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Mathias K Gehrmann,1 Melanie A Kimm,2 Stefan Stangl,1 Thomas E Schmid,1 Peter B Noël,2 Ernst J Rummeny,2 Gabriele Multhoff11Department of Radiation Oncology, 2Department of Diagnostic and Interventional Radiology, Klinikum rechts der Isar, Technische Universität München, Munich, GermanyAbstract: Real-time imaging of small tumors is still one of the challenges in cancer diagnosis, prognosis, and monitoring of clinical outcome. Targeting novel biomarkers that are selectively expressed on a large variety of different tumors but not normal cells has the potential to improve the imaging capacity of existing methods such as computed tomography. Herein, we present a novel technique using cmHsp70.1 monoclonal antibody-conjugated spherical gold nanoparticles for quantification of the targeted uptake of gold nanoparticles into membrane Hsp70-positive tumor cells. Upon binding, cmHsp70.1-conjugated gold nanoparticles but not nanoparticles coupled to an isotype-matched IgG1 antibody or empty nanoparticles are rapidly taken up by highly malignant Hsp70 membrane-positive mouse tumor cells. After 24 hours, the cmHsp70.1-conjugated gold nanoparticles are found to be enriched in the perinuclear region. Specificity for membrane Hsp70 was shown by using an Hsp70 knockout tumor cell system. Toxic side effects of the cmHsp70.1-conjugated nanoparticles are not observed at a concentration of 1–10 µg/mL. Experiments are ongoing to evaluate whether cmHsp70.1 antibody-conjugated gold nanoparticles are suitable for the detection of membrane-Hsp70-positive tumors in vivo.Keywords: heat shock protein 70, tumor biomarker, theranostics, multimodal CT, multispectral CT, k-edge

High-linear energy transfer (LET) radiation, such as heavy ions is associated with a higher relative biological effectiveness (RBE) than low-LET radiation, such as photons. Irradiation with low- and high-LET particles differ in the interaction with the cellular matter and therefore in the spatial dose distribution. When a single high-LET particle interacts with matter, it results in doses of up to thousands of gray (Gy) locally concentrated around the ion trajectory, whereas the mean dose averaged over the target, such as a cell nucleus is only in the range of a Gy. DNA damage therefore accumulates in this small volume. In contrast, up to hundreds of low-LET particle hits are required to achieve the same mean dose, resulting in a quasi-homogeneous damage distribution throughout the cell nucleus. In this study, we investigated the dependence of RBE from different spatial dose depositions using different focused beam spot sizes of proton radiation with respect to the induction of chromosome aberrations and clonogenic cell survival. Human-hamster hybrid (AL) as well as Chinese hamster ovary cells (CHO-K1) were irradiated with focused low LET protons of 20 MeV (LET = 2.6 keV/µm) beam energy with a mean dose of 1.7 Gy in a quadratic matrix pattern with point spacing of 5.4 × 5.4 µm2 and 117 protons per matrix point at the ion microbeam SNAKE using different beam spot sizes between 0.8 µm and 2.8 µm (full width at half maximum). The dose-response curves of X-ray reference radiation were used to determine the RBE after a 1.7 Gy dose of radiation. The RBE for the induction of dicentric chromosomes and cell inactivation was increased after irradiation with the smallest beam spot diameter (0.8 µm for chromosome aberration experiments and 1.0 µm for cell survival experiments) compared to homogeneous proton radiation but was still below the RBE of a corresponding high LET single ion hit. By increasing the spot size to 1.6-1.8 µm, the RBE decreased but was still higher than for homogeneously distributed protons. By further increasing the spot size to 2.7-2.8 µm, the RBE was no longer different from the homogeneous radiation. Our experiments demonstrate that varying spot size of low-LET radiation gradually modifies the RBE. This underlines that a substantial fraction of enhanced RBE originates from inhomogeneous energy concentrations on the µm scale (mean intertrack distances of low-LET particles below 0.1 µm) and quantifies the link between such energy concentration and RBE. The missing fraction of RBE enhancement when comparing with high-LET ions is attributed to the high inner track energy deposition on the nanometer scale. The results are compared with model results of PARTRAC and LEM for chromosomal aberration and cell survival, respectively, which suggest mechanistic interpretations of the observed radiation effects., (© 2024 by Radiation Research Society.)

