Search

The in vivo evolution of radiotherapy necessitates innovative platforms for preclinical investigation, bridging the gap between bench research and clinical applications. Understanding the nuances of radiation response, specifically tailored to proton and photon therapies, is critical for optimizing treatment outcomes. Within this context, preclinical in vivo experimental setups incorporating image guidance for both photon and proton therapies are pivotal, enabling the translation of findings from small animal models to clinical settings. The SAPPHIRE project represents a milestone in this pursuit, presenting the installation of the small animal radiation therapy integrated beamline (SmART+ IB, Precision X-Ray Inc., Madison, Connecticut, USA) designed for preclinical image-guided proton and photon therapy experiments at University Proton Therapy Dresden. Through Monte Carlo simulations, low-dose on-site cone beam computed tomography imaging and quality assurance alignment protocols, the project ensures the safe and precise application of radiation, crucial for replicating clinical scenarios in small animal models. The creation of Hounsfield lookup tables and comprehensive proton and photon beam characterizations within this system enable accurate dose calculations, allowing for targeted and controlled comparison experiments. By integrating these capabilities, SAPPHIRE bridges preclinical investigations and potential clinical applications, offering a platform for translational radiobiology research and cancer therapy advancements.

This repository contains the data shown in the results part of the paper entitled: SAPPHIRE - Establishment of small animal proton and photon image-guided radiation experiments.

Purpose: A better knowledge of the dependence of the tissue sparing effect at ultra-high dose rate (UHDR) on physical beam parameters (dose, dose rate, radiation quality) would be helpful towards a mechanistic understanding of the FLASH effect and for its broader clinical translation. To address this, a comprehensive study on the normal tissue sparing at UHDR using the zebrafish embryo (ZFE) model irradiated with protons and electrons was conducted. Methods: 1 day old ZFE were irradiated over a wide dose range (15-95 Gy) in three different beams (proton entrance channel, proton spread out Bragg peak and 30 MeV electrons) at UHDR and reference dose rate. After irradiation the ZFE were incubated for 4 days and then analyzed for their development and morphological characteristics. Results: Dose-effect curves for four different biological endpoints of ZFE (pericardial edema, curved spine, embryo length and eye diameter) were obtained and a sparing effect was observed for all three beams. It was demonstrated that proton relative biological effectiveness and UHDR sparing are both relevant to consider in order to predict the resulting dose response. Dose dependent FLASH modifying factors (FMF) for ZFE, calculated based on the obtained dose-effect curves, were found to be compatible with rodent data from the literature. It was found that the UHDR sparing effect saturates at doses above ~50 Gy with an FMF of ~0.7-0.8. Only a moderate dependence of the tissue sparing effect in ZFE on the biological endpoint, but a strong dose rate dependence were observed. Conclusion: The ZFE model was shown to be a suitable high-throughput pre-clinical model for radiobiological studies on FLASH radiotherapy, providing results comparable to rodent models. The obtained results emphasize to further clarify the nature of the observed dose rate dependence.

Böhlen et al. [1] recently proposed a model that describes the magnitude of the normal tissue sparing Flash effect as a function of dose based on available in vivo data. The newly introduced flash modifying factor FMF translates doses applied at ultra-high dose rate to equivalent doses at conventional dose rate similar to the concept of relative biological effectiveness [1]. Primarily founded on rodent data, the model [1] includes only one study that demonstrated a dose-dependent Flash effect by length differences measured at 5 days-old zebrafish embryo (ZFE) after irradiation with electron doses of 5 – 12 Gy [2]. We studied this systematic overview about the available Flash data with great interest and acknowledged it as a very useful guidance for future Flash research. Coincidentally, we have just recently measured ZFE data in the high dose range (15 – 50 Gy) that appear to match very well with the existing rodent data. Comparable to our previous studies at the ELBE accelerator [3, 4], one day-old ZFE were irradiated using electron beams of reference (mean dose rate 0.11 Gy/s) and ultra-high dose rate (UHDR; mean dose rate 0.9×105 Gy/s) (Fig. 1a). Normal tissue toxicity was quantified by analyzing the length deficit of the 5 days-old embryos compared to unirradiated controls. Since the controls grew on average 30% from irradiation to analysis this is the maximum length deficit that can be caused by irradiation. The derived FMF values extend the available ZFE data [2] and cover in a single experiment almost the entire dose range applied in the rodent studies for different tissues. Comparable to the rodent data (Fig. 1b) the ZFE FMF increases with dose. The good agreement of our ZFE data with the rodent data [1] demonstrates the feasibility of the ZFE model for basic Flash effect studies, e.g., on the influence of physical beam parameters [3–6]. Hence, the ZFE model could be deployed as a high-throughput alternative to rodent studies at this translational level [5]

