Enhanced RBE of Particle Radiation Depends on Beam Size in the Micrometer Range.

High-linear energy transfer (LET) radiation, such as heavy ions is associated with a higher relative biological effectiveness (RBE) than low-LET radiation, such as photons. Irradiation with low- and high-LET particles differ in the interaction with the cellular matter and therefore in the spatial dose distribution. When a single high-LET particle interacts with matter, it results in doses of up to thousands of gray (Gy) locally concentrated around the ion trajectory, whereas the mean dose averaged over the target, such as a cell nucleus is only in the range of a Gy. DNA damage therefore accumulates in this small volume. In contrast, up to hundreds of low-LET particle hits are required to achieve the same mean dose, resulting in a quasi-homogeneous damage distribution throughout the cell nucleus. In this study, we investigated the dependence of RBE from different spatial dose depositions using different focused beam spot sizes of proton radiation with respect to the induction of chromosome aberrations and clonogenic cell survival. Human-hamster hybrid (AL) as well as Chinese hamster ovary cells (CHO-K1) were irradiated with focused low LET protons of 20 MeV (LET = 2.6 keV/µm) beam energy with a mean dose of 1.7 Gy in a quadratic matrix pattern with point spacing of 5.4 × 5.4 µm2 and 117 protons per matrix point at the ion microbeam SNAKE using different beam spot sizes between 0.8 µm and 2.8 µm (full width at half maximum). The dose-response curves of X-ray reference radiation were used to determine the RBE after a 1.7 Gy dose of radiation. The RBE for the induction of dicentric chromosomes and cell inactivation was increased after irradiation with the smallest beam spot diameter (0.8 µm for chromosome aberration experiments and 1.0 µm for cell survival experiments) compared to homogeneous proton radiation but was still below the RBE of a corresponding high LET single ion hit. By increasing the spot size to 1.6-1.8 µm, the RBE decreased but was still higher than for homogeneously distributed protons. By further increasing the spot size to 2.7-2.8 µm, the RBE was no longer different from the homogeneous radiation. Our experiments demonstrate that varying spot size of low-LET radiation gradually modifies the RBE. This underlines that a substantial fraction of enhanced RBE originates from inhomogeneous energy concentrations on the µm scale (mean intertrack distances of low-LET particles below 0.1 µm) and quantifies the link between such energy concentration and RBE. The missing fraction of RBE enhancement when comparing with high-LET ions is attributed to the high inner track energy deposition on the nanometer scale. The results are compared with model results of PARTRAC and LEM for chromosomal aberration and cell survival, respectively, which suggest mechanistic interpretations of the observed radiation effects.

Dosimetry of heavy ion exposure to human cells using nanoscopic imaging of double strand break repair protein clusters.

The human body is constantly exposed to ionizing radiation of different qualities. Especially the exposure to high-LET (linear energy transfer) particles increases due to new tumor therapy methods using e.g. carbon ions. Furthermore, upon radiation accidents, a mixture of radiation of different quality is adding up to human radiation exposure. Finally, long-term space missions such as the mission to mars pose great challenges to the dose assessment an astronaut was exposed to. Currently, DSB counting using γH2AX foci is used as an exact dosimetric measure for individuals. Due to the size of the γH2AX IRIF of ~ 0.6 µm, it is only possible to count DSB when they are separated by this distance. For high-LET particle exposure, the distance of the DSB is too small to be separated and the dose will be underestimated. In this study, we developed a method where it is possible to count DSB which are separated by a distance of ~ 140 nm. We counted the number of ionizing radiation-induced pDNA-PKcs (DNA-PKcs phosphorylated at T2609) foci (size = 140 nm ± 20 nm) in human HeLa cells using STED super-resolution microscopy that has an intrinsic resolution of 100 nm. Irradiation was performed at the ion microprobe SNAKE using high-LET 20 MeV lithium (LET = 116 keV/µm) and 27 MeV carbon ions (LET = 500 keV/µm). pDNA-PKcs foci label all DSB as proven by counterstaining with 53BP1 after low-LET γ-irradiation where separation of individual DSB is in most cases larger than the 53BP1 gross size of about 0.6 µm. Lithium ions produce (1.5 ± 0.1) IRIF/µm track length, for carbon ions (2.2 ± 0.2) IRIF/µm are counted. These values are enhanced by a factor of 2-3 compared to conventional foci counting of high-LET tracks. Comparison of the measurements to PARTRAC simulation data proof the consistency of results. We used these data to develop a measure for dosimetry of high-LET or mixed particle radiation exposure directly in the biological sample. We show that proper dosimetry for radiation up to a LET of 240 keV/µm is possible.

Sub-micrometer 20MeV protons or 45MeV lithium spot irradiation enhances yields of dicentric chromosomes due to clustering of DNA double-strand breaks.