Microbeam radiation therapy (MRT) is a still pre-clinical form of spatially fractionated radiotherapy, which uses an array of micrometer-wide, planar beams of X-ray radiation. The dose modulation in MRT has proven effective in the treatment of tumors while being well tolerated by normal tissue. Research on understanding the underlying biological mechanisms mostly requires large third-generation synchrotrons. In this study, we aimed to develop a preclinical treatment environment that would allow MRT independent of synchrotrons. We built a compact microbeam setup for pre-clinical experiments within a small animal irradiator and present in vivo MRT application, including treatment planning, dosimetry, and animal positioning. The brain of an immobilized mouse was treated with MRT, excised, and immunohistochemically stained against γH2AX for DNA double-strand breaks. We developed a comprehensive treatment planning system by adjusting an existing dose calculation algorithm to our setup and attaching it to the open-source software 3D-Slicer. Predicted doses in treatment planning agreed within 10% with film dosimetry readings. We demonstrated the feasibility of MRT exposures in vivo at a compact source and showed that the microbeam pattern is observable in histological sections of a mouse brain. The platform developed in this study will be used for pre-clinical research of MRT.

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-β1, TNF-α, IL1α, and IL1β 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., (© 2024. The Author(s).)

Magnetic nanoparticles can be functionalized in many ways for biomedical applications. Here, we combine four advantageous features in a novel Fe-Pt-Yb 2 O 3 core-shell nanoparticle. (a) The nanoparticles have a size of 10 nm allowing them to diffuse through neuronal tissue. (b) The particles are superparamagnetic after synthesis and ferromagnetic after annealing, enabling directional control by magnetic fields, enhance NMRI contrast, and hyperthermia treatment. (c) After neutron-activation of the shell, they carry low-energetic, short half-life β-radiation from 175 Yb, 177 Yb, and 177 Lu. (d) Additionally, the particles can be optically visualized by plasmonic excitation and luminescence. To demonstrate the potential of the particles for cancer treatment, we exposed cultured human glioblastoma cells (LN-18) to non-activated and activated particles to confirm that the particles are internalized, and that the β-radiation of the radioisotopes incorporated in the neutron-activated shell of the nanoparticles kills more than 98% of the LN-18 cancer cells, promising for future anti-cancer applications., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Background: Inflammatory skin reactions and skin alterations are still a potential side effect in radiation therapy (RT), which also need attention for patients' health care., Method: In a pre-clinical study we consider alterations in irradiated in-vitro skin models of epidermal and dermal layers. Typical dose regimes in radiation therapy are applied for irradiation. For non-invasive imaging and characterization optical coherence tomography (OCT) is used. Histological staining method is additionally applied for comparison and discussion., Results: Structural features, such as keratinization, modifications in epidermal cell layer thickness and disorder in the layering-as indications for reactions to ionizing radiation and aging-could be observed by means of OCT and confirmed by histology. We were able to recognize known RT induced changes such as hyper-keratosis, acantholysis, and epidermal hyperplasia as well as disruption and/or demarcation of the dermo-epidermal junction., Conclusion: The results may pave the way for OCT to be considered as a possible adjunctive tool to detect and monitor early skin inflammation and side effects of radiotherapy, thus supporting patient healthcare in the future., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Bromberger et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy. However, structured knowledge to mitigate a patient's specific risk of developing adverse events are limited. Nevertheless, there is an exponential growth of clinical studies combining conventional therapies such as radiation therapy (RT) with ICIs. Cutaneous reactions are among the most common adverse events after monotherapy with either ICIs or RT. So far, little is known about interindividual differences for the risk of developing severe tissue toxicity after the combination of RT with ICIs, and the underlying biological mechanisms are ill defined. We used experimental models of RT-induced skin injury to analyze skin toxicity after simultaneous application of ICIs. We compared different RT regimens such as fractionated or stereotactic RT with varying dose intensity. Strikingly, we found that simultaneous application of RT and ICIs did not significantly aggravate acute skin injury in two different mouse strains. Detailed examination of long-term tissue damage of the skin revealed similar signs of epidermal hyperplasia, dermal fibrosis, and adnexal atrophy. In summary, we here present the first experimental study demonstrating the excellent safety profiles of concurrent treatment with RT and ICIs. These findings will help to interpret the development of adverse events of the skin after radioimmunotherapy and guide the design of new clinical trials and clinical decision-making in individual cases. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