Laser plasma-based proton accelerators (LPA) can contribute to research of ultra-high dose rate radiobiology as they provide pulse dose rates unprecedented at medical proton sources. Yet, LPAs pose challenges regarding precise dosimetry due to the high pulse dose rates, but also due to the sources' lower spectral stability and pulsed operation mode. For in-vivo models, further challenges arise from the necessary small field dosimetry for volumetric dose distributions. In this work, we present a dosimetry and beam monitoring concept for in-vivo irradiations of small target volumes with LPA protons, solving aforementioned challenges. The volumetric dose distribution in a sample (mean dose value and lateral/depth dose inhomogeneity) is provided by combining two independent dose measurements using radiochromic films (dose-rate independent) and ionization chambers (dose-rate dependent), respectively. The unique feature of the dosimetric setup is beam monitoring with a transmission time-of-flight spectrometer to quantify spectral fluctuations of the irradiating proton pulses. The resulting changes in the depth dose profile during irradiation of an in-vivo sample are hence accessible and enable pulse-resolved depth dose correction for each dose measurement. A first successful small animal pilot study using an LPA proton source serves as a testcase for the presented dosimetry approach and proves its performance in a realistic setting.

The University Proton Therapy facility in Dresden (UPTD), Germany, is equipped with an experimental room with a beamline providing a static pencil beam. High proton beam currents can be achieved at this beamline which makes it suitable for FLASH experiments. However, the established experimental setup uses only the entrance channel of the proton Bragg curve. In this work, a set of 3D-printed range modulators designed to generate spread out Bragg peaks (SOBPs) for radiobiological experiments at ultra-high dose rate at this beamline is described. A new method to optimize range modulators specifically for the case of a static pencil beam based on the central depth dose profile is introduced. Modulators for two different irradiation setups were produced and characterized experimentally by measurements of lateral and depth dose distributions using different detectors. In addition, Monte Carlo simulations were performed to assess profiles of the dose averaged linear energy transfer. These newly produced range modulators will allow future proton FLASH experiments in the SOBP at UPTD with two different experimental setups.

The recently observed FLASH effect provides normal tissue protection at a similar tumor treatment efficacy via ultra-high dose rate (UHDR) irradiation and promises great benefits for radiotherapy patients. Dedicated studies are now necessary to define a robust set of dose application parameters for FLASH radiotherapy and to identify underlying mechanisms. These studies require particle accelerators with variable temporal dose application characteristics for numerous radiation qualities, equipped for preclinical radiobiological research. Here we present the DRESDEN PLATFORM, a research hub for ultra-high dose rate radiobiology. By uniting clinical and research accelerators with radiobiology infrastructure and know-how, the DRESDEN PLATFORM offers a unique environment for studying the FLASH effect. We introduce its experimental capabilities and qualify the platform for systematic investigation of FLASH by presenting results from a concerted in vivo radiobiology study with zebrafish embryos. The comparative pre-clinical study was conducted across three accelerator facilities, including an advanced laser-driven proton source applied for FLASH-relevant in vivo irradiations for the first time. The data show a protective effect of UHDR irradiation up to 10^5 Gy/s and suggests consistency of the protective effect even at escalated dose rates of 10^9 Gy/s. With the first clinical FLASH studies underway, research facilities like the DRESDEN PLATFORM, addressing the open questions surrounding FLASH, are essential to accelerate FLASH’s translation into clinical practice.

Laser-plasma accelerated (LPA) proton bunches are now prepared and provided for research fields ranging from ultra-high dose rate radiobiology to material science and probing of extreme states of matter. Yet, the capabilities to fully characterize the spectrally and angularly broad LPA bunches lag behind the rapidly evolving applications. The Optical Cone beam TOmograph for Proton Online Dosimetry - short OCTOPOD - translates the angularly resolved spectral characterization of LPA proton bunches into the spatially resolved detection of the volumetric dose distribution deposited in a liquid scintillator. Up to 24 multi-pinhole arrays record projections of the volumetric scintillation light distribution and allow for tomographic reconstruction of the volumetric dose deposition pattern, from which proton spectra may be retrieved in arbitrary directions by spectral deconvolution. Applying the OCTOPOD at a cyclotron, we show the reliable retrieval of various spatial dose deposition patterns and detector sensitivity over a broad dose range from less than 1 Gy to more than 100 Gy, as required for LPA proton bunch characterization. With a dedicated vacuum housing, the OCTOPOD was installed at a LPA proton source, providing real-time data on proton acceleration performance for various laser-target interaction parameters and attesting the system optimal performance in the harsh laser plasma environment.

Comprehending cellular changes of radiation-induced brain injury is crucial to prevent and treat the pathology. We provide a unique open dataset of proton-irradiated mouse brains consisting of medical imaging, radiation dose simulations, and large-scale microscopy images, all registered into a common coordinate system. This allows dose-dependent analyses on single-cell level.

The data set comprises raw data, processed data and detector data for the OCTOPOD detector applied for characterization of proton bunches at a proton cyclotron and a laser-driven proton accelerator.

Source data, scripts and parts of figures to generate the figures in publication

The data set comprises raw data, processed data and detector data for the miniSCIDOM detector applied for characterization of proton bunches at a proton cyclotron and a laser-driven proton accelerator.

