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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) cells were subcutaneously implanted in mice. This was followed by CONV or FLASH IR at 15 Gy. Tumors were harvested at 6 or 48 hours after IR and stained for CD31, phosphorylated myosin light chain (p-MLC), γH2AX (a surrogate marker for DNA double strand break), 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 before 15 Gy CONV IR. Tumors were similarly harvested and analyzed.

Results: By immunostaining, we observed that CONV IR at 6 hours resulted in constricted vessel morphology, increased expression of p-MLC, and much higher numbers of γH2AX-positive cells in tumors, which were not observed with FLASH IR. Mechanistically, MLC activation by ROS is unlikely, because FLASH IR produced significantly more 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 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 the 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 , Myeoung Su Kim  3 , Dawit Oh  3 , Frederik M 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 Jr  9 , G-One Ahn  10
1 Department of Life Science, Pohang University of Science and Technology, Gyeongbuk, Korea.
2 Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Gyeongbuk, Korea.
3 College of Veterinary Medicine, Seoul National University, Seoul, Korea.
4 Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California.
5 Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California; Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, Houston, Texas.
6 Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea.
7 Institute of Stem Cell and Regenerative Medicine, Stanford University School of Medicine, Stanford, California.
8 Department of Radiation Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.
9 Department of Radiation Oncology, Stanford University School of Medicine, Stanford, California. Electronic address: bwloo@stanford.edu.
10 College of Veterinary Medicine, Seoul National University, Seoul, Korea. Electronic address: goneahn@snu.ac.kr.