Abstract 1802: Correlating flow-cytometric and 19F-NMR changes in fluorodeoxyglucose signal in response to the Raf-inhibitor RAF265
Authors: Dzik-Jurasz Andrzej , Lin Melissa , Dohoney Kathleen , McCormick Jason , Ising Mary , Stuart Darrin , Jespersen Diana 
Published: February 2011

Abstract
FDG-PET (2-Deoxy-2-fluoro-D-glucose-Positron-Emission-Tomography) is an established tool in the management of cancer patients and is increasingly used as an early response marker. Interpreting the cellular basis of the FDG signal however, remains a challenge. The aim of this study was to explore changes in FDG signal by leveraging the quantitative and chemical specificity of 19F-NMR (nuclear magnetic resonance) and correlating these with flow-cytometric (FC) measurement of cell count, apoptosis and cell cycle in A375M melanoma cells treated with the investigational Raf-kinase inhibitor RAF265.
Cell culture and flow cytometry: A375M cells (melanoma cell line expressing B-RafV600E) were grown in Eagle's Minimal Essential Media with 10% HI FCS (Hyclone), penicillin 100U/ml and streptomycin 100μg/ml. Cells were incubated in the presence of 1μM RAF265 or a 1:1000 dilution of DMSO as a control. After 24 hours 1.5 mM FDG was added to the culture media followed two hours later by a perchloric acid extraction of trypsinized cells. The neutralized lysate was analyzed via 19F-NMR. FC measurements were performed on trypsinized cells using a B-D Canto II with Diva software and analyzed with FlowJo Analysis software and Dean-Jett-Fox cell cycle modeling software.
19F-NMR: All experiments were performed at room temperature on a Bruker 300 MHz DPX spectrometer equipped with a QNP probe. All samples were doped with D2O and 5-fluorouracil as the internal 19F-reference. A non-1H-decoupled sequence was used to collect and quantitate spectra. Spectral assignments were based on prior reports.
The spectra and FC results differed qualitatively and quantitatively between groups. Spectral 19F-NMR resonances consistent with phosphorylated FDG metabolites were identified. The mean quantity of FDG metabolites in the treated group was 113.8 nmol ± 3.06 (SD) and 281.3 nmol ± 1.34 (SD) in the untreated group, representing a significant (p&lt;0.001) 59.5% difference. FC results indicated that RAF265 treatment resulted in a mean increase of 32% in the G0/G1 population and a decrease of 41% and 40% in the percentage of cells in the S and G2/M phases respectively. Correspondingly, there was a 27% decrease in the cell count between the treated and untreated groups. There was a four-fold increase in the proportion of Annexin-V staining cells.
In summary, we have identified and quantified FDG metabolites in cell extracts using 19FNMR following treatment with the investigational Raf kinase inhibitor RAF265. There was a qualitative and quantitative drop in FDG metabolites suggesting a modulation in the metabolism of FDG. The corresponding changes in cell cycle and number in the drug-treated samples suggest a less metabolically active profile. By inference, FDG is expected to behave similarly in the clinic and therefore this technique, subject to validation, could provide a cellular basis for the interpretation of response.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1802.