Background and Aims The Flash effect, i.e. the radiobiological observation of normal tissue sparing but efficient tumor killing by ultra-high dose rate (UHDR) irradiation, promise great benefits for cancer patient treatment. The translation process of the Flash effect should be accompanied by systematic studies on the necessary beam parameters for each clinical applied radiation. Using the zebrafish embryo model, the influence of partial oxygen level (https://doi.org/10.1016/j.radonc.2021.02.003), electron pulse structure and dose were studied thoroughly. To investigate a similar range of proton beam parameters UHDR experiments at different accelerators, but under comparable conditions, are required. Methods To cover a broad range of proton dose rates, experiments have been prepared at the University Proton Therapy Dresden (UPTD) and at the Draco laser accelerator (Helmholtz-Zentrum Dresden-Rossendorf) providing quasi-continuous and single-shot proton beam delivery in the range of 0.12 to 10^9 Gy/s. To fulfil the requirements of the model, i.e., irradiating a sufficiently high number of embryos at low oxygen level, dedicated setups were established that also allow for the online measurement of oxygen partial pressure and cope with the geometric restrictions of the respective accelerator. Results A comparison experiment at UPTD reveal a significant protecting Flash effect in both setups for zebrafish embryo treated with 300 Gy/s relative to conventional proton dose rate. Moreover, a higher protection of the embryos was indicated comparing the embryo length after irradiation with 10^9 Gy/s and conventional proton dose rate, respectively. Conclusions Experimental setups have been established that allow for systematic proton dose rate studies using the zebrafish embryo model at the clinical cyclotron of UPTD and at the Draco laser accelerator. Therewith, the experimental possibilities at the Dresden platform for UHDR radiobiology are extended providing electron and prot

Background and Aims A prominent explanation of the FLASH effect is the oxygen depletion hypothesis, in which the radiolysis of water or cytoplasm produces radicals that react with the O2 dissolved in the water or cytoplasm. This would result in an oxygen depletion leading to a hypoxic target and hence radiation protection effect based on the Oxygen Enhancement Ratio. The presented study aims to investigate the impact of beam pulse structure on oxygen depletion and its correlation with biological endpoints. Methods O2 depletion was measured using 30 MeV electron irradiation on a sealed water target. Read-out was performed using TROXSP5 sensors. The beam pulse structure was altered to assess 4 different regimes of average and beam pulse dose rate. At clinical doses, not enough O2 was consumed to explain a FLASH effect due to radiation-induced hypoxia. The amount of O2 depleted per dose depends on the dose rate, and slightly less O2 is removed at higher dose rates, suggesting radical-radical reactions as a possible mechanism of the FLASH effect. Furthermore, our results regarding the pulse structures showed that the average dose rate seems to dominate the pulse dose rate in terms of radical production and O2 depletion. The direct comparison of the depletion measurements presented here with biological experiments on zebrafish embryos from another study also showed that there was a strong correlation between O2 depletion and biological radiation response (FIG 1). The results emphasize that the FLASH effect in biological tissues is likely to be explained by decreased effective radical production at high dose rates. Conclusions In the tested beam parameters, the mean dose rate has the most pronounced effect on O2 depletion. Depletion measurements showed a clear correlation with biological data, from which FLASH effects can be largely explained by changes in radical production.

Purpose/Objective In a previous experiment at the HZDR research electron accelerator ELBE high mean dose rates of 105 Gy/s in combination with partial oxygen pressure below 5 mmHg protect zebrafish embryo from radiation damage compared to continuous reference irradiation (mean dose rate of 0.11 Gy/s) and higher oxygen pressure (Pawelke et al. Radiother Oncol 2021). However, the influence of beam pulse structure on the radiation response remains unanswered and should be resolved in an upcoming experiment. Material/Methods In addition to the Flash and the reference regime, the ELBE accelerator was used to mimic the pulse structure of a clinical electron linac delivering a dose of 28 Gy by 5 pulses at a frequency of 250 Hz. For comparison, a fourth regime of similar mean dose rate, but continuous beam (280 Gy/s) mimicking FLASH irradiation at a isochronous proton cyclotron (Beyreuther et al. Radiother Oncol 2019) was applied. Wild type zebrafish embryo (24 hpf) were irradiated and the radiation induced malformation were studied during the four day follow up for all four regimes. Zebrafish embryo irradiation was performed under low oxygen pressure and the depletion of depletion during irradiation was measured online. Results Compared to the reference regime a protecting Flash effect was found the three other pulse regimes for endpoints, except embryo survival. Analysing the radiation induced malformation more detailed significant correlations to mean and pulse dose rate are revealed. Surprisingly, the beam delivery in macro pulses (Linac regime) reduces the Flash effect relative to delivery at the same pulse dose rate but in a single pulse. Conclusion. The ELBE electron accelerator can be applied to study the influence of beam dose rate and pulse structure on the Flash effect by varying both parameters over several orders of magnitude. Hence, ELBE is an ideal tool for systematic studies on optimal electron beam parameters for Flash, including pulse structures that are r

