Ultra-High Dose Rate FLASH Irradiation Induced Radio-Resistance of Normal Fibroblast Cells Can Be Enhanced by Hypoxia and Mitochondrial Dysfunction Resulting From Loss of Cytochrome C.

Jintao Han,Jing Qian,Ye Zhao,Xueqing Yan,Ying Gao,Defeng Kong,Yinren Shou,Zhuo Pan,Jiaer Chen,Zhusong Mei,Yulan Liang,Guijun Qi,Yixing Geng,Chen Lin,Gen Yang,Xiaoyi Sun,Zhipeng Liu,Zhengxuan Cao,Shiyou Chen,Yanying Zhao,Songyun Xu ,Chun‐Yang Lu ,Wu Ma

doi:10.3389/fcell.2021.672929

Abstract

Ultra-high dose rate FLASH irradiation (FLASH-IR) has got extensive attention since it may provide better protection on normal tissues while maintain tumor killing effect compared with conventional dose rate irradiation. The FLASH-IR induced protection effect on normal tissues is exhibited as radio-resistance of the irradiated normal cells, and is suggested to be related to oxygen depletion. However, the detailed cell death profile and pathways are still unclear. Presently normal mouse embryonic fibroblast cells were FLASH irradiated (∼109 Gy/s) at the dose of ∼10–40 Gy in hypoxic and normoxic condition, with ultra-fast laser-generated particles. The early apoptosis, late apoptosis and necrosis of cells were detected and analyzed at 6, 12, and 24 h post FLASH-IR. The results showed that FLASH-IR induced significant early apoptosis, late apoptosis and necrosis in normal fibroblast cells, and the apoptosis level increased with time, in either hypoxic or normoxic conditions. In addition, the proportion of early apoptosis, late apoptosis and necrosis were significantly lower in hypoxia than that of normoxia, indicating that radio-resistance of normal fibroblast cells under FLASH-IR can be enhanced by hypoxia. To further investigate the apoptosis related profile and potential pathways, mitochondria dysfunction cells resulting from loss of cytochrome c (cyt c–/–) were also irradiated. The results showed that compared with irradiated normal cells (cyt c+/+), the late apoptosis and necrosis but not early apoptosis proportions of irradiated cyt c–/– cells were significant decreased in both hypoxia and normoxia, indicating mitochondrial dysfunction increased radio-resistance of FLASH irradiated cells. Taken together, to our limited knowledge, this is the first report shedding light on the death profile and pathway of normal and cyt c–/– cells under FLASH-IR in hypoxic and normoxic circumstances, which might help us improve the understanding of the FLASH-IR induced protection effect in normal cells, and thus might potentially help to optimize the future clinical FLASH treatment.

Highlights

Radiotherapy (RT) makes up an essential part of cancer treatment and more than half of cancer patients receive radiotherapy during treatment (Vozenin et al, 2019a)
Cells were harvested at the confluence of 70–80%, transfered to 96-well plate and stained with Calcin-AM and propidium iodide (PI)
We revealed early apoptosis, late apoptosis and necrosis induced by FLASH irradiation in both cyt c+/+ and cyt c−/− mouse embryonic fibroblast cells

Summary

Introduction

Radiotherapy (RT) makes up an essential part of cancer treatment and more than half of cancer patients receive radiotherapy during treatment (Vozenin et al, 2019a). Major advances in precise treatment delivery and multimodal imaging allowed radiotherapy to apply to more patients, radiation resistance still remains an unsolved clinical problem, more powerful and better tolerated radiotherapy was needed (Vozenin et al, 2019a). A novel modality of irradiation, named FLASH radiotherapy (FLASH-RT), has got extensive attention in recent years, since it provides better protection on normal tissues while maintains tumor killing effects (Durante et al, 2018). The increased radio-resistance and reduced normal tissue toxicity has been named as the FLASH protection effect (Montay-Gruel et al, 2018). Compared with conventional irradiation (∼1 Gy/min), FLASH-RT provides ultra-high dose rate (>40 Gy/s) and is always delivered in an ultra-short time (

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