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Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers. Innovative treatment concepts may enhance oncological outcome. Clinically relevant tumor models are essential in developing new therapeutic strategies. In the present study, we used two human PDAC cell lines for an orthotopic xenograft mouse model and compared treatment characteristics between this in vivo tumor model and PDAC patients. Tumor-bearing mice received stereotactic high-precision irradiation using arc technique after 3D-treatment planning. Induction of DNA damage in tumors and organs at risk (OARs) was histopathologically analyzed by the DNA damage marker γH2AX and compared with results after unprecise whole-abdomen irradiation. Our mouse model and preclinical setup reflect the characteristics of PDAC patients and clinical RT. It was feasible to perform stereotactic high-precision RT after defining tumor and OARs by CT imaging. After stereotactic RT, a high rate of DNA damage was mainly observed in the tumor but not in OARs. The calculated dose distributions and the extent of the irradiation field correlate with histopathological staining and the clinical example. We established and validated 3D-planned stereotactic RT in an orthotopic PDAC mouse model, which reflects the human RT. The efficacy of the whole workflow of imaging, treatment planning, and high-precision RT was proven by longitudinal analysis showing a significant improved survival. Importantly, this model can be used to analyze tumor regression and therapy-related toxicity in one model and will allow drawing clinically relevant conclusions.

Background and Purpose: Radiotherapy of thoracic tumours can lead to side effects in the lung, which may benefit from early diagnosis. We investigated the potential of X-ray dark-field computed tomography by a proof-of-principle murine study in a clinically relevant radiotherapeutic setting aiming at the detection of radiation-induced lung damage., Material and Methods: Six mice were irradiated with 20 Gy to the entire right lung. Together with five unirradiated control mice, they were imaged using computed tomography with absorption and dark-field contrast before and 16 weeks post irradiation. Mean pixel values for the right and left lung were calculated for both contrasts, and the right-to-left-ratio R of these means was compared. Radiologists also assessed the tomograms acquired 16 weeks post irradiation. Sensitivity, specificity, inter- and intra-reader accuracy were evaluated., Results: In absorption contrast the group-average of R showed no increase in the control group and increased by 7% (p = 0.005) in the irradiated group. In dark-field contrast, it increased by 2% in the control group and by 14% (p = 0.005) in the irradiated group. Specificity was 100% for both contrasts but sensitivity was almost four times higher using dark-field tomography. Two cases were missed by absorption tomography but were detected by dark-field tomography., Conclusions: The applicability of X-ray dark-field computed tomography for the detection of radiation-induced lung damage was demonstrated in a pre-clinical mouse model. The presented results illustrate the differences between dark-field and absorption contrast and show that dark-field tomography could be advantageous in future clinical settings., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Background: Treatment resistance of glioblastoma multiforme to chemo- and radiotherapy remains a challenge yet to overcome. In particular, the O 6 -methylguanine-DNA-methyltransferase (MGMT) promoter unmethylated patients have only little benefit from chemotherapy treatment using temozolomide since MGMT counteracts its therapeutic efficacy. Therefore, new treatment options in radiotherapy need to be developed to inhibit MGMT and increase radiotherapy response., Methods: Lomeguatrib, a highly specific MGMT inhibitor, was used to inactivate MGMT protein in vitro. Radiosensitivity of established human glioblastoma multiforme cell lines in combination with lomeguatrib was investigated using the clonogenic survival assay. Inhibition of MGMT was analyzed using Western Blot. Cell cycle distribution and apoptosis were investigated to determine the effects of lomeguatrib alone as well as in combination with ionizing radiation., Results: Lomeguatrib significantly decreased MGMT protein and reduced radiation-induced G2/M arrest. A radiosensitizing effect of lomeguatrib was observed when administered at 1 µM and increased radioresistance at 20 µM., Conclusion: Low concentrations of lomeguatrib elicit radiosensitization, while high concentrations mediate a radioprotective effect.

Objective: Assessing the advantage of x-ray dark-field contrast over x-ray transmission contrast in radiography for the detection of developing radiation-induced lung damage in mice., Methods: Two groups of female C57BL/6 mice (irradiated and control) were imaged obtaining both contrasts monthly for 28 weeks post irradiation. Six mice received 20 Gy of irradiation to the entire right lung sparing the left lung. The control group of six mice was not irradiated. A total of 88 radiographs of both contrasts were evaluated for both groups based on average values for two regions of interest, covering (irradiated) right lung and healthy left lung. The ratio of these average values, R, was distinguished between healthy and damaged lungs for both contrasts. The time-point when deviations of R from healthy lung exceeded 3σ was determined and compared among contrasts. The Wilcoxon-Mann-Whitney test was used to test against the null hypothesis that there is no difference between both groups. A selection of 32 radiographs was assessed by radiologists. Sensitivity and specificity were determined in order to compare the diagnostic potential of both contrasts. Inter-reader and intra-reader accuracy were rated with Cohen's kappa., Results: Radiation-induced morphological changes of lung tissue caused deviations from the control group that were measured on average 10 weeks earlier with x-ray dark-field contrast than with x-ray transmission contrast. Sensitivity, specificity, and accuracy doubled using dark-field radiography., Conclusion: X-ray dark-field radiography detects morphological changes of lung tissue associated with radiation-induced damage earlier than transmission radiography in a pre-clinical mouse model., Key Points: • Significant deviations from healthy lung due to irradiation were measured after 16 weeks with x-ray dark-field radiography (p = 0.004). • Significant deviations occur on average 10 weeks earlier for x-ray dark-field radiography in comparison to x-ray transmission radiography. • Sensitivity and specificity doubled when using x-ray dark-field radiography instead of x-ray transmission radiography.

