-muscle ratio.