The Effect of Dose Rate on Tumor Microenvironment in External Beam Radiotherapy

Abstract

The fact that treatment can be given at a narrow dose rate range in external radiotherapy (ERT) applications with crude delivery techniques has caused the dose rate to be neglected for a long time. Recently dose delivery technology has rapidly evolved and with the re-introduction of FLASH-RT to the radiation oncology stage, dose rate studies escalated. It is also known that interactions within the tumor microenvironment (TME) play a significant role in tumor prognosis. Herein we aimed to investigate the effect of three different dose rates on TME. To generate the tumor model, 1 million B16-F10 malignant melanoma cells were subcutaneously injected into the right flank of C57BL/6 mice. RT time was estimated to be 0.5 cm in diameter. Forty-two mice were randomly assigned to the control group, 2 different doses (2 Gy and 10 Gy), and 3 different dose rates (1 Gy/min, 6 Gy/min, and 14 Gy/min). Vernier scale measurements were taken every day to monitor the mice's tumor size. Euthanasia is induced on the seventh day after ERT. Tumor and metastatic organs were harvested, and evaluated histomorphological, and immunohistochemically. Intracardiac blood was collected for the comet assay. After radiation, the 10 Gy arm's mean tumor size was lower than that of the 2 Gy and control arms (p = 0.01 and p = 0.007). Tumor size did not differ between the 2 Gy and control arms (p = 0.53). Additionally, there was no difference in tumor diameters between low and high doses at different dose rates (p = 0.12, p = 0.21). Blood mononuclear cells in the control arm had less DNA damage than those in the 10 Gy arm (p = 0.04). The 2 Gy and 10 Gy arms did not differ in terms of DNA damage (p = 0.65). Each dose arm was evaluated in terms of dose rates and no significant difference was found (p = 0.79, p = 0.24). The number of mitoses per 10 HPF was assessed during the histological analysis. The number of mitoses in the 10 Gy arm was less than the 2 Gy arm (p = 0.001). There was no difference at different dose rates in the 2 Gy and 10 Gy arms (p = 0.28, p = 0.97). Pleomorphism score was evaluated as mild-moderate and severe (score 1-2-3). In comparison to the 2 Gy and control arms, the 10 Gy arm had a higher pleomorphism score (p = 0.01). The 2 Gy arm and the control arm did not vary from one another (p = 0.04). Pleomorphism scores were comparable across low and high doses at different dose rates (p = 0.35, p = 0.58). Different dose rates resulted in the same staining pattern with aSMA and SOX-10. In our study involving the B16-F10 syngeneic tumor model, we could not find a significant effect of three different dose rates on TME. With constantly improving treatment techniques, larger dose rates are going to be achieved. Owing to this, protocols should also list dose rate as a treatment variable. This project was supported by the TÜBİTAK, Short Term R&D Funding Program.