DNA double strand breaks (DSB) play a pivotal role for cellular damage, which is a hazard encountered in toxicology and radiation protection, but also exploited e.g. in eradicating tumors in radiation therapy. It is still debated whether and in how far clustering of such DNA lesions leads to an enhanced severity of induced damage. Here we investigate - using focused spots of ionizing radiation as damaging agent - the spatial extension of DNA lesion patterns causing cell inactivation. We find that clustering of DNA damage on both the nm and µm scale leads to enhanced inactivation compared to more homogeneous lesion distributions. A biophysical model interprets these observations in terms of enhanced DSB production and DSB interaction, respectively. We decompose the overall effects quantitatively into contributions from these lesion formation processes, concluding that both processes coexist and need to be considered for determining the resulting damage on the cellular level.

Purpose: Proton minibeam radiotherapy using submillimeter beam dimensions allows to enhance tissue sparing in the entrance channel by spatial fractionation additionally to advantageous proton depth dose distribution. In the entrance channel, spatial fractionation leads to reduced side effects compared to conventional proton therapy. The submillimeter sized beams widen with depth due to small angle scattering and enable therefore, in contrary to x-ray microbeam radiation therapy (MRT), the homogeneous irradiation of a tumor. Proton minibeams can either be applied as planar minibeams or pencil shaped with an additional possibility to vary between a quadratic and a hexagonal arrangement for pencil minibeams.The purpose of this work is to deduce interbeam distances to achieve a homogeneous dose distribution for different tumor depths and tumor thicknesses. Furthermore, we aim for a better understanding of the sparing effect on the basis of surviving cells calculated by the linear-quadratic model. Methods: Two-dimensional dose distributions are calculated for proton minibeams of different shapes and arrangements. For a tumor in 10-15 cm depth, treatment plans are calculated with initial beam size of r 0 = 0.2 mm in a water phantom. Proton minibeam depth dose distributions are finally converted into cell survival using a linear-quadratic model. Results: Inter proton beam distances are maximized under the constraint of dose homogeneity in the tumor for tumor depths ranging from 4 to 15 cm and thickness ranging from 0.5 to 10 cm. Cell survival calculations for a 5 cm thick tumor covered by 10 cm healthy tissue show less cell death by up to 85%, especially in the superficial layers, while keeping the cell death in the tumor as in conventional therapy. In the entrance channel, the pencil minibeams result in higher cell survival in comparison to the planar minibeams while all proton minibeam irradiations show higher cell survival than conventional broadbeam irradiation. Conclusion: The deduced constraints for interbeam distances simplify treatment planning for proton minibeam radiotherapy applications in future studies. The cell survival results indicate that proton minibeam radiotherapy reduces side effects but keeps tumor control as in conventional proton therapy. It makes proton minibeam, especially pencil minibeam radiotherapy a potentially attractive new approach for radiation therapy.

Proton radiotherapy using minibeams of sub-millimeter dimensions reduces side effects in comparison to conventional proton therapy due to spatial fractionation. Since the proton minibeams widen with depth, the homogeneous irradiation of a tumor can be ensured by adjusting the beam distances to tumor size and depth to maintain tumor control as in conventional proton therapy. The inherent advantages of protons in comparison to photons like a limited range that prevents a dosage of distal tissues are maintained by proton minibeams and can even be exploited for interlacing from different beam directions. A first animal study was conducted to systematically investigate and quantify the tissue-sparing effects of proton pencil minibeams as a function of beam size and dose distributions, using beam widths between σ = 95, 199, 306, 411, 561 and 883 μm (standard deviation) at a defined center-to-center beam distance (ctc) of 1.8 mm. The average dose of 60 Gy was distributed in 4x4 minibeams using 20 MeV protons (LET ~ 2.7 keV/μm). The induced radiation toxicities were measured by visible skin reactions and ear swelling for 90 days after irradiation. The largest applied beam size to ctc ratio (σ/ctc = 0.49) is similar to a homogeneous irradiation and leads to a significant 3-fold ear thickness increase compared to the control group. Erythema and desquamation was also increased significantly 3–4 weeks after irradiation. With decreasing beam sizes and thus decreasing σ/ctc, the maximum skin reactions are strongly reduced until no ear swelling or other visible skin reactions should occur for σ/ctc < 0.032 (extrapolated from data). These results demonstrate that proton pencil minibeam radiotherapy has better tissue-sparing for smaller σ/ctc, corresponding to larger peak-to-valley dose ratios PVDR, with the best effect for σ/ctc < 0.032. However, even quite large σ/ctc (e.g. σ/ctc = 0.23 or 0.31, i.e. PVDR = 10 or 2.7) show less acute side effects than a homogeneous dose distribution. This suggests that proton minibeam therapy spares healthy tissue not only in the skin but even for dose distributions appearing in deeper layers close to the tumor enhancing its benefits for clinical proton therapy.

Side effects caused by radiation are a limiting factor to the amount of dose that can be applied to a tumor volume. A novel method to reduce side effects in radiotherapy is the use of spatial fractionation, in which a pattern of sub-millimeter beams (minibeams) is applied to spare healthy tissue. In order to determine the skin reactions in dependence of single beam sizes, which are relevant for spatially fractionated radiotherapy approaches, single pencil beams of submillimeter to 6 millimeter size were applied in BALB/c mice ears at a Small Animal Radiation Research Platform (SARRP) with a plateau dose of 60 Gy. Radiation toxicities in the ears were observed for 25 days after irradiation. Severe radiation responses were found for beams ≥ 3 mm diameter. The larger the beam diameter the stronger the observed reactions. No ear swelling and barely reddening or desquamation were found for the smallest beam sizes (0.5 and 1 mm). The findings were confirmed by histological sections. Submillimeter beams are preferred in minibeam therapy to obtain optimized tissue sparing. The gradual increase of radiation toxicity with beam size shows that also larger beams are capable of healthy tissue sparing in spatial fractionation.

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