Recent oncological studies identified beneficial properties of radiation applied at ultra-high dose rates several orders of magnitude higher than the clinical standard of the order of Gy/min. Sources capable of providing these ultra-high dose rates are under investigation. Here, we show that a stable, compact laser-driven proton source with energies greater than 60 MeV enables radiobiological in vivo studies. We performed a pilot irradiation study on human tumors in a mouse model, showing the concerted preparation of mice and laser accelerator, the dose-controlled, tumor-conform irradiation using a laser-driven as well as a clinical reference proton source, and the radiobiological evaluation of irradiated and unirradiated mice for radiation-induced tumor growth delay. The prescribed homogeneous dose of 4 Gy was precisely delivered at the laser-driven source. The results demonstrate a complete laser-driven proton research platform for diverse user-specific small animal models, able to deliver tunable single-shot doses up to around 20 Gy to millimeter-scale volumes on nanosecond time scales, equivalent to around 1E9 Gy/s, spatially homogenized and tailored to the sample. The platform provides a unique infrastructure for translational research with protons at ultra-high dose rate.

Laser-plasma based proton accelerators as a novel accelerator technology have matured to a level at which laboratory-scale setups for the emerging topic of image-guided precision small animal irradiation studies come into reach [Bra2019]. Providing a high proton energy bandwidth [Sch2016] which is filtered in a tunable pulsed magnet beam transport, these accelerators enable flexibility in terms of scattering-free irradiation field formation [Mas2014, Mas2015]. Regarding temporal dose delivery, with single pulse doses reaching the Gy level at unprecedented peak dose rates of up to 1012 Gy/s [Sch2016], laser-plasma based proton accelerator setups can give access to the dose rate regime of FLASH [Fav2014, Voz2019]. The realization of a full-scale setup for image-guided precision radiobiological studies for small animals focusing on dose rate dependent effects is currently prepared at the PENELOPE laser-plasma based proton accelerator at HZDR and will be presented with a focus on the technological requirements and solutions for dose delivery at laser-plasma based sources. This development relies on our experience in performing dose-controlled radiobiological in vitro studies [Kra2010, Zei2013] and first in vivo irradiation experiments at the DRACO laser-plasma based proton accelerator at HZDR, where we have established a pulsed solenoid beamline for 3D irradiation field formation, yielding a (5 mm)³ homogeneous volumetric dose distribution at a Gy single pulse dose level [8]. [Bra2019] F.-E. Brack, F. Kroll, L. Gaus, C. Bernert, E. Beyreuther, T. E. Cowan, L. Karsch, S. D. Kraft, L. A. Kunz-Schughart, E. Lessmann, J. Metzkes-Ng, L. Obst-Hübl, J. Pawelke, M. Rehwald, H.-P. Schlenvoigt, U. Schramm, M. Sobiella, E. R. Szabó, T. Ziegler, K. Zeil, Spectral and spatial shaping of laser-driven proton beams using a pulsed high-field magnet beamline. arXiv:1910.08430 (2019) [Sch2016] J. Schreiber, P. Bolton, K. Parodi, “Hands-on” laser-driven ion acceleration: A primer for laser

Laser-driven plasma accelerators (LPA) are compact sources of ultra-short, intense proton pulses in the multi-10-MeV energy range. These unique parameters predestine LPAs as powerful tools for ultra-high dose rate radiobiology research. The sources’ capabilities were recently demonstrated in the first successful small animal pilot study on radiation-induced tumor growth delay in mice using an LPA proton source [1]. To promote further sophisticated radiobiological studies at LPAs, adapted approaches for primary LPA source characterization, beam monitoring and dosimeters are required. Here, most prominent challenges are LPA-inherent pulse-to-pulse fluctuation in terms of intensity as well as proton energy distribution, the ultra-high pulse dose rate and the harsh plasma environment, featuring a strong electromagnetic pulse (EMP) and an intense mixed radiation background. These conditions call for robust online monitoring solutions. We present the solutions for source-to-sample characterization implemented at the ALBUS-2S beamline [2] at the Draco Petawatt laser system [3] at Helmholtz-Zentrum Dresden-Rossendorf. These include firstly an online beam monitoring system based on a time-of-flight spectrometer (ToF BMS). A core feature of the ToF BMS method is a precise spectrum-based forward-calculation of the corresponding volumetric dose distribution via Monte-Carlo simulation. Secondly, a dosimetric system for volumetric mm-scale sample irradiations was conceptualized and tested during an in vivo irradiation study, showing a solution for precise dosimetric characterization of ultra-high dose rate pulses at the ~500 mGy pulse dose range. Lastly, with the OCTOPOD and MiniSCIDOM, two devices for online, single pulse characterization of volumetric dose distributions are presented, applicable for the primary LPA source and mm-scale dose distributions at the sample site, respectively. Both devices are based on volumetric scintillators as active detector material and rely on t

We report technological developments at DRACO-PW to monitor and improve laser-plasma conditions resulting in a stable particle-source >60MeV, which in combination with our transport-beamline and high-quality dosimetry enabled first dose-controlled “in-vivo” studies with laser-driven protons.

