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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 percentage of parent radiotracer in tumors compared to [ 18 F]FSPG. [ 18 F]FRPG was sensitive to redox manipulations and tumor retention was reduced following treatment with liposomal doxorubicin in mice bearing ovarian tumors. Conclusions : Given the fast clearance and low background retention of [ 18 F]FRPG throughout the body, this radiotracer holds promise for the imaging of system x C - activity and treatment response monitoring in tumors of the thorax, abdomen, and head and neck. [ 18 F]FRPG PET imaging provides a sensitive noninvasive measure of system x C - and excellent properties for cancer imaging., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Purpose: (S)-4-(3- 18 F-Fluoropropyl)-ʟ-Glutamic Acid ([ 18 F]FSPG) is a radiolabeled non-natural amino acid that is used for positron emission tomography (PET) imaging of the glutamate/cystine antiporter, system x C - , whose expression is upregulated in many cancer types. To increase the clinical adoption of this radiotracer, reliable and facile automated procedures for [ 18 F]FSPG production are required. Here, we report a cassette-based method to produce [ 18 F]FSPG at high radioactivity concentrations from low amounts of starting activity., Procedures: An automated synthesis and purification of [ 18 F]FSPG was developed using the GE FASTlab. Optimization of the reaction conditions and automated manipulations were performed by measuring the isolated radiochemical yield of [ 18 F]FSPG and by assessing radiochemical purity using radio-HPLC. Purification of [ 18 F]FSPG was conducted by trapping and washing of the radiotracer on Oasis MCX SPE cartridges, followed by a reverse elution of [ 18 F]FSPG in phosphate-buffered saline. Subsequently, the [ 18 F]FSPG obtained from the optimized process was used to image an animal model of non-small cell lung cancer., Results: The optimized protocol produced [ 18 F]FSPG in 38.4 ± 2.6 % radiochemical yield and >96 % radiochemical purity with a molar activity of 11.1 ± 7.7 GBq/μmol. Small alterations, including the implementation of a reverse elution and an altered Hypercarb cartridge, led to significant improvements in radiotracer concentration from <10 MBq/ml to >100 MBq/ml. The improved radiotracer concentration allowed for the imaging of up to 20 mice, starting with just 1.5 GBq of [ 18 F]Fluoride., Conclusions: We have developed a robust and facile method for [ 18 F]FSPG radiosynthesis in high radiotracer concentration, radiochemical yield, and radiochemical purity. This cassette-based method enabled the production of [ 18 F]FSPG at radioactive concentrations sufficient to facilitate large-scale preclinical experiments with a single prep of starting activity. The use of a cassette-based radiosynthesis on an automated synthesis module routinely used for clinical production makes the method amenable to rapid and widespread clinical translation., (© 2021. The Author(s).)

Tumour response to therapy is assessed primarily in the clinic by monitoring reductions in tumour size. However, this approach lacks sensitivity since in many cases several weeks may elapse before there is evidence of tumour shrinkage. There is therefore a need to develop non-invasive imaging techniques for monitoring tumour treatment response in the clinic. Here, we assessed the pre-clinical utility of (18)F-ICMT-11 positron emission tomography--a method for detecting caspase 3/7 activation--in non-small cell lung cancer (NSCLC). (18)F-ICMT-11 uptake was compared to molecular biochemical measures of cell death in PC9 and A549 NSCLC cells following treatment with carboplatin in vitro and in vivo. Carboplatin-induced apoptosis in the ERCC1 low/mutant EGFR PC9 cells was characterised by time and dose-related increased caspase-3/7 activation, poly-ADP-ribose polymerase cleavage and Annexin V staining. 18F-ICMT-11 uptake was consequently increased up to 14-fold at 200 µM carboplatin compared to vehicle treated cells (P<0.01). In contrast, necrosis was the predominant death mechanism in ERCC1 high/wt EGFR A549 cells and no change in (18)F-ICMT-11 uptake was detected. In vivo, histological analysis of PC9 tumour xenografts indicated high pre-therapy necrosis. A 4.6-fold increase in cleaved caspase-3/7 was measured in non-necrotic regions of PC9 tumours at 48 h post carboplatin therapy. Average PET-derived tumour (18)F-ICMT-11 uptake was insensitive to changes in apoptosis in the presence of substantial pre-existing necrosis. PET-based voxel intensity sorting however, identified intra-tumoural regions of high (18)F-ICMT-11 uptake, enabling accurate assessment of apoptosis and therefore therapy response. In A549 tumours that lacked high pre-therapy necrosis, carboplatin induced growth inhibition that was only minimally associated with apoptosis and thus not detectable by (18)F-ICMT-11 PET.