Hted 1H anatomical image (upper left), hyperpolarized [1-13C]lactate image and [1-13C]Title Loaded From File pyruvate image (lower left and right, respectively) acquired from the MDA-MB-231 rat xenograft model. Signals from lactate and pyruvate measured from the tumor were also plotted as a function of time (upper right). doi:10.1371/journal.pone.0056551.gFigure 2. Parameters obtained from hyperpolarized 13C metabolic imaging of MDA-MB-231 tumors. Left: ratio of lactate to pyruvate signals measured in the 1655472 rat tumors and kidneys (* 25033180 P,0.05). Right, Total 13C signals in the tumors (pyruvate+lactate) normalized by total 13C signals in the kidneys. doi:10.1371/journal.pone.0056551.gRadiation Therapy Response and 13C Metabolic MRIFigure 3. Apoptosis, senescence and vascularity of the tumors were assessed by TUNEL, SA-b-galactosidase and CD31 staining, respectively. A) Representative photographs of TUNEL, b-galactosidase and CD31 staining of control or radiation treated MDA-MB-231 tumors. B) Significantly increased cell apoptosis and senescence were Title Loaded From File observed for tumors treated with radiation (* p,0.05). Lower MVD was observed for the treated tumors but the difference was not significant. doi:10.1371/journal.pone.0056551.gSignificantly lower average lactate to pyruvate ratios were observed in radiation treated tumors as compared to the control tumors (0.32 vs. 0.46, p,0.05, Fig. 2, left), while slightly higher lactate over pyruvate ratios were observed in the rat kidneys for the treated animals (0.18 vs. 0.15, not statistically significant, Fig. 2). Total 13C signal in the tumor (pyruvate+lactate) normalized by the total 13C signal in the kidney were similar between the control and the treated animals (Fig. 2 right). Average tumor volumes for the treated cohort were larger than for thecontrol cohort at the time of imaging (6.6 ml vs. 4.7 ml) but the difference was not significant (P.0.3). TUNEL and b-galactosidase assays were performed on harvested tumors following the imaging studies to assess apoptosis and senescence, respectively. A significant increase in apoptosis was observed in the irradiated tumors as compared to the untreated tumors (16.1 vs. 6.5 , p,0.05, Fig. 3). A larger and also significant increase in b-galactosidase staining was found in the radiation treated tumors as compared to the controls (22.6Radiation Therapy Response and 13C Metabolic MRIRadiation Therapy Response and 13C Metabolic MRIFigure 4. Cell apoptosis, senescence and flux between pyruvate and lactate were investigated in MDA-MB-231 cells after radiation treatment in vitro. A) Representative flow cytometry data from the Annexin 5 and PI assay, b-galactosidase staining and 13C MRS spectra from cells at 96 hours post 16 Gy radiation as well as control cells are shown. B) Significant increase in cell apoptosis and senescence were observed in treated cells as compared to control cells (* P,0.05). doi:10.1371/journal.pone.0056551.gvs. 3.2 , p,0.05, Fig. 3). CD31 staining was also performed to assess any changes in tumor microvasculature post radiation therapy. Slightly lower MVD was observed in radiation treated tumors as compared to controls, and the difference was not statistically significantly (14.7 vs. 12.0, Fig. 3). Long segments of the tubules formed by the MS1 cells [23] were observed in the tumor histopathologic slides but showed virtually no TUNEL or bgalactosidase staining, both in the radiation treated tumors and the controls, indicating that the observed changes were not likely.Hted 1H anatomical image (upper left), hyperpolarized [1-13C]lactate image and [1-13C]pyruvate image (lower left and right, respectively) acquired from the MDA-MB-231 rat xenograft model. Signals from lactate and pyruvate measured from the tumor were also plotted as a function of time (upper right). doi:10.1371/journal.pone.0056551.gFigure 2. Parameters obtained from hyperpolarized 13C metabolic imaging of MDA-MB-231 tumors. Left: ratio of lactate to pyruvate signals measured in the 1655472 rat tumors and kidneys (* 25033180 P,0.05). Right, Total 13C signals in the tumors (pyruvate+lactate) normalized by total 13C signals in the kidneys. doi:10.1371/journal.pone.0056551.gRadiation Therapy Response and 13C Metabolic MRIFigure 3. Apoptosis, senescence and vascularity of the tumors were assessed by TUNEL, SA-b-galactosidase and CD31 staining, respectively. A) Representative photographs of TUNEL, b-galactosidase and CD31 staining of control or radiation treated MDA-MB-231 tumors. B) Significantly increased cell apoptosis and senescence were observed for tumors treated with radiation (* p,0.05). Lower MVD was observed for the treated tumors but the difference was not significant. doi:10.1371/journal.pone.0056551.gSignificantly lower average lactate to pyruvate ratios were observed in radiation treated tumors as compared to the control tumors (0.32 vs. 0.46, p,0.05, Fig. 2, left), while slightly higher lactate over pyruvate ratios were observed in the rat kidneys for the treated animals (0.18 vs. 0.15, not statistically significant, Fig. 2). Total 13C signal in the tumor (pyruvate+lactate) normalized by the total 13C signal in the kidney were similar between the control and the treated animals (Fig. 2 right). Average tumor volumes for the treated cohort were larger than for thecontrol cohort at the time of imaging (6.6 ml vs. 4.7 ml) but the difference was not significant (P.0.3). TUNEL and b-galactosidase assays were performed on harvested tumors following the imaging studies to assess apoptosis and senescence, respectively. A significant increase in apoptosis was observed in the irradiated tumors as compared to the untreated tumors (16.1 vs. 6.5 , p,0.05, Fig. 3). A larger and also significant increase in b-galactosidase staining was found in the radiation treated tumors as compared to the controls (22.6Radiation Therapy Response and 13C Metabolic MRIRadiation Therapy Response and 13C Metabolic MRIFigure 4. Cell apoptosis, senescence and flux between pyruvate and lactate were investigated in MDA-MB-231 cells after radiation treatment in vitro. A) Representative flow cytometry data from the Annexin 5 and PI assay, b-galactosidase staining and 13C MRS spectra from cells at 96 hours post 16 Gy radiation as well as control cells are shown. B) Significant increase in cell apoptosis and senescence were observed in treated cells as compared to control cells (* P,0.05). doi:10.1371/journal.pone.0056551.gvs. 3.2 , p,0.05, Fig. 3). CD31 staining was also performed to assess any changes in tumor microvasculature post radiation therapy. Slightly lower MVD was observed in radiation treated tumors as compared to controls, and the difference was not statistically significantly (14.7 vs. 12.0, Fig. 3). Long segments of the tubules formed by the MS1 cells [23] were observed in the tumor histopathologic slides but showed virtually no TUNEL or bgalactosidase staining, both in the radiation treated tumors and the controls, indicating that the observed changes were not likely.