Overview on in vitro experiment possibilities and Flash experiments

Introduction Due to the beneficial inverse physical depth-dose profile, proton radiotherapy (RT) offers the potential to reduce normal tissue toxicity by depositing the maximum dose within the tumor volume while sparing the surrounding tissue. However, range uncertainties and necessary clinical safety margins in combination with varying relative biological effectiveness (RBE) may result in a critical dose in tumor-surrounding normal tissue. Dedicated preclinical studies have been proposed to assess and better understand potential adverse effects of proton RT using image-guided proton irradiation of mouse brain. Here, we present the entire workflow from pre-treatment imaging, over treatment planning, mouse brain irradiation as established at the University Proton Therapy Center Dresden as well as first results from subsequent DNA damage analysis. Materials & Methods An experimental setup was designed and characterized to shape proton beams with 7 mm range in water and 3 mm diameter allowing for irradiation of the mouse brain´s right hemisphere. To simulate the dose distributions in vivo, a Monte Carlo model of the proton beam was designed in the simulation toolkit TOPAS, experimentally commissioned and validated. Cone-beam computed tomography (CT) and orthogonal X-ray imaging were used to delineate the hippocampus as target and position the mice at the proton beam. Mouse brains of C3H and C57BL/6 mice were irradiated with 4 Gy or 8 Gy in a single fraction and excised at different timepoints after irradiation. The number of remaining DNA double-strand break repair proteins was visualized by staining brain sections for cell nuclei and H2AX. Imaged sections were analyzed with an automated and validated processing pipeline to provide quantitative data on spatially resolved radiation damage distributions. Results Animals were planned and treated for proton irradiation of the right hippocampus with a proton beam stopping in the center of the brain. The analysis of irradia

Background and purpose: Proton therapy has become a popular treatment modality in the field of radiooncology due to higher spatial dose conformity compared to conventional radiotherapy, which holds the potential to spare normal tissue. Nevertheless, unresolved research questions, such as the much debated relative biological effectiveness (RBE) of protons, call for preclinical research, especially regarding in vivo studies. To mimic clinical workflows, high-precision small animal irradiation setups with image-guidance are needed. Material and methods: A preclinical experimental setup for small animal brain irradiation using proton radiographies was established to perform planning, repositioning, and irradiation of mice. The image quality of proton radiographies was optimized regarding the resolution, contrast-to-noise ratio (CNR), and minimal dose deposition in the animal. Subsequently, proof-of-concept histological analysis was conducted by staining for DNA double-strand breaks that were then correlated to the delivered dose. Results: The developed setup and workflow allow precise brain irradiation with a lateral target positioning accuracy of <0.26mm for in vivo experiments at minimal imaging dose of <23mGy per mouse. The custom-made software for image registration enables the fast and precise animal positioning at the beam with low observer-variability. DNA damage staining validated the successful positioning and irradiation of the mouse hippocampus. Conclusion: Proton radiography enables fast and effective high-precision lateral alignment of proton beam and target volume in mouse irradiation experiments with limited dose exposure. In the future, this will enable irradiation of larger animal cohorts as well as fractionated proton irradiation.

Background and purpose Continuing recent experiments at the research electron accelerator ELBE at the Helmholtz-Zentrum Dresden-Rossendorf the influence of beam pulse time structure on the Flash effect should be investigated in a zebrafish embryo model. Materials and methods The pulse structures of an isochronous and a synchrocyclotron were mimicked at ELBE with mean dose rates of 287 Gy/s and 177 Gy/s and pulse dose rates of 106 Gy/s and 109 Gy/s, respectively; and a macro pulsing for the latter. For comparison, a maximum (mean dose rate 2.5 x 105 Gy/s, pulse dose rate ~109 Gy/s) and a reference (mean dose rate of ~8 Gy/min) regime were applied. Radiation induced changes were assessed in zebrafish embryos over four days post irradiation. Results A significant protecting Flash effect with a clear dependence on mean dose rate was revealed for almost all endpoints and all electron pulse regimes relative to the reference. The macro pulse dependent prolongation of treatment time of the synchrotron-like regime reduce the protecting effect compared to the maximum regime delivered at same pulse but higher mean dose rate. The protecting Flash effect of the cyclotron-like regime was confirmed at a clinical isochronous proton cyclotron comparing the effects induced by 300 Gy/s relative to conventional proton beam delivery. Conclusion The mean dose rate or treatment time are more important than pulse dose rate for the extent of the normal tissue protecting Flash effect.