Purpose: Radioresistance in pancreatic cancer patients remains a critical obstacle to overcome. Understanding the molecular mechanisms underlying radioresistance may achieve better response to radiotherapy and thereby improving the poor treatment outcome. The aim of the present study was to elucidate the mechanisms leading to radioresistance by detailed characterization of isogenic radioresistant and radiosensitive cell lines., Methods: The human pancreatic cancer cell lines, Panc-1 and MIA PaCa-2 were repeatedly exposed to radiation to generate radioresistant (RR) isogenic cell lines. The surviving cells were expanded, and their radiosensitivity was measured using colony formation assay. Tumor growth delay after irradiation was determined in a mouse pancreatic cancer xenograft model. Gene and protein expression were analyzed using RNA sequencing and Western blot, respectively. Cell cycle distribution and apoptosis (Caspase 3/7) were measured by FACS analysis. Reactive oxygen species generation and DNA damage were analyzed by detection of CM-H 2 DCFDA and γH2AX staining, respectively. Transwell chamber assays were used to investigate cell migration and invasion., Results: The acquired radioresistance of RR cell lines was demonstrated in vitro and validated in vivo. Ingenuity pathway analysis of RNA sequencing data predicted activation of cell viability in both RR cell lines. RR cancer cell lines demonstrated greater DNA repair efficiency and lower basal and radiation-induced reactive oxygen species levels. Migration and invasion were differentially affected in RR cell lines., Conclusions: Our data indicate that repeated exposure to irradiation increases the expression of genes involved in cell viability and thereby leads to radioresistance. Mechanistically, increased DNA repair capacity and reduced oxidative stress might contribute to the radioresistant phenotype., Competing Interests: Declaration of Competing Interest Prof. Combs reports grants from Deutsche Forschungsgemeinschaft, non-financial support from Deutsches Konsortium für Translationale Krebsforschung, during the conduct of the study; personal fees and non-financial support from Roche, personal fees and non-financial support from AstraZeneca, personal fees and non-financial support from Medac, personal fees and nonfinancial support from Dr. Sennewald Medizintechnik, personal fees and non-financial support from Elekta, personal fees and non-financial support from Accuray, personal fees and nonfinancial support from BMS, personal fees and non-financial support from Brainlab, personal fees and non-financial support from Daiichi Sankyo, personal fees and non-financial support from Icotec, outside the submitted work., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: Microbeam radiation therapy is a preclinical concept in radiation oncology. It spares normal tissue more effectively than conventional radiation therapy at equal tumor control. The radiation field consists of peak regions with doses of several hundred gray, whereas doses between the peaks (valleys) are below the tissue tolerance level. Widths and distances of the beams are in the submillimeter range for microbeam radiation therapy. A similar alternative concept with beam widths and distances in the millimeter range is presented by minibeam radiation therapy. Although both methods were developed at large synchrotron facilities, compact alternative sources have been proposed recently., Methods and Materials: A small-animal irradiator was fitted with a special 3-layered collimator that is used for preclinical research and produces microbeams of flexible width of up to 100 μm. Film dosimetry provided measurements of the dose distributions and was compared with Monte Carlo dose predictions. Moreover, the micronucleus assay in Chinese hamster CHO-K1 cells was used as a biological dosimeter. The focal spot size and beam emission angle of the x-ray tube were modified to optimize peak dose rate, peak-to-valley dose ratio (PVDR), beam shape, and field homogeneity. An equivalent collimator with slit widths of up to 500 μm produced minibeams and allowed for comparison of microbeam and minibeam field characteristics., Results: The setup achieved peak entrance dose rates of 8 Gy/min and PVDRs >30 for microbeams. Agreement between Monte Carlo simulations and film dosimetry is generally better for larger beam widths; qualitative measurements validated Monte Carlo predicted results. A smaller focal spot enhances PVDRs and reduces beam penumbras but substantially reduces the dose rate. A reduction of the beam emission angle improves the PVDR, beam penumbras, and dose rate without impairing field homogeneity. Minibeams showed similar field characteristics compared with microbeams at the same ratio of beam width and distance but had better agreement with simulations., Conclusion: The developed setup is already in use for in vitro experiments and soon for in vivo irradiations. Deviations between Monte Carlo simulations and film dosimetry are attributed to scattering at the collimator surface and manufacturing inaccuracies and are a matter of ongoing research., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., (Copyright © 2020. Published by Elsevier Inc.)

