The mean, standard deviation and coefficient of variation for all repetitions (N = 7) of the dynamic phantom.

<p>Lac and Pyr refer to the total volume under the spectral and temporal curves for each tracer; <i>k_PL</i> is the forward reaction rate (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071274#pone.0071274.e006" target="_blank">Equations 6</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071274#pone.0071274.e010" target="_blank">8</a>).</p

A schematic view of the dynamic chemical phantom structure.

<p>The injection and exhaust ports were fitted with catheters to facilitate rapid mixture of reagents at isocenter. A thin acrylic sheet was attached to the top to seal the fill cavity. This top could be removed to allow cleaning after injection. The phantom rested on a sled that allowed convenient removal and insertion of the phantom and included warm circulating water to maintain constant temperature.</p

Dynamic signal evolution across (N = 7) injections.

<p>The mean signal for lactate and pyruvate, normalized to peak carbon signal for each injection, are displayed with error bars that indicate the minimum and maximum values at each time over all injections. Total HP ¹³C was estimated by summing signal from HP ¹³C Lactate and HP ¹³C Pyruvate. The average linewidth for pyruvate and lactate peaks were 19±5 Hz and 17±5 Hz, respectively.</p

Spectroscopic images of the reaction carried out in a standard imaging phantom.

<p>Proton imaging (top left) shows the phantom structure in high resolution. Spectroscopic imaging data acquired using a radial EPSI sequence allows metabolite-specific visualization of tracer distribution (bottom row). Spectroscopic data can be intrinsically registered to high-resolution proton images (top center and right).</p

IR cytotoxicity in ATC is driven by changes in ROS levels.

<p>A) ATC (U-HTH83) intra-cellular ROS levels can be manipulated through the addition of exogenous ROS sources (H₂O₂) or ROS scavenging NAC. B) IR induces a dose dependent increase in intra-cellular ROS levels, which is neutralized by the addition of NAC. C) IR cytoxicity as measured using surviving fraction can be potentiated by the addition of H₂O₂ or reversed by NAC. Data are presented as averages with error bars representing standard deviation. Each experiment was performed at least in duplicate. *indicates p-value <0.05 compared to corresponding control condition unless otherwise indicated as in panel C. All experiments were conducted using the U-HTH83 cell line. (CNT = control, DCFDA = 2′,7′-dichlorodihydrofluorescein diacetate).</p

HP-MRS can detect both acute and chronic IR induced changes in ATC reducing potential.

<p>A) Control (n = 4) and irradiated (5 Gy) (n = 3) tumors were imaged pre- and post- IR (or sham). Generation of labeled lactate (nLac) and conversion rate constants (Kpl) were calculated and changes from first to second measurement were recorded. B) Control and irradiated ATC tumors demonstrating difference in size using anatomic imaging (T2 weighted sequential slices). C) Reducing potential levels in control (n = 3) and irradiated (n = 3) tumors at 2 weeks post irradiation (5 Gy).</p

ATC tumors display significant metabolic heterogeneity.

<p>A) Control and irradiated tumors (IR) were serially sectioned and H&E staining was used to evaluate overall tumor architecture. Control tumors were substantially larger, but the majority of the tumor core consisted of non viable tissue (NVT). In contrast the majority of the irradiated tumor volume consisted of apparently viable tissue (VT). Irradiated tumors exhibited a high degree of aberrant cellular morphology as illustrated in the right lower panel inset. B) ATC tumor imaged at 2 weeks following tumor cell injection. Single snapshot imaging was performed 20 seconds after injection of labeled pyruvate. Spatial heat maps were generated from raw data and superimposed onto T2 weighted anatomic images for both pyruvate and lactate. Chemical spectra obtained in two separate voxels demonstrating differential conversion of pyruvate into lactate are shown.</p

Perturbations in oxidative stress and reducing potential are reflected in altered lactate generation.

<p>A) Endogenous reactive oxygen species (ROS) are generated by multiple cellular processes in both the mitochondria and cytoplasm. Exogenous ROS can increase the free radical burden inside the cell. Reducing equivalents in the form of NAD and NADP moieties are generated through multiple metabolic pathways and can cycle rapidly throughout various cellular compartments. Reducing equivalents are utilized by the cell to neutralize ROS. The conversion of pyruvate into lactate requires the presence of NADH; the conversion rate of labeled pyruvate into lactate therefore is an indirect measure of global cellular reducing potential and ROS stress. B) ATC cells were exposed to varying dose of radiation. Cells were harvested at indicated time points and lactate production was assayed biochemically. Data are presented as means, with error bars indicating standard deviation. *indicates p-value compared to control time point <0.05 using a two-tailed Student’s t test.</p

Glucose catabolism controls ATC reducing potential and ROS levels.

<p>A) ATC (U-HTH7) cellular reducing potential is maintained largely through glucose catabolism. B) ATC (U-HTH83) inhibition of glucose catabolism using 2-deoxyglucose (2-DG) increases intra-cellular ROS levels in a dose dependent fashion. C) ROS perturbations trigger changes in cellular reducing potential. D) Inhibition of glucose catabolism radiosensitizes ATC cells (U-HTH83) in a dose dependent manner. These effects are reversed by NAC. Data are presented as averages with error bars representing standard deviation. Each experiment was performed at least in duplicate. *indicates p-value <0.05 compared to corresponding control condition unless otherwise indicated. (CNT = control, DCFDA = 2′,7′-dichlorodihydrofluorescein diacetate).</p