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.

Elemental Depth Profiling of Chlorinated Polyamide-Based Thin-Film Composite Membranes with Elastic Recoil Detection.

The chlorine resistance of nanofiltration and reverse osmosis membranes is of high importance in the water treatment industry. Elastic recoil detection (ERD) is now presented as a powerful tool to uniquely provide elemental depth profiles, including hydrogen, of NaOCl-treated polyamide-based thin-film composite (TFC) membranes. The influence of pressure, pH, and chlorine feed concentration on the volume-averaged Cl uptake, the location of chlorine throughout the membrane, and the z-gradient in the Cl/N ratio is demonstrated. The results suggest that (i) higher volume-averaged Cl uptakes are achieved at higher chlorine doses and at acidic pH; (ii) chlorination is mostly restricted to the top layer; (iii) a gradient in the Cl/N ratio exists along the membrane depth; and (iv) the shape of this gradient is influenced by the chlorination pH and the applied pressure. Conclusions on the chlorination mechanisms could also be deduced. Conversely, no conclusive relationships between H fractions and Cl uptake could be drawn, even though changes in the H content after chlorination were observed. To corroborate these results and fully exploit the potential of ERD, the exact microstructure of the (chlorinated) TFC membranes should be better understood.

Superresolution light microscopy shows nanostructure of carbon ion radiation-induced DNA double-strand break repair foci.

Carbon ion radiation is a promising new form of radiotherapy for cancer, but the central question about the biologic effects of charged particle radiation is yet incompletely understood. Key to this question is the understanding of the interaction of ions with DNA in the cell's nucleus. Induction and repair of DNA lesions including double-strand breaks (DSBs) are decisive for the cell. Several DSB repair markers have been used to investigate these processes microscopically, but the limited resolution of conventional microscopy is insufficient to provide structural insights. We have applied superresolution microscopy to overcome these limitations and analyze the fine structure of DSB repair foci. We found that the conventionally detected foci of the widely used DSB marker γH2AX (Ø 700-1000 nm) were composed of elongated subfoci with a size of ∼100 nm consisting of even smaller subfocus elements (Ø 40-60 nm). The structural organization of the subfoci suggests that they could represent the local chromatin structure of elementary DSB repair units at the DSB damage sites. Subfocus clusters may indicate induction of densely spaced DSBs, which are thought to be associated with the high biologic effectiveness of carbon ions. Superresolution microscopy might emerge as a powerful tool to improve our knowledge of interactions of ionizing radiation with cells.-Lopez Perez, R., Best, G., Nicolay, N. H., Greubel, C., Rossberger, S., Reindl, J., Dollinger, G., Weber, K.-J., Cremer, C., Huber, P. E. Superresolution light microscopy shows nanostructure of carbon ion radiation-induced DNA double-strand break repair foci.

Nanoscopic exclusion between Rad51 and 53BP1 after ion irradiation in human HeLa cells.

Many proteins involved in detection, signalling and repair of DNA double-strand breaks (DSB) accumulate in large number in the vicinity of DSB sites, forming so called foci. Emerging evidence suggests that these foci are sub-divided in structural or functional domains. We use stimulated emission depletion (STED) microscopy to investigate localization of mediator protein 53BP1 and recombination factor Rad51 after irradiation of cells with low linear energy transfer (LET) protons or high LET carbon ions. With a resolution better than 100 nm, STED microscopy and image analysis using a newly developed analyzing algorithm, the reduced product of the differences from the mean, allowed us to demonstrate that with both irradiation types Rad51 occupies spherical regions of about 200 nm diameter. These foci locate within larger 53BP1 accumulations in regions of local 53BP1 depletion, similar to what has been described for the localization of Brca1, CtIP and RPA. Furthermore, localization relative to 53BP1 and size of Rad51 foci was not different after irradiation with low and high LET radiation. As expected, 53BP1 foci induced by low LET irradiation mostly contained one Rad51 focal structure, while after high LET irradiation, most foci contained >1 Rad51 accumulation.

The influence of the channel size on the reduction of side effects in microchannel proton therapy.

The potential of proton microchannel radiotherapy to reduce radiation effects in the healthy tissue but to keep tumor control the same as in conventional proton therapy is further elucidated. The microchannels spread on their way to the tumor tissue resulting in different fractions of the healthy tissue covered with doses larger than the tumor dose, while the tumor gets homogeneously irradiated. The aim of this study was to evaluate the effect of increasing channel width on potential side effects in the normal tissue. A rectangular 180 × 180 µm(2) and two Gaussian-type dose distributions of σ = 260 µm and σ = 520 µm with an interchannel distance of 1.8 mm have been applied by 20-MeV protons to a 3D human skin model in order to simulate the widened channels and to compare the irradiation effects at different endpoints to those of a homogeneous proton irradiation. The number of protons applied was kept constant at all irradiation modes resulting in the same average dose of 2 Gy. All kinds of proton microchannel irradiation lead to higher cell viability and produce significantly less genetic damage than homogeneous proton irradiation, but the reduction is lower for the wider channel sizes. Our findings point toward the application of microchannel irradiation for clinical proton or heavy ion therapy to further reduce damage of normal tissues while maintaining tumor control via a homogeneous dose distribution inside the tumor.