Purpose: The MSc Radiation Biology course is a highly interdisciplinary degree program placing radiation biology at the interface between biology, medicine, and physics, as well as their associated technologies. The goal was to establish an internationally acknowledged program with diverse and heterogeneous student cohorts, who benefit from each other academically as well as culturally. We have completed a Five-Year evaluation of the program to assess our qualification profile and the further direction we want to take., Materials and Methods: We evaluated the student cohort's data from the last 5 years regarding gender, age, and nationality as well as the highest degree before applying and career path after graduation., Results: Data shows a great diversity regarding nationalty as well as undergraduate background. Cohort sizes could be increased and future prospects mainly aimed to a PhD. Measures after regular quality meetings and students' feedback led to improving the curriculum and workload, teacher's training, and changes to examination regulations., Conclusions: After 5 years, statistics show that our expectations have been met exceedingly. All graduates had excellent career opportunities reflecting the necessity of this MSc and its topics. We are continuously working on improving the program and adapting the curriculum to the requirements in radiation sciences. The future vision includes an expansion of the program as well as undergraduate education opportunities in this field.

Today, pancreatic cancer is the seventh leading cause of cancer-related deaths worldwide with a five-year overall survival rate of less than 7%. Only 15-20% of patients are eligible for curative intent surgery at the time of diagnosis. Therefore, neoadjuvant treatment regimens have been introduced in order to downsize the tumor by chemotherapy and radiotherapy. To further increase the efficacy of radiotherapy, novel molecular biomarkers are urgently needed to define the subgroup of pancreatic cancer patients who would benefit most from radiotherapy. MicroRNAs (miRNAs) could have the potential to serve as novel predictive and prognostic biomarkers in patients with pancreatic cancer. In the present article, the role of miRNAs as blood biomarkers, which are associated with either radioresistance or radiation-induced changes of miRNAs in pancreatic cancer, is discussed. Furthermore, the manuscript provides own data of miRNAs identified in a pancreatic cancer mouse model as well as radiation-induced miRNA changes in the plasma of tumor-bearing mice.

Purpose: X-ray microbeam radiation therapy is a preclinical concept for tumor treatment promising tissue sparing and enhanced tumor control. With its spatially separated, periodic micrometer-sized pattern, this method requires a high dose rate and a collimated beam typically available at large synchrotron radiation facilities. To treat small animals with microbeams in a laboratory-sized environment, we developed a dedicated irradiation system at the Munich Compact Light Source (MuCLS)., Methods: A specially made beam collimation optic allows to increase x-ray fluence rate at the position of the target. Monte Carlo simulations and measurements were conducted for accurate microbeam dosimetry. The dose during irradiation is determined by a calibrated flux monitoring system. Moreover, a positioning system including mouse monitoring was built., Results: We successfully commissioned the in vivo microbeam irradiation system for an exemplary xenograft tumor model in the mouse ear. By beam collimation, a dose rate of up to 5.3 Gy/min at 25 keV was achieved. Microbeam irradiations using a tungsten collimator with 50 μm slit size and 350 μm center-to-center spacing were performed at a mean dose rate of 0.6 Gy/min showing a high peak-to-valley dose ratio of about 200 in the mouse ear. The maximum circular field size of 3.5 mm in diameter can be enlarged using field patching., Conclusions: This study shows that we can perform in vivo microbeam experiments at the MuCLS with a dedicated dosimetry and positioning system to advance this promising radiation therapy method at commercially available compact microbeam sources. Peak doses of up to 100 Gy per treatment seem feasible considering a recent upgrade for higher photon flux. The system can be adapted for tumor treatment in different animal models, for example, in the hind leg., (© 2020 The Authors. Medical Physics published by Wiley Periodicals LLC on behalf of American Association of Physicists in Medicine.)

