Chromatin Organization after High-LET Irradiation Revealed by Super-Resolution STED Microscopy.

Ion-radiation-induced DNA double-strand breaks can lead to severe cellular damage ranging from mutations up to direct cell death. The interplay between the chromatin surrounding the damage and the proteins responsible for damage recognition and repair determines the efficiency and outcome of DNA repair. The chromatin is organized in three major functional compartments throughout the interphase: the chromatin territories, the interchromatin compartment, and the perichromatin lying in between. In this study, we perform correlation analysis using super-resolution STED images of chromatin; splicing factor SC35, as an interchromatin marker; and the DNA repair factors 53BP1, Rad51, and γH2AX in carbon-ion-irradiated human HeLa cells. Chromatin and interchromatin overlap only in protruding chromatin branches, which is the same for the correlation between chromatin and 53BP1. In contrast, between interchromatin and 53BP1, a gap of (270 ± 40) nm is visible. Rad51 shows overlap with decondensed euchromatic regions located at the borders of condensed heterochromatin with further correlation with γH2AX. We conclude that the DNA damage is repaired in decondensed DNA loops in the perichromatin, located in the periphery of the DNA-dense chromatin compartments containing the heterochromatin. Proteins like γH2AX and 53BP1 serve as supporters of the chromatin structure.

Proton-FLASH: effects of ultra-high dose rate irradiation on an in-vivo mouse ear model.

FLASH-radiotherapy may provide significant sparing of healthy tissue through ultra-high dose rates in protons, electrons, and x-rays while maintaining the tumor control. Key factors for the FLASH effect might be oxygen depletion, the immune system, and the irradiated blood volume, but none could be fully confirmed yet. Therefore, further investigations are necessary. We investigated the protective (tissue sparing) effect of FLASH in proton treatment using an in-vivo mouse ear model. The right ears of Balb/c mice were irradiated with 20 MeV protons at the ion microprobe SNAKE in Garching near Munich by using three dose rates (Conv = 0.06 Gy/s, Flash9 = 9.3 Gy/s and Flash930 = 930 Gy/s) at a total dose of 23 Gy or 33 Gy. The ear thickness, desquamation, and erythema combined in an inflammation score were measured for 180 days. The cytokines TGF-ß1, TNF-α, IL1α, and IL1ß were analyzed in the blood sampled in the first 4 weeks and at termination day. No differences in inflammation reactions were visible in the 23 Gy group for the different dose rates. In the 33 Gy group, the ear swelling and the inflammation score for Flash9 was reduced by (57 ± 12) % and (67 ± 17) % and for Flash930 by (40 ± 13) % and (50 ± 17) % compared to the Conv dose rate. No changes in the cytokines in the blood could be measured. However, an estimation of the irradiated blood volume demonstrates, that 100-times more blood is irradiated when using Conv compared to using Flash9 or Flash930. This indicates that blood might play a role in the underlying mechanisms in the protective effect of FLASH.

Longitudinally Heterogeneous Tumor Dose Optimizes Proton Broadbeam, Interlaced Minibeam, and FLASH Therapy.

The prerequisite of any radiation therapy modality (X-ray, electron, proton, and heavy ion) is meant to meet at least a minimum prescribed dose at any location in the tumor for the best tumor control. In addition, there is also an upper dose limit within the tumor according to the International Commission on Radiation Units (ICRU) recommendations in order to spare healthy tissue as well as possible. However, healthy tissue may profit from the lower side effects when waving this upper dose limit and allowing a larger heterogeneous dose deposition in the tumor, but maintaining the prescribed minimum dose level, particularly in proton minibeam therapy. Methods: Three different longitudinally heterogeneous proton irradiation modes and a standard spread-out Bragg peak (SOBP) irradiation mode are simulated for their depth-dose curves under the constraint of maintaining a minimum prescribed dose anywhere in the tumor region. Symmetric dose distributions of two opposing directions are overlaid in a 25 cm-thick water phantom containing a 5 cm-thick tumor region. Interlaced planar minibeam dose distributions are compared to those of a broadbeam using the same longitudinal dose profiles. Results and Conclusion: All longitudinally heterogeneous proton irradiation modes show a dose reduction in the healthy tissue compared to the common SOBP mode in the case of broad proton beams. The proton minibeam cases show eventually a much larger mean cell survival and thus a further reduced equivalent uniform dose (EUD) in the healthy tissue than any broadbeam case. In fact, the irradiation mode using only one proton energy from each side shows better sparing capabilities in the healthy tissue than the common spread-out Bragg peak irradiation mode with the option of a better dose fall-off at the tumor edges and an easier technical realization, particularly in view of proton minibeam irradiation at ultra-high dose rates larger than ~10 Gy/s (so-called FLASH irradiation modes).