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.

Reduced side effects by proton microchannel radiotherapy: study in a human skin model.

The application of a microchannel proton irradiation was compared to homogeneous irradiation in a three-dimensional human skin model. The goal is to minimize the risk of normal tissue damage by microchannel irradiation, while preserving local tumor control through a homogeneous irradiation of the tumor that is achieved because of beam widening with increasing track length. 20 MeV protons were administered to the skin models in 10- or 50-µm-wide irradiation channels on a quadratic raster with distances of 500 µm between each channel (center to center) applying an average dose of 2 Gy. For comparison, other samples were irradiated homogeneously at the same average dose. Normal tissue viability was significantly enhanced after microchannel proton irradiation compared to homogeneous irradiation. Levels of inflammatory parameters, such as Interleukin-6, TGF-Beta, and Pro-MMP1, were significantly lower in the supernatant of the human skin tissue after microchannel irradiation than after homogeneous irradiation. The genetic damage as determined by the measurement of micronuclei in keratinocytes also differed significantly. This difference was quantified via dose modification factors (DMF) describing the effect of each irradiation mode relative to homogeneous X-ray irradiation, so that the DMF of 1.21 ± 0.20 after homogeneous proton irradiation was reduced to 0.23 ± 0.11 and 0.40 ± 0.12 after microchannel irradiation using 10- and 50-µm-wide channels, respectively. Our data indicate that proton microchannel irradiation maintains cell viability while significantly reducing inflammatory responses and genetic damage compared to homogeneous irradiation, and thus might improve protection of normal tissue after irradiation.

Range verification of a clinical proton beam in an abdominal phantom by co-registration of ionoacoustics and ultrasound.

Objective.The range uncertainty in proton radiotherapy is a limiting factor to achieve optimum dose conformity to the tumour volume. Ionoacoustics is a promising approach forin siturange verification, which would allow to reduce the size of the irradiated volume relative to the tumour volume. The energy deposition of a pulsed proton beam leads to an acoustic pressure wave (ionoacoustics), the detection of which allows conclusion about the distance between the Bragg peak and the acoustic detector. This information can be transferred into a co-registered ultrasound image, marking the Bragg peak position relative to the surrounding anatomy.Approach.A CIRS 3D abdominal phantom was irradiated with 126 MeV protons at a clinical proton therapy centre. Acoustic signals were recorded on the beam axis distal to the Bragg peak with a Cetacean C305X hydrophone. The ionoacoustic measurements were processed with a correlation filter using simulated filter templates. The hydrophone was rigidly attached to an ultrasound device (Interson GP-C01) recording ultrasound images of the irradiated region.Main results.The time of flight obtained from ionoacoustic measurements were transferred to an ultrasound image by means of an optoacoustic calibration measurement. The Bragg peak position was marked in the ultrasound image with a statistical uncertainty ofσ= 0.5 mm of 24 individual measurements depositing 1.2 Gy at the Bragg peak. The difference between the evaluated Bragg peak position and the one obtained from irradiation planning (1.0 mm) is smaller than the typical range uncertainty (≈4 mm) at the given penetration depth (10 cm).Significance.The measurements show that it is possible to determine the Bragg peak position of a clinical proton beam with submillimetre precision and transfer the information to an ultrasound image of the irradiated region. The dose required for this is smaller than that used for a typical irradiation fraction.

Double-strand break-induced transcriptional silencing is associated with loss of tri-methylation at H3K4.

Epigenetic alterations induced by ionizing radiation may contribute to radiation carcinogenesis. To detect relative accumulations or losses of constitutive post-translational histone modifications in chromatin regions surrounding DNA double-strand breaks (DSB), we developed a method based on ion microirradiation and correlation of the signal intensities after immunofluorescence detection of the histone modification in question and the DSB marker γ-H2AX. We observed after ionizing irradiation markers for transcriptional silencing, such as accumulation of H3K27me3 and loss of active RNA polymerase II, at chromatin regions labeled by γ-H2AX. Confocal microscopy of whole nuclei and of ultrathin nuclear sections revealed that the histone modification H3K4me3, which labels transcriptionally active regions, is underrepresented in γ-H2AX foci. While some exclusion of H3K4me3 is already evident at the earliest time amenable to this kind of analysis, the anti-correlation apparently increases with time after irradiation, suggesting an active removal process. Focal accumulation of the H3K4me3 demethylase, JARID1A, was observed at damaged regions inflicted by laser irradiation, suggesting involvement of this enzyme in the DNA damage response. Since no accumulation of the repressive mark H3K9me2 was found at damaged sites, we suggest that DSB-induced transcriptional silencing resembles polycomb-mediated silencing rather than heterochromatic silencing.