Glioblastoma multiforme (GBM) is the most common high-grade intracranial tumor in adults. It is characterized by uncontrolled proliferation, diffuse infiltration due to high invasive and migratory capacities, as well as intense resistance to chemo- and radiotherapy. With a five-year survival of less than 3% and an average survival rate of 12 months after diagnosis, GBM has become a focus of current research to urgently develop new therapeutic approaches in order to prolong survival of GBM patients. The methylation status of the promoter region of the O 6 -methylguanine-DNA methyltransferase (MGMT) is nowadays routinely analyzed since a methylated promoter region is beneficial for an effective response to temozolomide-based chemotherapy. Furthermore, several miRNAs were identified regulating MGMT expression, apart from promoter methylation, by degrading MGMT mRNA before protein translation. These miRNAs could be a promising innovative treatment approach to enhance Temozolomide (TMZ) sensitivity in MGMT unmethylated patients and to increase progression-free survival as well as long-term survival. In this review, the relevant miRNAs are systematically reviewed.

Microbeam radiation therapy (MRT), a preclinical form of spatially fractionated radiotherapy, uses an array of microbeams of hard synchrotron X-ray radiation. Recently, compact synchrotron X-ray sources got more attention as they provide essential prerequisites for the translation of MRT into clinics while overcoming the limited access to synchrotron facilities. At the Munich compact light source (MuCLS), one of these novel compact X-ray facilities, a proof of principle experiment was conducted applying MRT to a xenograft tumor mouse model. First, subcutaneous tumors derived from the established squamous carcinoma cell line FaDu were irradiated at a conventional X-ray tube using broadbeam geometry to determine a suitable dose range for the tumor growth delay. For irradiations at the MuCLS, FaDu tumors were irradiated with broadbeam and microbeam irradiation at integral doses of either 3 Gy or 5 Gy and tumor growth delay was measured. Microbeams had a width of 50 µm and a center-to-center distance of 350 µm with peak doses of either 21 Gy or 35 Gy. A dose rate of up to 5 Gy/min was delivered to the tumor. Both doses and modalities delayed the tumor growth compared to a sham-irradiated tumor. The irradiated area and microbeam pattern were verified by staining of the DNA double-strand break marker γH2AX. This study demonstrates for the first time that MRT can be successfully performed in vivo at compact inverse Compton sources.

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

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., Competing Interests: The authors have declared that no competing interests exist.

Introduction: The major stress-inducible heat shock protein 70 (Hsp70) is induced after different stress stimuli. In tumors, elevated intracellular Hsp70 levels were associated on the one hand with radio- and chemotherapy resistance and on the other hand with a favorable outcome for patients. This study was undertaken to investigate cytosolic Hsp70 (cHsp70) as a potential biomarker for progression free (PFS) and overall survival (OS) in patients with primary glioblastomas (GBM)., Methods: The cHsp70 expression in tumor tissue of 60 patients diagnosed with primary GBM was analyzed by immunohistochemistry. The cHsp70 expression was correlated to the PFS and OS of the patients., Results: A high cHsp70 expression was associated with a prolonged PFS (hazard ratio = 0.374, p = 0.001) and OS (hazard ratio = 0.416, p = 0.014) in GBM patients treated according to the standard Stupp protocol with surgery, radiotherapy and temozolomide., Conclusions: These data suggest that the intracellular Hsp70 expression might serve as a prognostic marker in patients with primary GBM., Competing Interests: The authors have declared that no competing interests exist.

Supplementation with the antioxidant selenium is frequently performed in breast cancer patients to protect the normal tissue from radiation-induced side effects. However, concerns exist whether selenium also protects tumor cells from radiation-induced cell kill and thereby reduces the efficacy of radiotherapy. In this work, the effect of selenium administration on the radiosensitivity of breast cancer cells was evaluated in vitro. Physiological relevant selenium concentrations (70 and 140 µg/l) did not affect DNA double-strand breaks (γH2AX foci) after 4-Gy X-ray irradiation. Also apoptosis (caspase 3/7) after irradiation with 10 Gy was not influenced by selenium treatment in MDA-MB-231 and MCF7 cells. Most importantly, selenium supplementation did not impair the clonogenic survival of the breast cancer cell lines after irradiation (0, 2, 4, 6, 8 Gy). The data suggest that physiological relevant selenium concentrations administered in combination with radiation therapy do not deteriorate the efficacy of radiotherapy in breast cancer patients. However, randomized clinical trials comparing the effectiveness of radiotherapy and the associated side effects in patients with and without selenium supplementation are recommended.