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

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.

CeCILE - An Artificial Intelligence Based Cell-Detection for the Evaluation of Radiation Effects in Eucaryotic Cells.

The fundamental basis in the development of novel radiotherapy methods is in-vitro cellular studies. To assess different endpoints of cellular reactions to irradiation like proliferation, cell cycle arrest, and cell death, several assays are used in radiobiological research as standard methods. For example, colony forming assay investigates cell survival and Caspase3/7-Sytox assay cell death. The major limitation of these assays is the analysis at a fixed timepoint after irradiation. Thus, not much is known about the reactions before or after the assay is performed. Additionally, these assays need special treatments, which influence cell behavior and health. In this study, a completely new method is proposed to tackle these challenges: A deep-learning algorithm called CeCILE (Cell Classification and In-vitro Lifecycle Evaluation), which is used to detect and analyze cells on videos obtained from phase-contrast microscopy. With this method, we can observe and analyze the behavior and the health conditions of single cells over several days after treatment, up to a sample size of 100 cells per image frame. To train CeCILE, we built a dataset by labeling cells on microscopic images and assign class labels to each cell, which define the cell states in the cell cycle. After successful training of CeCILE, we irradiated CHO-K1 cells with 4 Gy protons, imaged them for 2 days by a microscope equipped with a live-cell-imaging set-up, and analyzed the videos by CeCILE and by hand. From analysis, we gained information about cell numbers, cell divisions, and cell deaths over time. We could show that similar results were achieved in the first proof of principle compared with colony forming and Caspase3/7-Sytox assays in this experiment. Therefore, CeCILE has the potential to assess the same endpoints as state-of-the-art assays but gives extra information about the evolution of cell numbers, cell state, and cell cycle. Additionally, CeCILE will be extended to track individual cells and their descendants throughout the whole video to follow the behavior of each cell and the progeny after irradiation. This tracking method is capable to put radiobiologic research to the next level to obtain a better understanding of the cellular reactions to radiation.

Optimizing proton minibeam radiotherapy by interlacing and heterogeneous tumor dose on the basis of calculated clonogenic cell survival.

Proton minibeam radiotherapy (pMBRT) is a spatial fractionation method using sub-millimeter beams at center-to-center (ctc) distances of a few millimeters to widen the therapeutic index by reduction of side effects in normal tissues. Interlaced minibeams from two opposing or four orthogonal directions are calculated to minimize side effects. In particular, heterogeneous dose distributions applied to the tumor are investigated to evaluate optimized sparing capabilities of normal tissues at the close tumor surrounding. A 5 cm thick tumor is considered at 10 cm depth within a 25 cm thick water phantom. Pencil and planar minibeams are interlaced from two (opposing) directions as well as planar beams from four directions. An initial beam size of σ0 = 0.2 mm (standard deviation) is assumed in all cases. Tissue sparing potential is evaluated by calculating mean clonogenic cell survival using a linear-quadratic model on the calculated dose distributions. Interlacing proton minibeams for homogeneous irradiation of the tumor has only minor benefits for the mean clonogenic cell survival compared to unidirectional minibeam irradiation modes. Enhanced mean cell survival, however, is obtained when a heterogeneous dose distribution within the tumor is permitted. The benefits hold true even for an elevated mean tumor dose, which is necessary to avoid cold spots within the tumor in concerns of a prescribed dose. The heterogeneous irradiation of the tumor allows for larger ctc distances. Thus, a high mean cell survival of up to 47% is maintained even close to the tumor edges for single fraction doses in the tumor of at least 10 Gy. Similar benefits would result for heavy ion minibeams with the advantage of smaller minibeams in deep tissue potentially offering even increased tissue sparing. The enhanced mean clonogenic cell survival through large ctc distances for interlaced pMBRT with heterogeneous tumor dose distribution results in optimum tissue sparing potential. The calculations show the largest enhancement of the mean cell survival in normal tissue for high-dose fractions. Thus, hypo-fractionation or even single dose fractions become possible for tumor irradiation. A widened therapeutic index at big cost reductions is offered by interlaced proton or heavy ion minibeam therapy.

Normal Tissue Response of Combined Temporal and Spatial Fractionation in Proton Minibeam Radiation Therapy.