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).

Proton beam range verification by means of ionoacoustic measurements at clinically relevant doses using a correlation-based evaluation.

Purpose: The Bragg peak located at the end of the ion beam range is one of the main advantages of ion beam therapy compared to X-Ray radiotherapy. However, verifying the exact position of the Bragg peak within the patient online is a major challenge. The goal of this work was to achieve submillimeter proton beam range verification for pulsed proton beams of an energy of up to 220 MeV using ionoacoustics for a clinically relevant dose deposition of typically 2 Gy per fraction by i) using optimal proton beam characteristics for ionoacoustic signal generation and ii) improved signal detection by correlating the signal with simulated filter templates. Methods: A water tank was irradiated with a preclinical 20 MeV proton beam using different pulse durations ranging from 50 ns up to 1 µs in order to maximise the signal-to-noise ratio (SNR) of ionoacoustic signals. The ionoacoustic signals were measured using a piezo-electric ultrasound transducer in the MHz frequency range. The signals were filtered using a cross correlation-based signal processing algorithm utilizing simulated templates, which enhances the SNR of the recorded signals. The range of the protons is evaluated by extracting the time of flight (ToF) of the ionoacoustic signals and compared to simulations from a Monte Carlo dose engine (FLUKA). Results: Optimised SNR of 28.0 ± 10.6 is obtained at a beam current of 4.5 µA and a pulse duration of 130 ns at a total peak dose deposition of 0.5 Gy. Evaluated ranges coincide with Monte Carlo simulations better than 0.1 mm at an absolute range of 4.21 mm. Higher beam energies require longer proton pulse durations for optimised signal generation. Using the correlation-based post-processing filter a SNR of 17.8 ± 5.5 is obtained for 220 MeV protons at a total peak dose deposition of 1.3 Gy. For this clinically relevant dose deposition and proton beam energy, submillimeter range verification was achieved at an absolute range of 303 mm in water. Conclusion: Optimal proton pulse durations ensure an ideal trade-off between maximising the ionoacoustic amplitude and minimising dose deposition. In combination with a correlation-based post-processing evaluation algorithm, a reasonable SNR can be achieved at low dose levels putting clinical applications for online proton or ion beam range verification into reach.

Scanning irradiation device for mice in vivo with pulsed and continuous proton beams.

A technical set-up for irradiation of subcutaneous tumours in mice with nanosecond-pulsed proton beams or continuous proton beams is described and was successfully used in a first experiment to explore future potential of laser-driven particle beams, which are pulsed due to the acceleration process, for radiation therapy. The chosen concept uses a microbeam approach. By focusing the beam to approximately 100 × 100 µm(2), the necessary fluence of 10(9) protons per cm(2) to deliver a dose of 20 Gy with one-nanosecond shot in the Bragg peak of 23 MeV protons is achieved. Electrical and mechanical beam scanning combines rapid dose delivery with large scan ranges. Aluminium sheets one millimetre in front of the target are used as beam energy degrader, necessary for adjusting the depth-dose profile. The required procedures for treatment planning and dose verification are presented. In a first experiment, 24 tumours in mice were successfully irradiated with 23 MeV protons and a single dose of 20 Gy in pulsed or continuous mode with dose differences between both modes of 10%. So far, no significant difference in tumour growth delay was observed.

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.

Magnetically focused 70 MeV proton minibeams for preclinical experiments combining a tandem accelerator and a 3 GHz linear post-accelerator.