To optimize neoadjuvant radiochemotherapy of pancreatic ductal adenocarcinoma (PDAC), the value of new irradiation modalities such as proton therapy needs to be investigated in relevant preclinical models. We studied individual treatment responses to RCT using patient-derived PDAC organoids (PDO). Four PDO lines were treated with gemcitabine, 5-fluorouracile (5FU), photon and proton irradiation and combined RCT. Therapy response was subsequently measured via viability assays. In addition, treatment-naive PDOs were characterized via whole exome sequencing and tumorigenicity was investigated in NMRI Foxn1nu/nu mice. We found a mutational pattern con- taining common mutations associated with PDAC within the PDOs. Although we could unravel potential complications of the viability assay for PDOs in radiobiology, distinct synergistic effects of gemcitabine and 5FU with proton irradiation were observed in two PDO lines that may lead to further mechanistical studies. We could demonstrate that PDOs are a powerful tool for translational proton radiation research.

Novel particle therapy was implemented into standard-of-care for cancer patients during the last years. However, experimental studies investigating cellular and molecular mechanisms are lacking and prognostic biomarker are urgently needed. Cancer stem cell (CSC)-related biomarkers such as aldehyde dehydrogenase (ALDH) are known to cellular radiosensitivity by affecting defense against reactive oxygen species, DNA damage repair and cell survival. Within a previous study, we found that ionizing radiation itself enriches for ALDH-positive CSCs. Within the present study, we investigated CSC marker dynamics in prostate cancer, head and neck cancer and glioblastoma cells upon proton beam irradiation. We found that proton irradiation has an increased CSC targeting potential, reduced methylation of activating histone marks and a lower induction of cellular senescence compared to conventional photon irradiation. Interestingly, mathematical modeling indicated to differences in plasticity rates among ALDH-positive CSCs and ALDH-negative cancer cells between the two irradiation types.

Pathological complete response (pCR) has been correlated with overall survival in several cancer entities including colorectal cancer. Novel total neoadjuvant treatment (TNT) in rectal cancer has achieved pathological complete response in one‐third of the patients. To define better treatment options for nonresponding patients, we used patient‐derived organoids (PDOs) as avatars of the patient´s tumor to apply both photon‐ and proton‐based irradiation as well as single and combined chemo(radio)therapeutic treatments. While response to photon and proton therapy was similar, PDOs revealed heterogeneous responses to irradiation and different chemotherapeutic drugs. Radiotherapeutic response of the PDOs was significantly correlated with their ability to repair irradiation‐induced DNA damage. The classical combination of 5‐FU and irradiation could not sensitize radioresistant tumor cells. Ataxia‐telangiectasia mutated (ATM) kinase was activated upon radiation, and by inhibition of this central sensor of DNA damage, radioresistant PDOs were resensitized. The study underlined the capability of PDOs to define nonresponders to irradiation and could delineate therapeutic approaches for radioresistant patients.

The influence of different average and bunch dose rates in electron beams on the FLASH effect was investigated. The present study measures O2 content in water at different beam pulse patterns and finds strong correlation with biological data, strengthening the hypothesis of radical-related mechanisms as a reason for the FLASH effect.

Ultra-high dose rate radiobiology at the "Dresden platform for high dose-rate radiobiology"

The recent rediscovery of the “Flash Effect” revived the interest in high and ultra-high dose-rate radiation effects throughout the radiobiology community, promising protection of normal tissue, while simultaneously not altering tumour control. Systematic preclinical studies at (modified) clinical accelerators resulted in a recipe of necessary beam parameters for the induction of electron Flash effect (doi:10.3389/fonc.2019.01563), whereas for protons the optimal parameter setting is still under investigation. Expanding the clinical parameter range the “Dresden platform for high-dose rate radiobiology” enables electron and proton experiments with doserates of up to 109 Gy/s and more flexible beam pulse structures. For systematic studies of the available electron and proton beam parameters, the zebrafish embryo model was irradiated under similar conditions at the different accelerators. The irradiation setup was adapted with respect to model requirements, i.e. a certain partial oxygen pressure, and the respective beam parameters. Making use of the flexible pulse structure of the research electron accelerator ELBE, the mean dose rate was identified as the factor that defines the electron Flash effect with decreasing radiation damage for electron mean dose rate from 0.1 to 10^5 Gy/s. To cover a similar range of dose rates for protons, irradiations at the University Proton Therapy Dresden (UPTD) were combined with proton treatment at the laser proton accelerator DRACO. In doing so, the effects of proton dose rates in the range of 0.1 to 10^9 Gy/s could be investigated. To sum up, using the zebrafish embryo model as showcase the possibilities of the “Dresden platform” were demonstrated, which opens the possibility for systematic studies on the mechanisms of the Flash effect in tissue, on physico-chemical or molecular level.

Overview over preclinical proton flash experiments

The recent rediscovery of the “Flash-effect” revived the interest in high dose-rate radiation effects throughout the radiobiology community, promising protection of normal tissue, while simultaneously not altering tumour control. Several preclinical and clinical studies are presently dedicated to electron FLASH with Linacs. However, the mechanisms of Flash is still unresolved albeit its clear impact in tissue sparing to radiation toxicity. For protons, the Flash effect was confirmed in a few animal experiments using the beam parameters available at clinical cyclotrons. Laser-driven proton accelerators offer the possibility to extend the range of proton beam parameters to higher dose rates enabling the investigation in vivo of high dose-rate effects for up to 10^9 Gy/s. The general applicability of these beams for radiobiological studies was proven with simple cellular models and zebrafish embryos. One-step further, systematic in vivo experiments were prepared with a proof-of-principle mouse irradiation campaign at the Draco laser accelerator and, for comparison, at the University Proton Therapy Dresden (UPTD). Moreover, to investigate the interplay of oxygen tension, oxygen consumption and proton dose rate in more detail, an experiment with zebrafish embryos was performed at both proton sources. At the conference, we will give an overview of our radiobiological experiments performed at both DRACO and Oncoray/UPTD, for different dose rates and varying oxygen pressures.

