Radiotracer stereochemistry affects substrate affinity and kinetics for improved imaging of system x_C^- in tumors

Abstract

Amino acid utilization is perturbed in cancer cells, which rewire their metabolism to support cell survival and proliferation. This metabolic reprogramming can be exploited for diagnostic purposes through positron emission tomography imaging of fluorine-18 labeled amino acids. Despite its promise, little is known regarding transporter-recognition of non-natural amino acid stereoisomers or their utility for cancer imaging. We report here the synthesis and in vivo characterization of a radiolabeled amino acid ( R )-4-(3- 18 F-fluoropropyl)-ʟ-glutamate ([ 18 F]FRPG) and compared its tumor imaging properties to the 4 S -isomer, [ 18 F]FSPG. Methods : [ 18 F]FRPG and [ 18 F]FSPG uptake was assessed in H460 lung cancer cells, with efflux measured 30 min after removal of exogenous activity. Specificity of [ 18 F]FRPG for system x C - was further examined following transporter inhibition and blocking studies with system x C - substrates. [ 18 F]FRPG and [ 18 F]FSPG pharmacokinetics was next quantified in mice bearing subcutaneous A549, H460, VCAP and PC3 tumors, with mice bearing A549 tumors imaged by PET/CT. To better-understand differential tumor retention, radiometabolite analysis was performed on tissue and blood samples after imaging. Next, [ 18 F]FRPG and [ 18 F]FSPG retention in lipopolysaccharide-treated lungs were compared to an orthotopic H460 lung cancer model. Finally, the sensitivity of [ 18 F]FRPG to manipulation of the redox environment was examined in cell and in vivo models. Results : [ 18 F]FRPG was specifically transported across the plasma membrane by the cystine/glutamate antiporter system x C - and retained at high levels in multiple tumor models. Conversely, [ 18 F]FRPG was rapidly extracted from the blood and cleared from tissues with low system x C - expression. Due to its favorable imaging properties, tumor-to-blood ratios ≥10 were achieved with [ 18 F]FRPG, which were either equal to or greater than [ 18 F]FSPG. In addition, [ 18 F]FRPG retention in orthotopic lung tumors with high system x C - expression was 2.5-fold higher than inflamed tissue, allowing for clear tumor visualization. In vivo , [ 18 F]FRPG and [ 18 F]FSPG were metabolized to a single species, with [ 18 F]FRPG showing a higher ...

mRNA therapy corrects defective glutathione metabolism and restores ureagenesis in preclinical argininosuccinic aciduria

“…Metabolic reprogramming in cancer cells generates oxidative stress, which is balanced by increased glutathione biosynthesis via enhanced x c − -mediated cystine import ( 30 ). Using PET, the radiolabeled glutamate analog [ 18 F]FSPG provides an in vivo functional readout of de novo glutathione biosynthesis and has been used both in the clinic for cancer diagnosis ( 29 , 31 , 32 ) and used preclinically to assess cancer drug resistance ( 33 ) and disease progression in multiple sclerosis ( 34 ).…”

mRNA therapy restores ureagenesis and corrects glutathione metabolism in argininosuccinic aciduria

“…Western blot analysis for was carried out using the iBind Flex system (ThermoFisher Scientific), a previously published method (26), for antibody immunoblotting. To produce lysates frozen liver and skin tissue was processed and the protein concentration determined as above, except, the prechilled ice cold 400 μL RIPA buffer, with 1% Proteinase and phosphatase inhibitors (HALT) was used instead of 1X passive lysis buffer.…”

“…Western blot: Western blot analysis for was carried out using the iBind Flex system (ThermoFisher Scientific), a previously published method (26), for antibody immunoblotting.…”

“…Cystine is subsequently reduced to cysteine for de novo glutathione biosynthesis (19). Owing to the overexpression of system x C in malignancy, [ 18 F]FSPG has shown utility in clinical trials for cancer diagnosis (20)(21)(22)(23), and preclinically [ 18 F]FSPG has been used to non-invasively assess redox status both in tumors (24)(25)(26) and in a mouse model of multiple sclerosis (27).…”

A prodrug strategy for the in vivo imaging of aldehyde dehydrogenase activity

“…Western blot analysis was carried out using an iBind Flex system (ThermoFisher Scientific) for primary and secondary antibody immunoblotting following a previously described method. 29 For cell lysate collection SKOV3-ip1 and SKOV3-TRip2 cells were seeded in 6-well plates for 24 h at a density of 1.25 × 10 5 mL −1 and 1.75 × 10 5 mL −1 , respectively, in 2 mL of media. Lysates were collected in RIPA buffer, protein quantified by Pierce BCA assay and 20–30 μg loaded on a 10% polyacrylamide gel which was run at 200 V. Rabbit monoclonal primary antibodies against anti-ALDH1A1 (Cell Signaling Technology) was used at a concentration of 1 : 1000 and anti-rabbit IgG secondary antibody (1 : 200 dilution; Cell Signaling Technology) for cell lysates analysis.…”