PURPOSE: Radiotherapy plays an important role for the treatment of tumor diseases in two-thirds of all cases, but it is limited by side effects in the surrounding healthy tissue. Proton minibeam radiotherapy (pMBRT) is a promising option to widen the therapeutic window for tumor control at reduced side effects. An accelerator concept based on an existing tandem Van de Graaff accelerator and a linac enables the focusing of 70 MeV protons to form minibeams with a size of only 0.1 mm for a preclinical small animal irradiation facility, while avoiding the cost of an RFQ injector. METHODS: The tandem accelerator provides a 16 MeV proton beam with a beam brightness of B = 4 nA mm 2 mrad 2 as averaged from 5 µs long pulses with a flat top current of 17 µA at 200 Hz repetition rate. Subsequently, the protons are accelerated to 70 MeV by a 3 GHz linear post-accelerator consisting of two Side Coupled Drift Tube Linac (SCDTL) structures and four Coupled Cavity Linac (CCL) structures [design: AVO-ADAM S.A (Geneva, Switzerland)]. A 3 GHz buncher and four magnetic quadrupole lenses are placed between the tandem and the post-accelerator to maximize the transmission through the linac. A quadrupole triplet situated downstream of the linac structure focuses the protons into an area of (0.1 × 0.1) mm2 . The beam dynamics of the facility is optimized using the particle optics code TRACE three-dimensional (3D). Proton transmission through the facility is elaborated using the particle tracking code TRAVEL. RESULTS: A study about buncher amplitude and phase shift between buncher and linac is showing that 49% of all protons available from the tandem can be transported through the post-accelerator. A mean beam current up to 19 nA is expected within an area of (0.1 × 0.1) mm2 at the beam focus. CONCLUSION: An extension of existing tandem accelerators by commercially available 3 GHz structures is able to deliver a proton minibeam that serves all requirements to obtain proton minibeams to perform preclinical minibeam irradiations as it would be the case for a complete commercial 3 GHz injector-RFQ-linac combination. Due to the modularity of the linac structure, the irradiation facility can be extended to clinically relevant proton energies up to or above 200 MeV.

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.

Concept and performance evaluation of two 3 GHz buncher units optimizing the dose rate of a novel preclinical proton minibeam irradiation facility.

To demonstrate the large potential of proton minibeam radiotherapy (pMBRT) as a new method to treat tumor diseases, a preclinical proton minibeam radiation facility was designed. It is based on a tandem Van-de-Graaff accelerator providing a 16 MeV proton beam and a 3 GHz linac post-accelerator (designs: AVO-ADAM S.A, Geneva, Switzerland and ENEA, Frascati, Italy). To enhance the transmission of the tandem beam through the post-accelerator by a factor of 3, two drift tube buncher units were designed and constructed: A brazed 5-gap structure (adapted SCDTL tank of the TOP-IMPLART project (ENEA)) and a non-brazed low budget 4-gap structure. Both are made of copper. The performance of the two differently manufactured units was evaluated using a 16 MeV tandem accelerator beam and a Q3D magnetic spectrograph. Both buncher units achieve the required summed voltage amplitude of 42 kV and amplitude stability at a power feed of less than 800 W.

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.

Membrane Hsp70-supported cell-to-cell connections via tunneling nanotubes revealed by live-cell STED nanoscopy.

Heat shock protein Hsp70 (Hsp70) is found on the cell surface of a large variety of human and mouse tumor cell types including U87, GL261 glioblastoma, and 4T1 mammary carcinoma cells. We studied the role of membrane-bound Hsp70 (mHsp70) in the formation of cell-to-cell connections via tunneling nanotubes (TNTs) using live-cell STED nanoscopy. This technique allows the visualization of microstructures in the 100-nm range in the living cells. We could show that the presence of tumor-derived mHsp70 in TNTs with a diameter ranging from 120 to 140 nm predominantly originates from cholesterol-rich-microdomains containing the lipid compound globoyltriaosylceramide (Gb3). Under non-stress conditions, Hsp70 and Gb3 are structurally clustered in the membrane of TNTs of tumor cells that showed tumor type specific variations in the amount of cell-to-cell connection networks. Furthermore depletion of cholesterol and ionizing radiation as a stress factor results in a complete loss of Hsp70-containing TNTs.

Nanoscopic analysis of 53BP1, BRCA1 and Rad51 reveals new insights in temporal progression of DNA-repair and pathway choice.

The accumulation and spatial distribution of 53BP1, BRCA1 and Rad51, key proteins in DNA double-strand break (DSB) repair, was investigated with high temporal resolution over a time span of 24 h, using STED nanoscopy. DNA lesions were induced by irradiation with high-LET (linear energy transfer) α-particles. We show that 53BP1 IRIF formation occurs quickly in almost all cells and after about 6 h the fraction of 53BP1 IRIF positive cells slowly declines. Against the expectations BRCA1 and Rad51 IRIF formation is only shortly delayed but with the maximum of cells showing foci after 6 and 8 h after irradiation. At this stage, almost all IRIF in a given Rad51-positive cell show Rad51 accumulation, suggesting that repair via homologous recombination is attempted at almost all residual DSB sites. The frequency of BRCA1 IRIF positive cells increases much earlier and remains high after Rad51 positive cells start to decline, supporting models claiming that functional roles of BRCA1 change over time. Correlation analysis showed a high degree of correlation of Rad51 with BRCA1, while the exclusion of 53BP1 from the actual resection-zone is demonstrated by anti-correlation of Rad51 and 53BP1. Interestingly, these correlation and anti-correlation patterns exhibit complementary temporal variation.