The Need for a Research Room in a Proton Therapy Centre – Dresden perspective

Durch Hochleistungslaser getriebene Protonenquellen stellen eine interessante Ergänzung zu konventionellen Protonenbeschleunigern dar, insbesondere für die radiobiologische Forschung. Unserem interdisziplinären Forschungsteam ist es erstmals gelungen, eine radiobiologische Kleintierstudie mit laserbeschleunigten Protonen durchzuführen.

This dataset contains the raw and evaluated data of the paper plus the script for plotting the results.

The dataset contains comprehensive image data for a total of nine mice, which underwent normal tissue brain irradiation with 90 MeV protons.              In particular, the image data comprise cone-bem computed tomographies (CBCT), Monte Carlo beam transport simulations based on those CTs, regular magnetic resonance imaging (MRI) follow-up (≥ 26 weeks), a co-aligned DSURQE mouse brain atlas and scanned whole-brain tissue sections with histochemical and immunofluorescent markers for morphology (H&E), cell nuclei (DAPI), astrocytes (GFAP), microglia (Iba1), the intermediate filament protein Nestin, proliferation (Ki67), neurons (NeuN) and oligodendrocytes (OSP).           The volumetric image data (i.e. CBCT, MRI and brain atlas) were co-aligned using the ImageJ plugin Big Warp. The CBCT data was used as spatial reference to allow for mask-based, slice-wise alignment of CBCT and light microscopy image data in 3D with the scriptable registration tool Elastix.   We provide the data in raw format and as aligned data sets, as well as their spatial transformations.

Source data for all figures of publication: "Tumor irradiation in mice with a laser-accelerated proton beam". The folder structure is adapted to match the figures in the publication.

Daten, die für die Veröffentlichung "ExponatONE: eine hochpräzise Kleintierbestrahlungsanlage mit Protonenradiographie" verwendet wurden. Das Repository enthält alle Daten, die zur Erstellung der quantitativen Ergebnisse und Abbildungen im eingereichten Manuskript verwendet wurden. Satz von Skripten zur Aufnahme und Verarbeitung von Radiografiebildern/CTs, wie im zugehörigen Paper beschrieben. Bei dem Datensatz handelt es sich um alle verwendeten Bilder und Grafen (CT, Röntgenbilder, Radiografiebilder, Simulationen, Mikroskopiebilder) für die Auswertung der Ergebnisse und die Darstellung in den Figures.

Background and Aims After the rediscovery of the normal tissue sparing FLASH effect of high dose rate radiation, research activities on this topic have been revived. But especially for protons, the portfolio of accelerators capable of performing studies at ultra-high dose rates is limited. Laser-plasma accelerators (LPA) can generate extremely intense proton bunches of many 10 MeV kinetic energy. In combination with dedicated dose delivery systems, LPA proton sources facilitate peak dose rates well above 10^8 Gy/s in a pulse structure regime complementary to conventional accelerators. Methods The reliable generation of proton spectra beyond 60 MeV at DRACO-PW [Ziegler et al, SciRep2021], combined with a dedicated energy selective pulsed magnet beam transport system [Brack et al, SciRep2020], allows tailored sample-specific dose distributions. Adapted on-shot dosimetry enables the required spectral monitoring of every proton bunch. Two irradiation series on volumetric biological samples were performed at DRACO-PW, accompanied by reference irradiations at the University Proton Therapy Dresden. Results The first small animal pilot study at a laser-driven proton source was conducted successfully. The mouse-ear tumor model’s requirements [Beyreuther et al, PLoS One2018] were fulfilled and verified at high precision (+/- 5%) concerning predefined dose value and conformity. Complementary, a study investigating dose-rate effects such as FLASH was performed irradiating zebrafish embryos with above 10^9 Gy/s. Conclusions We present a laser-based irradiation platform at the DRACO-PW facility that enables systematic radiobiological studies, laying the foundations for further studies at LPA sources exploring ultra-high dose-rate effects, such as FLASH, over previously unreachable parameter space.

