바로가기  >

Abstract
Purpose: To investigate whether the vascular collapse in tumors by conventional dose rate (CONV) irradiation (IR) would also occur by the ultra-high dose rate FLASH IR.

Methods and materials: Lewis lung carcinoma (LLC) were subcutaneously implanted in mice followed by CONV or FLASH IR at 15 Gy. Tumors were harvested at 6 or 48 hr post-IR and stained for CD31, phosphorylated myosin-light chain (p-MLC), γH2AX, intracellular reactive oxygen species (ROS), or immune cells such as myeloid and CD8α T cells. Cell lines were irradiated with CONV IR for Western blot analyses. ML-7 was intraperitoneally administered daily to LLC-bearing mice for 7 days prior to 15 Gy CONV IR. Tumors were similarly harvested and analyzed as above.

Results: By immunostaining, we observed that CONV IR at 6 hr post-IR resulted in constricted vessel morphology, increased expression of phosphorylated myosin light chain (p-MLC), and much higher numbers of γH2AX (surrogate marker for DNA double strand break)-positive cells in tumors, which were not observed with FLASH IR. Mechanistically, we found that MLC activation by reactive oxygen species (ROS) is unlikely since FLASH IR produced significantly higher ROS than CONV IR in tumors. In vitro studies demonstrated that ML-7, an inhibitor of MLC kinase abrogated IR-induced γH2AX formation and disappearance kinetics. Lastly, we observed that CONV IR when combined with ML-7 produced some effects similar to FLASH IR including the reduction in the vasculature collapse, fewer γH2AX-positive cells, and increased immune cell influx to the tumors.

Conclusions: FLASH IR produced novel changes in the tumor microenvironment that were not observed with CONV IR. We believe that MLC activation in tumors may be responsible for some of those microenvironmental changes differentially regulated between CONV and FLASH IR.

Affiliations

Young-Eun Kim  1 , Seung-Hee Gwak  1 , Beom-Ju Hong  2 , Jung-Min Oh  2 , Hyung-Seok Choi  1 , Myeong Su Kim  3 , Dawit Oh  3 , Fred Lartey  4 , Marjan Rafat  4 , Emil Schüler  5 , Hyo-Soo Kim  6 , Rie von Eyben  4 , Irving L Weissman  7 , Cameron J Koch  8 , Peter G Maxim  4 , Billy W Loo  9 , G-One Ahn  10
1 Department of Life Science, Pohang 37673, Gyeongbuk, Korea.
2 Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang 37673, Gyeongbuk, Korea.
3 College of Veterinary Medicine, Seoul National University, Seoul 08826, Korea.
4 Department of Radiation Oncology Stanford University School of Medicine, Stanford, CA94305, USA.
5 Department of Radiation Oncology Stanford University School of Medicine, Stanford, CA94305, USA; Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, Houston, TX77030, USA.
6 Department of Internal Medicine, Seoul National University College of Medicine, Seoul 03080, Korea.
7 Institute of Stem Cell and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA94305, USA.
8 Department of Radiation Oncology, University of Pennsylvania School of Medicine, PA19104, USA.
9 Department of Radiation Oncology Stanford University School of Medicine, Stanford, CA94305, USA. Electronic address: bwloo@stanford.edu.
10 College of Veterinary Medicine, Seoul National University, Seoul 08826, Korea. Electronic address: goneahn@snu.ac.kr.