The increased number of cell divisions undergone by spermatogonia of older fathers cannot fully account for the observed increase in germline genetic damage. Studies have shown that the mechanisms induced in germ cells in response to oxidative damage varies with age, that DNA repair efficiency declines, and both sperm DNA damage and spontaneous mutations increase. However, it is not known whether the altered response with age is a cause, or consequence, of an age-associated change in cell susceptibility to genetic damage. Following a single 150 mg/kg dose of cyclophosphamide (CP), young (8-weeks old) and aged (17-month old) male mice were examined 24 h later for induced genetic damage in epididymal spermatozoa using the alkaline comet and sperm chromatin stability assays. Apoptosis among testicular cells was examined on tissue cross-sections using the TUNEL assay. Sperm showed no significant increase in DNA strand breaks with age (detected by the comet assay) and no change in sperm chromatin stability (detected by the SCSA assay). Following CP treatment, there was no effect on DNA-strand breakage but sperm chromatin instability was significantly higher. Furthermore, it was also significantly elevated in old treated, compared with young treated, animals suggesting that increased age affects the sensitivity of epididymal sperm to chromatin damage. There was no difference in apoptosis in testicular germ cells from either young or old control animals, while CP administration resulted in a significant increase in apoptosis among young animals but not old animals. Following genotoxin exposure, an increase in chromatin instability in the spermatozoa of old animals and a decrease in the ability of their testicular germ cells undergo apoptosis suggests an age-related decrease in genome protection mechanisms. Since those germ cells are only transiently present in the testis, it is likely that this age-related deterioration originates in the spermatogonial stem cells. The findings are also evidence that the safety evaluation of reproductive genotoxins should consider young and old individuals separately., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 (TGFβ1)-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 TGFβ1-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 TGFβ1-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.

The biophysical simulation tool PARTRAC contains modules for DNA damage response representing non-homologous end joining of DNA double-strand breaks (DSB) and the formation of chromosomal aberrations. Individual DNA ends from the induced DSB are followed regarding both their enzymatic processing and spatial mobility, as is needed for chromosome aberrations to arise via ligating broken ends from different chromosomes. In particular, by tracking the genomic locations of the ligated fragments and the positions of centromeres, the induction of dicentrics can be modeled. In recent experiments, the impact of spatial clustering of DNA damage on dicentric yields has been assessed in AL human-hamster hybrid cells: Defined numbers of 20 MeV protons (linear energy transfer, LET 2.6 keV/μm), 45 MeV Li ions (60 keV/μm) and 55 MeV C ions (310 keV/μm) focused to sub-μm spot sizes were applied with the ion microbeam SNAKE in diverse grid modes, keeping the absorbed dose constant. The impact of the μm-scaled spatial distribution of DSB (focusing effect) has thus been separated from nm-scaled DSB complexity (LET effect). The data provide a unique benchmark for the model calculations. Model and parameter refinements are described that enabled the simulations to largely reproduce both the LET-dependence and the focusing effect as well as the usual biphasic rejoining kinetics. The predictive power of the refined model has been benchmarked against dicentric yields for photon irradiation., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Extraordinary normal tissue response to highly spatially fractionated X-ray beams has been explored for over 25 years. More recently, alternative radiation sources have been developed and utilized with the aim to evoke comparable effects. These include protons, which lend themselves well for this endeavour due to their physical depth dose characteristics as well as corresponding variable biological effectiveness. This paper addresses the motivation for using protons to generate spatially fractionated beams and reviews the technological implementations and experimental results to date. This includes simulation and feasibility studies, collimation and beam characteristics, dosimetry and biological considerations as well as the results of in vivo and in vitro studies. Experimental results are emerging indicating an extraordinary normal tissue sparing effect analogous to what has been observed for synchrotron generated X-ray microbeams. The potential for translational research and feasibility of spatially modulated proton beams in clinical settings is discussed.

Gliomas are primary brain tumors that present the majority of malignant adult brain tumors. Gliomas are subdivided into low- and high-grade tumors. Despite extensive research in recent years, the prognosis of malignant glioma patients remains poor. This is caused by naturally highly infiltrative capacities as well as high levels of radio- and chemoresistance. Additionally, it was shown that low linear energy transfer (LET) irradiation enhances migration and invasion of several glioma entities which might counteract today's treatment concepts. However, this finding is discussed controversially. In the era of personalized medicine, this controversial data might be attributed to the patient-specific heterogeneity that ultimately could be used for treatment. Thus, current developments in glioma therapy should be seen in the context of intrinsic and radiation-enhanced migration and invasion. Due to the natural heterogeneity of glioma cells and different radiation responses, a personalized radiation treatment concept is suggested and alternative radiation concepts are discussed.

