PARENT SESSION
Work-in-Progress
(WIP-378) Methods for non-invasive monitoring of response to radiation and hyperthermia with 13C NMR spectroscopy and polarographic oxygen probes.
Mancuso, Anthony*,1, Pickup, Stephen1, Kuhn, Nancy1, Glickson, Jerry1, 1 University of Pennsylvania, Dept. of Radiology/4283, Philadelphia, PA
ABSTRACT-
Introduction: 13C NMR spectroscopy can be used to monitor cellular energetics and may be useful in detecting changes that occur with therapy, before death occurs by apoptosis or necrosis. For example, radiation therapy may cause free radical damage to the enzymes of oxidative phosphosphorylation before DNA damage is manifested (1). This may change the relative fluxes for pathways interconnected with the TCA cycle, which will in turn change the rate appearance of 13C-label in glutamate, when cells are fed [1-13C] glucose (2). Because NMR is a relatively insensitive technique, cells must be grown at high densities to detect labeling kinetics. Materials and Methods: EMT6/SF adenocarcinoma cells were grown on the surface of collagen-coated non-porous plastic microcarriers. Because the cells grew only on the outside of the microcarriers as a bi-layer, the diffusion distance for metabolites was very short and heterogeneity in metabolite concentrations was minimized. The microcarriers were perfused inside a 20-mm NMR tube as a tightly-packed fixed bed, to avoid inter-microcarrier collisions and consequent cellular damage. Cells were perfused with pH and temperature controlled DMEM medium containing 10 mM [1-13C] glucose. Oxygen consumption was monitored with polarographic probes in both the inlet and outlet perfusion lines. NMR spectra were acquired with a 9.4 T, 8.9 cm vertical bore magnet and 400 MHz console. Results: 31P NMR of ATP levels indicated that the number of cells in the NMR-detectable window was 1 x 10 9. Similarly, oxygen consumption rates indicated that the cell number was 8 x 10 8 cells. The rate of accumulation of label in glutamate-4 was approximately 0.9 mM/109cells/hr and was much higher than that observed for glutamate-2 and 3. Discussion: The data indicate that TCA cycle labeling kinetics and oxygen consumption can be monitored simultaneously. Cells in this system can be treated with hyperthermia and/or radiation without being removed from the NMR tube, to allow direct assessment of the effects of these therapies on metabolic fluxes. References: (1) Cenciotti L. et al. Radiobiol. Radioter. Fis. Med. 21:390 (1966). (2) Weiss, RG et al., PNAS 86:6426 (1989).
KEYWORDS: NMR , microcarriers, oxygen