This study aimed to evaluate whether high-energy X-rays (HEXs) of the PARTER (platform for advanced radiotherapy research) platform built on CTFEL (Chengdu THz Free Electron Laser facility) can produce ultrahigh dose rate (FLASH) X-rays and trigger the FLASH effect. Materials and methods: EBT3 radiochromic film and fast current transformer (FCT) devices were used to measure absolute dose and pulsed beam current of HEXs. Subcutaneous tumor-bearing mice and healthy mice were treated with sham, FLASH, and conventional dose rate radiotherapy (CONV), respectively to observe the tumor control efficiency and normal tissue damage. Results: The maximum dose rate of HEXs of PARTER was up to over 1000 Gy/s. Tumor-bearing mice experiment showed a good result on tumor control (p < 0.0001) and significant difference in survival curves (p < 0.005) among the three groups. In the thorax-irradiated healthy mice experiment, there was a significant difference (p = 0.038) in survival among the three groups, with the risk of death decreased by 81% in the FLASH group compared to that in the CONV group. The survival time of healthy mice irradiated in the abdomen in the FLASH group was undoubtedly higher (62.5% of mice were still alive when we stopped observation) than that in the CONV group (7 days). Conclusion: This study confirmed that HEXs of the PARTER system can produce ultrahigh dose rate X-rays and trigger a FLASH effect, which provides a basis for future scientific research and clinical application of HEX in FLASH radiotherapy.

DNA-damaging treatments such as radiotherapy (RT) have become promising to improve the efficacy of immune checkpoint inhibitors by enhancing tumor immunogenicity. However, accompanying treatment-related detrimental events in normal tissues have posed a major obstacle to radioimmunotherapy and present new challenges to the dose delivery mode of clinical RT. In the present study, ultrahigh dose rate FLASH X-ray irradiation was applied to counteract the intestinal toxicity in the radioimmunotherapy. In the context of programmed cell death ligand-1 (PD-L1) blockade, FLASH X-ray minimized mouse enteritis by alleviating CD8 + T cell-mediated deleterious immune response compared with conventional dose rate (CONV) irradiation. Mechanistically, FLASH irradiation was less efficient than CONV X-ray in eliciting cytoplasmic double-stranded DNA (dsDNA) and in activating cyclic GMP-AMP synthase (cGAS) in the intestinal crypts, resulting in the suppression of the cascade feedback consisting of CD8 + T cell chemotaxis and gasdermin E-mediated intestinal pyroptosis in the case of PD-L1 blocking. Meanwhile, FLASH X-ray was as competent as CONV RT in boosting the antitumor immune response initiated by cGAS activation and achieved equal tumor control in metastasis burdens when combined with anti–PD-L1 administration. Together, the present study revealed an encouraging protective effect of FLASH X-ray upon the normal tissue without compromising the systemic antitumor response when combined with immunological checkpoint inhibitors, providing the rationale for testing this combination as a clinical application in radioimmunotherapy.

Background Ultrahigh dose‐rate irradiation (FLASH‐IR) was reported to be efficient in tumor control while reducing normal tissue radiotoxicity. However, the mechanism of such phenomenon is still unclear. Besides, the FLASH experiments using high energy X‐ray, the most common modality in clinical radiotherapy, are rarely reported. This study aims to investigate the radiobiological response using 6 MV X‐ray FLASH‐IR or conventional dose‐rate IR (CONV‐IR). Methods The superconducting linac of Chengdu THz Free Electron Laser (CTFEL) facility was used for FLASH‐IR, a diamond radiation detector and a CeBr3 scintillation detector were used to monitor the time structure and dose rate of FLASH pulses. BALB/c nude mice received whole abdominal 6 MV X‐ray FLASH‐IR or CONV‐IR, the prescribed dose was 15 Gy or 10 Gy and the delivered absolute dose was monitored with EBT3 films. The mice were either euthanized 24 h post‐IR to evaluate acute tissue responses or followed up for 6 weeks to observe late‐stage responses and survival probability. Complete blood count, histological analyses, and measurement of cytokine expression and redox status were performed. Results The mean dose rate of >150 Gy/s and instantaneous dose rate of >5.5 × 105 Gy/s was reached in FLASH‐IR at the center of mice body. After 6 weeks’ follow‐up of mice that received 15 Gy IR, the FLASH group showed faster body weight recovery and higher survival probability than the CONV group. Histological analysis showed that FLASH‐IR induced less acute intestinal damage than CONV‐IR. Complete blood count and cytokine concentration measurement found that the inflammatory blood cell counts and pro‐inflammatory cytokine concentrations were elevated at the acute stage after both FLASH‐IR and CONV‐IR. However, FLASH irradiated mice had significantly fewer inflammatory blood cells and diminished pro‐inflammatory cytokine at the late stage. Moreover, higher reactive oxygen species (ROS) signal intensities but significantly reduced lipid peroxidation were found in the FLASH group than in the CONV group in the acute stage. Conclusions The radioprotective effect of 6 MV X‐ray FLASH‐IR was observed. The differences in inflammatory responses and redox status between the two groups may be the factors responsible for reduced radiotoxicities following FLASH‐IR. Further studies are required to thoroughly evaluate the impact of ROS on FLASH effect.

The ultrahigh dose-rate (FLASH) radiotherapy, which is efficient in tumor control while sparing healthy tissue, has attracted intensive attention due to its revolutionary application prospect. This so-called FLASH effect has been reported in preclinical experiments with electrons, kilo-voltage X-rays, and protons, thus making FLASH a promising revolutionary radiotherapy modality. High energy X-ray (HEX) should be the ideal radiation type for clinical applications of FLASH due to its advantages in deep penetration, small divergence, and cost-friendly. In this work, we report the first implementation of HEXs with ultrahigh dose-rate (HEX-FLASH) and corresponding application of in vivo study of the FLASH effect produced by a high-current (10 mA), high-energy (6-8 MeV) superconducting linac. Joint measurements using radiochromic film, scintillator and Fast Current Transformer device validated that a maximum dose rate of over 1000 Gy/s was achieved in the mice and the mean value within several square centimeters keeps higher than 50 Gy/s within a depth of over 15 cm. The performance of the present HEX can satisfy the requirement of the FLASH study on animals. Breast cancer (EMT6) inoculated into BAL b/c mice was found efficiently controlled by HEX-FLASH. The radio-protective effect of normal tissue was observed on the C57BL/6 mice after thorax/abdomen irradiation by HEX-FLASH. Theoretical analyses of cellular response following HEX-FLASH irradiation based on the radiolytic oxygen depletion hypothesis were performed to interpret experimental results and future experiment design. This work provided the first demonstration of the FLASH effect triggered by HEX, which paved the way for future preclinical research and clinical application of HEX-FLASH.

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