PURPOSE: Proton minibeam radiation therapy, a spatial fractionation concept, widens the therapeutic window. By reducing normal tissue toxicities, it allows a temporally fractionated regime with high daily doses. However, an array shift between daily fractions can affect the tissue-sparing effect by decreasing the total peak-to-valley dose ratio. Therefore, combining temporal fractions with spatial fractionation raises questions about the impact of daily applied dose modulations, reirradiation accuracies, and total dose modulations. METHODS AND MATERIALS: Healthy mouse ear pinnae were irradiated with 4 daily fractions of 30 Gy mean dose, applying proton pencil minibeams (pMB) of Gaussian σ = 222 µm in 3 different schemes: a 16 pMB array with a center-to-center distance of 1.8 mm irradiated the same position in all sessions (FS1) or was shifted by 0.9 mm to never hit the previously irradiated tissue in each session (FS2), or a 64 pMB array with a center-to-center distance of 0.9 mm irradiated the same position in all sessions (FS3), resulting in the same total dose distribution as FS2. Reirradiation positioning and its accuracy were obtained from image guidance using the unique vessel structure of ears. Acute toxicities (swelling, erythema, and desquamation) were evaluated for 153 days after the first fraction. Late toxicities (fibrous tissue, inflammation) were analyzed on day 153. RESULTS: Reirradiation of highly dose-modulated arrays at a positioning accuracy of 110 ± 52 µm induced the least severe acute and late toxicities. A shift of the same array in FS2 led to significantly inducted acute toxicities, a higher otitis score, and a slight increase in fibrous tissue. FS3 led to the strongest increase in acute and late toxicities. CONCLUSIONS: The highest normal-tissue sparing is achieved after accurate reirradiation of a highly dose modulated pMB array, although high positioning accuracies are challenging in a clinical environment. Nevertheless, the same integral dose applied in highly dose-modulated fractions is superior to low daily dose-modulated fractions.

Author Correction: DNA damage interactions on both nanometer and micrometer scale determine overall cellular damage.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Proton pencil minibeam irradiation of an in-vivo mouse ear model spares healthy tissue dependent on beam size.

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.

Beam size limit for pencil minibeam radiotherapy determined from side effects in an in-vivo mouse ear model.

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.

Local inhibition of rRNA transcription without nucleolar segregation after targeted ion irradiation of the nucleolus.

Nucleoli have attracted interest for their role as cellular stress sensors and as potential targets for cancer treatment. The effect of DNA double-strand breaks (DSBs) in nucleoli on rRNA transcription and nucleolar organisation appears to depend on the agent used to introduce DSBs, DSB frequency and the presence (or not) of DSBs outside the nucleoli. To address the controversy, we targeted nucleoli with carbon ions at the ion microbeam SNAKE. Localized ion irradiation with 1-100 carbon ions per point (about 0.3-30 Gy per nucleus) did not lead to overall reduced ribonucleotide incorporation in the targeted nucleolus or other nucleoli of the same cell. However, both 5-ethynyluridine incorporation and Parp1 protein levels were locally decreased at the damaged nucleolar chromatin regions marked by γH2AX, suggesting localized inhibition of rRNA transcription. This locally restricted transcriptional inhibition was not accompanied by nucleolar segregation, a structural reorganisation observed after inhibition of rRNA transcription by treatment with actinomycin D or UV irradiation. The presented data indicate that even multiple complex DSBs do not lead to a pan-nucleolar response if they affect only a subnucleolar region.

Acute Skin Damage and Late Radiation-Induced Fibrosis and Inflammation in Murine Ears after High-Dose Irradiation.

The use of different scoring systems for radiation-induced toxicity limits comparability between studies. We examined dose-dependent tissue alterations following hypofractionated X-ray irradiation and evaluated their use as scoring criteria. Four dose fractions (0, 5, 10, 20, 30 Gy/fraction) were applied daily to ear pinnae. Acute effects (ear thickness, erythema, desquamation) were monitored for 92 days after fraction 1. Late effects (chronic inflammation, fibrosis) and the presence of transforming growth factor beta 1 (TGFß1)-expressing cells were quantified on day 92. The maximum ear thickness displayed a significant positive correlation with fractional dose. Increased ear thickness and erythema occurred simultaneously, followed by desquamation from day 10 onwards. A significant dose-dependency was observed for the severity of erythema, but not for desquamation. After 4 × 20 and 4 × 30 Gy, inflammation was significantly increased on day 92, whereas fibrosis and the abundance of TGFß1-expressing cells were only marginally increased after 4 × 30 Gy. Ear thickness significantly correlated with the severity of inflammation and fibrosis on day 92, but not with the number of TGFß1-expressing cells. Fibrosis correlated significantly with inflammation and fractional dose. In conclusion, the parameter of ear thickness can be used as an objective, numerical and dose-dependent quantification criterion to characterize the severity of acute toxicity and allow for the prediction of late effects.

DNA damage interactions on both nanometer and micrometer scale determine overall cellular damage.

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.

Optimization of beam arrangements in proton minibeam radiotherapy by cell survival simulations.

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 σ0  = 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.