Ultra-high dose rate radiation has been reported to produce a more favorable toxicity and tumor control profile compared to conventional dose rates that are used for patient treatment. So far, the so-called FLASH effect has been validated for electron, photon and scattered proton beam, but not yet for proton pencil beam scanning (PBS). Because PBS is the state-of-the-art delivery modality for proton therapy and constitutes a wide and growing installation base, we determined the benefit of FLASH PBS on skin and soft tissue toxicity. Using a pencil beam scanning nozzle and the plateau region of a 250 MeV proton beam, a uniform physical dose of 35 Gy (toxicity study) or 15 Gy (tumor control study) was delivered to the right hind leg of mice at various dose rates: Sham, Conventional (Conv, 1 Gy/s), Flash60 (57 Gy/s) and Flash115 (115 Gy/s). Acute radiation effects were quantified by measurements of plasma and skin levels of TGF-β1 and skin toxicity scoring. Delayed irradiation response was defined by hind leg contracture as a surrogate of irradiation-induced skin and soft tissue toxicity and by plasma levels of 13 different cytokines (CXCL1, CXCL10, Eotaxin, IL1-beta, IL-6, MCP-1, Mip1alpha, TNF-alpha, TNF-beta, VEGF, G-CSF, GM-CSF and TGF- β1). Plasma and skin levels of TGF-β1, skin toxicity and leg contracture were all significantly decreased in FLASH compared to Conv groups of mice. FLASH and Conv PBS had similar efficacy with regards to growth control of MOC1 and MOC2 head and neck cancer cells transplanted into syngeneic, immunocompetent mice. These results demonstrate consistent delivery of FLASH PBS radiation from 1 to 115 Gy/s in a clinical gantry. Radiation response following delivery of 35 Gy indicates potential benefits of FLASH versus conventional PBS that are related to skin and soft tissue toxicity.

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In vitro studies allow evaluation of normal or cancer cell responses to radiation, either alone or in combination with agents used to modify these biological responses. Ionizing radiation can be produced by a variety of particles and sources, with varying energy spectra, interaction probabilities, linear energy transfer, dose uniformity, dose rates, and delivery methods. Multiple radiation sources have been used to irradiate cells in the published literature. However, the equivalence of response in cell culture models across radiation sources has not been rigorously established. Moreover, current reporting of radiation source parameters lacks consistency and rigor which may impact the reproducibility of pre-clinical data between laboratories. Relevant choices of radiation source are also of high importance due to growing interest in comparing photon versus particle radiation effect on biological responses. Therefore, this study robustly evaluates the cellular response (cell survival, apoptosis, and DNA damage) of three distinct cell lines using four unique photon generating radiation sources. We hypothesize there may be subtle differences across the radiation sources, without an appreciable difference in cellular response. The four photon irradiation energies investigated, 662 keV, 100 kVp, 220 kVp, 6 MV, did produce subtle differences in DNA damage and cell survival when treating three distinct tumor cell lines. These variations in cellular response emphasize the need to carefully consider irradiation source, energy, and dose rate depending on study goal and endpoint.

To investigate the role and mechanism of the circadian clock gene BMAL1 in regulating nasopharyngeal carcinoma (NPC) radiosensitivity Materials/Methods: The regulatory role of BMAL1 in NPC radiotherapy was investigated using 44 NPC patients and the NPC cell line CNE2 which was genetically engineered to overexpress or silence BMAL1 gene. Its correlation with NPC survival outcome was determined using immunohistochemistry and Kaplan-Meier survival curves. Infected CNE2 cells were divided into four groups: BMAL1 overexpression and control groups; and BMAL1 RNA interference (RNAi) and control groups; and their responses to different doses of radiotherapy were examined. In addition, the association between BMAL1 and the ATM/ATR pathway was determined using western blot and co-immunoprecipitation assays. Results: The 5-year survival rates of the BMAL1 high expression group were significantly higher than that of the low expression groups (p<0.05). Moreover, the BMAL1 overexpression group showed higher tumor growth inhibition rate (p<0.001) and apoptotic rate (p Z 0.004) after radiotherapy; a lower proportion of S phase cells (p<0.001) but higher proportion of G2/M phase cells (p Z 0.001) after 24 h of 8Gy irradiation; reduced number of colonies (p Z 0.042), and lower survival score (p Z 0.037); and upregulation of ATM/ATR pathway proteins (p<0.05). The reverse results were observed for the RNAi group. Of note, co-immunoprecipitation revealed the interaction between BMAL1 and ATM. Conclusion: Our findings demonstrated the positive correlation between BMAL1 expression and NPC survival and suggest that BMAL1 promotes NPC radiosensitivity by controlling cell cycle distribution of NPC cells through the ATM pathway.