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Synchrotron Microbeam Radiation Therapy (MRT) is a pioneering radiotherapy technique that spatially fractionates the beam of synchrotron X-rays into an array of quasi-parallel micro-planar beamlets. MRT is effective at delaying and even suppressing the growth of several types of tumors, with minor collateral effects on the normal tissue. One of our studies, conducted in rodents, demonstrated that MRT elicited a minor degree of pulmonary fibrosis compared to synchrotron Broad Beam (BB) radiotherapy even at 600 Gy peak-dose and one year post-irradiation (pi). The aim of this project is to elucidate the early cellular and molecular mechanisms responsible for comprehensive tissue repair and minor degree lung fibrosis after MRT.

To identify the underlying mechanisms, we established an ex-vivo mouse model of lung tissue slices (Fig.1). To the latter synchrotron MRT was applied (50 μm wide beams; 400 μm center to center) with peak-doses of 100, 200, 400 and 800 Gy. Immunostaining and confocal microscopy were performed at different time points: 1h, 4h, 12h, 24h and 48h pi.

The γH2A.X staining revealed persistent robust DNA damage in the cellular compartments exclusively within the beam paths after all doses up to 24h pi. Staining for type IV collagen does not reveal any structural damage to the basement membrane and ECM at all time points (Fig.2). Accumulation of macrophages negative for γH2A.X were observed along the beam paths over time suggesting that macrophages migrated from the “valley” regions between the beams.

The presented data support our hypothesis: (i) microbeams damage the cellular components within the beam path. (ii) The intact macrophages from the surrounding “valley” regions infiltrate and remove cell debris from the beam path. (iii) In the next step, the unaffected lung cells repopulate the “beam” region using the basal membranes as a scaffold.