Decline of Tumor Vascular Function as Assessed by Dynamic Contrast-Enhanced Magnetic Resonance Imaging Is Associated With Poor Responses to Radiation.

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Decline of Tumor Vascular Function as Assessed by Dynamic Contrast-Enhanced Magnetic Resonance Imaging Is Associated With Poor Responses to Radiation Therapy and Chemotherapy Fang-Hsin Chen, PhD, Chun-Chieh Wang, MD, PhD, Ho-Ling Liu, PhD, Sheng-Yung Fu, BSc, Ching-Fang Yu, PhD, Chen Chang, PhD, Chi-Shiun Chiang, PhD, Ji-Hong Hong, MD, PhD International Journal of Radiation Oncology • Biology • Physics Volume 95, Issue 5, Pages 1495-1503 (August 2016) DOI: 10.1016/j.ijrobp.2016.03.051 Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 1 Tumor growth and pathologic changes. Serial magnetic resonance imaging scans were performed on 3 mice in 1 group after tumor implantation and repeated in triplicate (n=9). (A) Representative tumor growth curve measured from T2-weighted images. (B) The percentage of tumor necrosis and hypoxia in tumors against time (n=5 in each time point). (C) Representative microscopic images illustrating the necrotic area (delineated by black line) in tumors (hematoxylin and eosin; bar, 500 μm). International Journal of Radiation Oncology • Biology • Physics 2016 95, 1495-1503DOI: (10.1016/j.ijrobp.2016.03.051) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 2 Spatial distribution of the Ktrans profile. Serial magnetic resonance imaging scans were performed on 3 mice after tumor implantation, and repeated in triplicate (n=9). (A) Representative Ktrans map from the biggest tumor region. (B) Temporal evolution of Ktrans values, which showed an initial increase from day 1 to day 7 and a subsequent decrease thereafter and the stable Ktrans values from the contralateral leg without tumor implantation. (C) Regional evolution of Ktrans fold changes in tumor core and periphery when the value on day 3 was used as the reference (*P<.05). International Journal of Radiation Oncology • Biology • Physics 2016 95, 1495-1503DOI: (10.1016/j.ijrobp.2016.03.051) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 3 Spatial distribution of tumor vasculature, and temporal changes of microvascular density (MVD) and blood flow. (A) Representative immunohistochemical images of CD31-positive tumor vessels (red) from day 3 to day 19. (Bar, 100 μm). (B) Quantitative analysis of MVD among tumor sections from day 3 to day 19 to reflect changes in MVD over time (n=5 in each time point). (C) Comparisons of MVD between tumor core and periphery in tumors of day 7 and day 12. (D) Representative immunohistochemical images of CD31-positive tumor vessels (red) and Hoesch33342 perfusion (blue) in tumors of day 7 and day 12 (bar, 100 μm). International Journal of Radiation Oncology • Biology • Physics 2016 95, 1495-1503DOI: (10.1016/j.ijrobp.2016.03.051) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 4 Tumor growth delay and DNA double-strand break induced by radiation therapy. (A) The responding pattern of TRAMP-C1 tumors to a dose of 25 Gy when irradiated before and after the change of Ktrans (n=5 in each group). (B) Representative profiles of γH2AX signal among tumors irradiated on day 7 (left) and day 12 (right). (C) Quantitative analysis of γH2AX-positive cells in the core and peripheral regions of the tumors irradiated at day 7 and day 12 (n=5 in each group). (D) Statistical correlation between Ktrans and the yield of DNA double-strand break. International Journal of Radiation Oncology • Biology • Physics 2016 95, 1495-1503DOI: (10.1016/j.ijrobp.2016.03.051) Copyright © 2016 Elsevier Inc. Terms and Conditions

Fig. 5 Heterogeneous distribution of doxorubicin in TRAMP C-1 tumors and its cytotoxicity to tumor cells. (A) Left, autofluorescence signal of doxorubicin (red) in day 7 and day 12 tumors (bar, 500 μm). Right, corresponding microscopic views of the very next cryosection (hematoxylin and eosin). (M = muscle; T = tumor) (B) Quantitative analysis of the apoptotic cell percentages by TUNEL staining within the whole tumor, core, and periphery (n=5 in each group). International Journal of Radiation Oncology • Biology • Physics 2016 95, 1495-1503DOI: (10.1016/j.ijrobp.2016.03.051) Copyright © 2016 Elsevier Inc. Terms and Conditions