Planar Proton Minibeam Irradiation Elicits Spatially Confined DNA Damage in a Human Epidermis Model.

Abstract

Simple SummaryRadiotherapy can lead to severe side effects involving the skin. Proton minibeam radiotherapy (pMBRT) can avoid such side effects by sparing healthy tissue between radiation beams. Here, we spatially mapped DNA damage in response to different widths of proton minibeams (66, 408 and 920 µm) at escalating times after pMBRT, and discovered that focused 66 µm pMBRT induced severe DNA damage at the dose peaks, while damage in the spared tissue was not apparent. Wider proton minibeams (408, 920 µm) damaged all cells. Seventy-two hours after irradiation, DNA damage was repaired, while apoptotic cell death (active caspase-3+) increased significantly 24 and 72 h after pMBRT. Hence, highly focused minibeam-irradiation spares healthy tissue from DNA damage and cell death, which may add to the tissue-sparing effect observed at the macro scale. Thus, highly focused pMBRT may be used in radiotherapy to reduce side effects to the skin.Purpose: High doses of ionizing radiation in radiotherapy can elicit undesirable side effects to the skin. Proton minibeam radiotherapy (pMBRT) may circumvent such limitations due to tissue-sparing effects observed at the macro scale. Here, we mapped DNA damage dynamics in a 3D tissue context at the sub-cellular level. Methods: Epidermis models were irradiated with planar proton minibeams of 66 µm, 408 µm and 920 µm widths and inter-beam-distances of 2.5 mm at an average dose of 2 Gy using the scanning-ion-microscope SNAKE in Garching, GER. γ-H2AX + 53BP1 and cleaved-caspase-3 immunostaining revealed dsDNA damage and cell death, respectively, in time courses from 0.5 to 72 h after irradiation. Results: Focused 66 µm pMBRT induced sharply localized severe DNA damage (pan-γ-H2AX) in cells at the dose peaks, while damage in the dose valleys was similar to sham control. pMBRT with 408 µm and 920 µm minibeams induced DSB foci in all cells. At 72 h after irradiation, DNA damage had reached sham levels, indicating successful DNA repair. Increased frequencies of active-caspase-3 and pan-γ-H2AX-positive cells revealed incipient cell death at late time points. Conclusions: The spatially confined distribution of DNA damage appears to underlie the tissue-sparing effect after focused pMBRT. Thus, pMBRT may be the method of choice in radiotherapy to reduce side effects to the skin.