Abstract
1093
Objectives: Oxidative stress has been discovered as one of the key factors in cancer progression.1 Cancer cells cope with this by upregulating system xC- transporter, the primary source of intracellular cystine,2 which is crucial for glutathione biosynthesis.3 System xC- thus presents a promising imaging biomarker, with several different radiotracers reported previously of which radiofluorinated glutamate analog ([18F]FSPG) and aminosuberic acid analog ([18F]FASu) are the most studied. The goal of this work was to directly compare these two system xC- targeting tracers, in terms of uptake specificity, biodistribution profile and imaging utility.
Methods: Both tracers were synthesized according to previously reported procedures.4-6 Cell uptake assays were conducted using prostate (PC-3), glioblastoma (U-87), colorectal (HT-29), ovarian (SKOV3) and non-small cell lung cancer (A549) cell lines at 20, 40 and 60min in HEPES buffer with or without 1 mM sulfasalazine (SSZ). Additional in vitro specificity and competition assays were conducted using A549, U-87 and MDA-MB-231 cells in the presence of HEPES buffer (control) or 1 mM SSZ, L-trans-pyrrolidine-2,4-dicarboxylate (PDC), L-serine or 2-amino-bicyclo[2.2.1]heptanes-2-carboxylic acid (BCH). PET imaging and biodistribution studies were conducted in immunodeficient mice bearing U-87 or A549 xenografts.
Results: [18F]FASu and [18F]FSPG were synthesized with comparable decay-corrected radiochemical yield (d.c. RCY 18±6% (n=6) and 28±7% (n=4), respectively), radiochemical purity (RCP > 98% in both cases) and molar activities (17.5±7 GBq/mmol for [18F]FASu and 15±5 GBq/mmol for [18F]FSPG). In vitro study showed [18F]FSPG had several fold higher uptake compared to [18F]FASu (%uptake/mg protein: ranging from 5.8±0.5 in U-87 cells to 46.6±1.4 in SKOV3 cells for [18F]FSPG at 60min, and from 1.8±0.1 in U-87 cells to 12.2±1.0 in A549 cells for [18F]FASu , n=3). [18F]FASu uptake was blocked by system xC- inhibitor SSZ but not by system L inhibitor BCH, system xAG- inhibitor PDC or by L-Ser, which is a substrate for transporter systems A, ACS, B0 and B0,+. Conversely, [18F]FSPG uptake decreased significantly in excess of PDC in two out of three tested cell lines, indicating reliance on system xAG- in these cells. In an in vivo setting, [18F]FASu and [18F]FSPG generated good contrast PET images in U-87 and A549 tumor-bearing mice. The tracers had similar biodistribution profiles, with rapid blood clearance (<0.85 %ID/g at 1h p.i.) and predominant renal excretion pathway. Tracer accumulation in A549 tumors at 1h p.i. was 5.0±0.8%ID/g (n≥5 for [18F]FASu) and 6.3±1.3%ID/g (n≥6, [18F]FSPG, adjusted p value = 0.7786) and 6.1±2.4%ID/g (n≥4, [18F]FASu) and 11.2±4.1%ID/g (n≥4 for [18F]FSPG, adjusted p value = 0.0321) in U-87 xenografts.
Conclusions: [18F]FSPG had higher in vitro uptake than [18F]FASu in all tested cell lines; however, our results did indicate that [18F]FSPG relies not only on system xC- to enter the cells, but also system xAG-, and likely other modes of glutamate transport. In vivo studies showed equally good ability of both tracers to image glioblastoma (U-87) and non-small lung cancer (A549) xenografts. References: 1) Gorrini, C., Harris, I. S. & Mak, T. W. Nat. Rev. Drug Discov. 12, 931-947 (2013). 2) Bridges, R. J., Natale, N. R. & Patel, S. A. Br. J. Pharmacol. 165, 20-34 (2012). 3) Conrad, M. & Sato, H. Amino Acids 42, 231-246 (2012). 4) Webster, J. M. et al. J. Nucl. Med. 55, 657-664 (2014). 5) Yang, H. et al. J. Nucl. Med. 58, 367-373 (2017). 6) Koglin, N. et al. Clin. Cancer Res. 17, 6000-6011 (2011).