In conventional experiments on biological effects of radiation types of diverse quality, micrometer-scale double-strand break (DSB) clustering is inherently interlinked with clustering of energy deposition events on nanometer scale relevant for DSB induction. Due to this limitation, the role of the micrometer and nanometer scales in diverse biological endpoints cannot be fully separated. To address this issue, hybrid human-hamster AL cells have been irradiated with 45MeV (60keV/µm) lithium ions or 20MeV (2.6keV/µm) protons quasi-homogeneously distributed or focused to 0.5×1µm(2) spots on regular matrix patterns (point distances up to 10.6×10.6µm), with pre-defined particle numbers per spot to provide the same mean dose of 1.7Gy. The yields of dicentrics and their distribution among cells have been scored. In parallel, track-structure based simulations of DSB induction and chromosome aberration formation with PARTRAC have been performed. The results show that the sub-micrometer beam focusing does not enhance DSB yields, but significantly affects the DSB distribution within the nucleus and increases the chance to form DSB pairs in close proximity, which may lead to increased yields of chromosome aberrations. Indeed, the experiments show that focusing 20 lithium ions or 451 protons per spot on a 10.6µm grid induces two or three times more dicentrics, respectively, than a quasi-homogenous irradiation. The simulations reproduce the data in part, but in part suggest more complex behavior such as saturation or overkill not seen in the experiments. The direct experimental demonstration that sub-micrometer clustering of DSB plays a critical role in the induction of dicentrics improves the knowledge on the mechanisms by which these lethal lesions arise, and indicates how the assumptions of the biophysical model could be improved. It also provides a better understanding of the increased biological effectiveness of high-LET radiation.

Improved normal tissue protection by proton and X-ray microchannels compared to homogeneous field irradiation.

The risk of developing normal tissue injuries often limits the radiation dose that can be applied to the tumour in radiation therapy. Microbeam Radiation Therapy (MRT), a spatially fractionated photon radiotherapy is currently tested at the European Synchrotron Radiation Facility (ESRF) to improve normal tissue protection. MRT utilizes an array of microscopically thin and nearly parallel X-ray beams that are generated by a synchrotron. At the ion microprobe SNAKE in Munich focused proton microbeams ("proton microchannels") are studied to improve normal tissue protection. Here, we comparatively investigate microbeam/microchannel irradiations with sub-millimetre X-ray versus proton beams to minimize the risk of normal tissue damage in a human skin model, in vitro. Skin tissues were irradiated with a mean dose of 2 Gy over the irradiated area either with parallel synchrotron-generated X-ray beams at the ESRF or with 20 MeV protons at SNAKE using four different irradiation modes: homogeneous field, parallel lines and microchannel applications using two different channel sizes. Normal tissue viability as determined in an MTT test was significantly higher after proton or X-ray microchannel irradiation compared to a homogeneous field irradiation. In line with these findings genetic damage, as determined by the measurement of micronuclei in keratinocytes, was significantly reduced after proton or X-ray microchannel compared to a homogeneous field irradiation. Our data show that skin irradiation using either X-ray or proton microchannels maintain a higher cell viability and DNA integrity compared to a homogeneous irradiation, and thus might improve normal tissue protection after radiation therapy.

Subdiffusion supports joining of correct ends during repair of DNA double-strand breaks.

The mobility of damaged chromatin regions in the nucleus may affect the probability of mis-repair. In this work, live-cell observation and distance tracking of GFP-tagged DNA damage response protein MDC1 was used to study the random-walk behaviour of chromatin domains containing radiation-induced DNA double-strand breaks (DSB). Our measurements indicate a subdiffusion-type random walk process with similar time dependence for isolated and clustered DSBs that were induced by 20 MeV proton or 43 MeV carbon ion micro-irradiation. As compared to normal diffusion, subdiffusion enhances the probability that both ends of a DSB meet, thus promoting high efficiency DNA repair. It also limits their probability of long-range movements and thus lowers the probability of mis-rejoining and chromosome aberrations.

Low LET protons focused to submicrometer shows enhanced radiobiological effectiveness.

This study shows that enhanced radiobiological effectiveness (RBE) values can be generated focusing low linear energy transfer (LET) radiation and thus changing the microdose distribution. 20 MeV protons (LET = 2.65 keV µm(-1)) are focused to submicrometer diameter at the ion microprobe superconducting nanoprobe for applied nuclear (Kern) physics experiments of the Munich tandem accelerator. The RBE values, as determined by measuring micronuclei (RBE(MN) = 1.48 ± 0.07) and dicentrics (RBE(D) = 1.92 ± 0.15), in human-hamster hybrid (A(L)) cells are significantly higher when 117 protons were focused to a submicrometer irradiation field within a 5.4 × 5.4 µm(2) matrix compared to quasi homogeneous in a 1 × 1 µm(2) matrix applied protons (RBE(MN) = 1.28 ± 0.07; RBE(D) = 1.41 ± 0.14) at the same average dose of 1.7 Gy. The RBE values are normalized to standard 70 kV (dicentrics) or 200 kV (micronuclei) x-ray irradiation. The 117 protons applied per point deposit the same amount of energy like a (12)C ion with 55 MeV total energy (4.48 MeV u(-1)). The enhancements are about half of that obtained for (12)C ions (RBE(MN) = 2.20 ± 0.06 and RBE(D) = 3.21 ± 0.10) and they are attributed to intertrack interactions of the induced damages. The measured RBE values show differences from predictions of the local effect model (LEM III) that is used to calculate RBE values for irradiation plans to treat tumors with high LET particles.