Department of Medical Physics and Biomedical Engineering, Tehran University of Medical Sciences, Tehran, Iran.
Radiation Oncology Research Center, Cancer institute, Tehran University of Medical Sciences, Tehran, Iran.
Int J Radiat Biol. 2020 Dec;96(12):1585-1596. doi: 10.1080/09553002.2020.1834163. Epub 2020 Nov 12.
The classical dogma that restricted the radiation effect to the directly irradiated cells has been challenged by the bystander effect. This off-target phenomenon which was manifested in adjacent cells via signaling of fully exposed cells might be involved in high-dose Grid therapy as well. Here, an in-vitro study was performed to examine the possible extent of carcinoma cells response to the inhomogeneous dose distribution of Grid irradiation in the context of the bystander effect.
Bystander effect was investigated in human carcinoma cell lines of HeLa and HN5 adjacent to those received high-dose Grid irradiation using 'medium transfer' and 'cell-to-cell contact' strategies. Based on the Grid peak-to-valley dose profile, medium transfer was exerted from 10 Gy uniformly exposed donors to 1.5 Gy uniformly irradiated recipients. Cell-contact bystander was evaluated after nonuniform dose distribution of 10 Gy Grid irradiation using cloning cylinders. GammaH2AX foci, micronucleus and clonogenic assays besides gene expression analysis were performed.
Various parameters (ɑ/β, D37, D50) extracted from survival curve which fitted to the Linear Quadratic model, verified more radioresistance of HN5. Survival fraction at 2 Gy (SF2) indicated as 0.42 ± 0.06 in HeLa and 0.5 ± 0.03 in HN5. The level of survival decrease, DNA damages and micronucleus of cells located in the Grid shielded areas (1.5 Gy cell-to-cell contact bystander cells) were significantly more than the values obtained from cells which were irradiated by merely uniform dose of 1.5 Gy. The gH2AX foci and micronuclei frequencies were enhanced in cell-contact bystander approximately more than 1.8 times. Relative expression of DNA damage repair pathway genes ( and ) in bystander cells increased significantly. The most cell survival reduction (11.6 times) was revealed in the Grid bystander cells of radioresistant cell line (HN5). No statistically significant difference between 10 Gy uniform beam and Grid non-uniform beam was observed.
Various endpoints confirmed an augmented response of cells in the valley dose region of the Grid block significantly (compared with the cells irradiated by identical dose of uniform beam), suggesting the role of high-dose bystander effect which was more pronounced in resistant carcinoma cell lines. These findings could provide a partial explanation for the Grid beneficial response seen in a number of pre-clinical and clinical studies.
受照射细胞的经典辐射效应理论受到了旁观者效应的挑战。这种非靶效应通过完全暴露细胞的信号传递到邻近细胞，这一现象可能也与高剂量栅格治疗有关。本研究旨在通过旁观者效应，检测在栅格照射不均匀剂量分布的情况下，癌细胞对高剂量栅格治疗的反应程度。
利用“介质转移”和“细胞间接触”策略，在与接受高剂量栅格照射的人宫颈癌细胞系 HeLa 和 HN5 相邻的细胞中，研究旁观者效应。基于栅格峰谷剂量分布，将 10Gy 均匀照射的供体介质转移至 1.5Gy 均匀照射的受体。采用克隆圆柱体进行非均匀剂量 10Gy 栅格照射后，评估细胞间接触的旁观者效应。进行了γH2AX 焦点、微核和克隆形成分析以及基因表达分析。
从符合线性二次模型的生存曲线中提取出各种参数（ɑ/β、D37、D50），验证了 HN5 的辐射抗性更高。HeLa 中的生存分数为 2Gy（SF2）为 0.42±0.06，HN5 为 0.5±0.03。位于栅格屏蔽区（细胞间接触旁观者细胞 1.5Gy）的细胞的生存减少程度、DNA 损伤和微核水平明显高于仅接受 1.5Gy 均匀剂量照射的细胞。细胞间接触旁观者的γH2AX 焦点和微核频率增加了约 1.8 倍。旁观者细胞中 DNA 损伤修复途径基因（和）的相对表达显著增加。在辐射抗性细胞系（HN5）的栅格旁观者细胞中，细胞存活率下降最为明显（降低了 11.6 倍）。在 10Gy 均匀束和栅格非均匀束之间没有观察到统计学上的显著差异。
各种终点证实，栅格阻断谷区细胞的反应明显增强（与接受相同剂量均匀束照射的细胞相比），表明高剂量旁观者效应的作用更为显著，这种效应在耐药性癌细胞系中更为明显。这些发现可以为一些临床前和临床研究中观察到的栅格治疗有益反应提供部分解释。