Recent years have shown that particle therapy offers highly conformal brain irradiation and optimized healthy tissue sparing. Nevertheless, elevated dose levels in healthy tissue, particularly in the distal beam region, can lead to undesirable long-term side effects. The biological mechanisms of such effects, however, remain unclear. A major obstacle towards correlating effects on clinical and cellular imaging levels is the mapping of radiation dose to specific brain regions or individual cell populations. We present a publicly available dataset of registered, multimodal imaging data of nine mice that received proton brain irradiation of different doses in a clinically relevant setting. It is available open access (https://rodare.hzdr.de/record/801) and comprises a baseline computed tomography (CT) scan, simulated distributions of dose and linear energy transfer, a co-aligned mouse brain atlas as well as magnetic resonance imaging (MRI) follow-up of up to six months. Additionally, we provide registered histological brain sections with eight histological stainings, reflecting all major cell types in adult mice brains. We used the self-developed tool Slice2Volume together with existing methods (Elastix & Big Warp) to fuse image data. The software is available open source: https://github.com/jo-mueller/Slice2Volume The provided image data spans several orders of magnitude of scale. Images of all modalities can be freely overlaid for every mouse as is demonstrated on Figure 1. This, for instance, allows tracing MRI image changes to specific cell populations. Hence, the dataset enables direct correlations and mechanistic observations regarding effects of proton radiation on the anatomical (atlas), clinical (MRI) and microscopic level (histology).

Further development of proton therapy takes place largely through in-vivo experiments, which are subject to increasingly strict ethical and technical requirements. Proton irradiation of small animal organs demand for highly suitable experimental setups and intelligent protocols to mimic clinical workflows as closely as possible. This includes the possibility of on-beam imaging for precise positioning and treatment. To meet these requirements, proton radiography was integrated into an existing beam setup for mouse brain proton irradiation at the University Proton Therapy Dresden (UPTD). For the realization of the radiography a flat panel detector was installed on proton beam axis behind mouse position. Transmission imaging was achieved by increasing the proton energy significantly above that required for brain irradiation, i.e. the protons pass through the mouse and reach the detector with minimal dose deposition. The final workflow includes the acquisition of cone-beam computed tomography scans and planar X-ray images before the irradiation to receive anatomical information and perform a treatment planning. The hippocampus as target region for current mouse brain irradiation experiments can be determined by registration with mouse brain atlas data. Proton radiography is acquired with a scattered proton beam right before the irradiation, resulting in a radiograph, which can be registered to the X-ray image. Finally, the target volume can be aligned to the collimated proton beam for accurate brain irradiation, which was validated by immunohistochemical staining of DNA damage region. The setup for proton radiography and mouse brain irradiation, parameters influencing the radiography quality and experimental data will be presented.

Background and aims In FLASH radiotherapy (RT), a protective effect of healthy tissue was observed, while tumor control remains comparable to conventional RT[1]. One possible explanation is the oxygen depletion hypothesis, in which radiolysis of water/cytoplasma causes the production of radicals that then react with O2 dissolved in water. This would cause a reduction in O2, which results in a hypoxic target and thus a radio-protective effect. In a previous study[2], we measured O2 depletion in sealed water phantoms during irradiation at high dose rates (<300 Gy/s) for protons, carbon ions and photons. Methods In the study presented here, this experiment was conducted further to ultra-high dose rates (10^9 Gy/s) with protons at DRACO and electrons at ELBE, where also the impact of different pulse structures on O2 depletion was tested. In addition, various settings were tested in order to irradiate zebrafish embryos with FLASH while simultaneously measuring O2. Results and Conclusion We were able to confirm the results of the previous study even at ultra-high dose rates and with electrons and came to two conclusions: 1. not enough O2 was depleted at clinical doses to explain a FLASH effect based on radiation-induced hypoxia 2. the amount of O2 depleted per dose depends on dose rate, and higher dose rates deplete slightly less O2. Furthermore, it was possible to measure O2 depletion during zebrafish embryo irradiation making a simultaneous study of biological response and O2 depletion possible. [1]Favaudon, et al. (2014) https://doi.org/10.1126/scitranslmed.3008973 [2]Jansen, et al. (2021) https://doi.org/10.1002/mp.14917

The recent rediscovery of the “Flash-effect” revived the interest in high dose-rate radiation effects throughout the radiobiology community, promising protection of normal tissue, while simultaneously not altering tumour control. Several preclinical and clinical studies are presently dedicated to electron FLASH with Linacs. However, the mechanisms of Flash is still unresolved albeit its clear impact in tissue sparing to radiation toxicity. For protons, the Flash effect was confirmed in a few animal experiments using the beam parameters available at clinical cyclotrons. Laser-driven proton accelerators offer the possibility to extend the range of proton beam parameters to higher dose rates enabling the investigation in vivo of high dose-rate effects for up to 10^9 Gy/s. The general applicability of these beams for radiobiological studies was proven with simple cellular models and zebrafish embryos. One-step further, systematic in vivo experiments were prepared with a proof-of-principle mouse irradiation campaign at the Draco laser accelerator and, for comparison, at the University Proton Therapy Dresden (UPTD). Moreover, to investigate the interplay of oxygen tension, oxygen consumption and proton dose rate in more detail, an experiment with zebrafish embryos was performed at both proton sources. At the conference, we will give an overview of our radiobiological experiments performed at both DRACO and Oncoray/UPTD, for different dose rates and varying oxygen pressures.