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

Purpose: Glioblastoma multiforme (GBM) is the most common primary brain tumor and has a very poor overall prognosis. Multimodal treatment is still inefficient and one main reason is the invasive nature of GBM cells, enabling the tumor cells to escape from the treatment area causing tumor progression. This experimental study describes the effect of low- and high-LET irradiation on the invasion of primary GBM cells with a validation in established cell systems., Methods: Seven patient derived primary GBM as well as three established cell lines (LN229, LN18 and U87) were used in this study. Invasion was investigated using Matrigel® coated transwell chambers. Irradiation was performed with low- (X-ray) and high-LET (alpha particles) radiation. The colony formation assay was chosen to determine the corresponding alpha particle dose equivalent to the X-ray dose., Results: 4 Gy X-ray irradiation increased the invasive potential of six patient derived GBM cells as well as two of the established lines. In contrast, alpha particle irradiation with an equivalent dose of 1.3 Gy did not show any effect on the invasive behavior. The findings were validated with established cell lines., Conclusion: Our results show that in contrast to low-LET irradiation high-LET irradiation does not enhance the invasion of established and primary glioblastoma cell lines. We therefore suggest that high-LET irradiation could become an alternative treatment option. To fully exploit the benefits of high-LET irradiation concerning the invasion of GBM further molecular studies should be performed.

Tumor cells frequently overexpress heat shock protein 70 (Hsp70) and present it on their cell surface, where it can be recognized by pre-activated NK cells. In our retrospective study the expression of Hsp70 was determined in relation to tumor-infiltrating CD56 + NK cells in formalin-fixed paraffin embedded (FFPE) tumor specimens of patients with SCCHN (N = 145) as potential indicators for survival and disease recurrence. All patients received radical surgery and postoperative cisplatin-based radiochemotherapy (RCT). In general, Hsp70 expression was stronger, but with variable intensities, in tumor compared to normal tissues. Patients with high Hsp70 expressing tumors (scores 3-4) showed significantly decreased overall survival (OS; p = 0.008), local progression-free survival (LPFS; p = 0.034) and distant metastases-free survival (DMFS; p = 0.044), compared to those with low Hsp70 expression (scores 0-2), which remained significant after adjustment for relevant prognostic variables. The adverse prognostic value of a high Hsp70 expression for OS was also observed in patient cohorts with p16- (p = 0.001), p53- (p = 0.0003) and HPV16 DNA-negative (p = 0.001) tumors. The absence or low numbers of tumor-infiltrating CD56 + NK cells also correlated with significantly decreased OS (p = 0.0001), LPFS (p = 0.0009) and DMFS (p = 0.0001). A high Hsp70 expression and low numbers of tumor-infiltrating NK cells have the highest negative predictive value (p = 0.00004). In summary, a strong Hsp70 expression and low numbers of tumor-infiltrating NK cells correlate with unfavorable outcome following surgery and RCT in patients with SCCHN, and thus serve as negative prognostic markers., (© 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background: Proton radiotherapy is a form of charged particle therapy that is preferentially applied for the treatment of tumors positioned near to critical structures due to their physical characteristics, showing an inverted depth-dose profile. The sparing of normal tissue has additional advantages in the treatment of pediatric patients, in whom the risk of secondary cancers and late morbidity is significantly higher. Up to date, a fixed relative biological effectiveness (RBE) of 1.1 is commonly implemented in treatment planning systems with protons in order to correct the physical dose. This value of 1.1 comes from averaging the results of numerous in vitro experiments, mostly conducted in the middle of the spread-out Bragg peak, where RBE is relatively constant. However, the use of a constant RBE value disregards the experimental evidence which clearly demonstrates complex RBE dependency on dose, cell- or tissue type, linear energy transfer and biological endpoints. In recent years, several in vitro studies indicate variations in RBE of protons which translate to an uncertainty in the biological effective dose delivery to the patient. Particularly for regions surrounding the Bragg peak, the more localized pattern of energy deposition leads to more complex DNA lesions. These RBE variations of protons bring the validity of using a constant RBE into question., Main Body: This review analyzes how RBE depends on the dose, different biological endpoints and physical properties. Further, this review gives an overview of the new insights based on findings made during the last years investigating the variation of RBE with depth in the spread out Bragg peak and the underlying differences in radiation response on the molecular and cellular levels between proton and photon irradiation. Research groups such as the Klinische Forschergruppe Schwerionentherapie funded by the German Research Foundation (DFG, KFO 214) have included work on this topic and the present manuscript highlights parts of the preclinical work and summarizes the research activities in this context., Short Conclusion: In summary, there is an urgent need for more coordinated in vitro and in vivo experiments that concentrate on a realistic dose range of in clinically relevant tissues like lung or spinal cord.

Background and Purpose: High-precision radiotherapy (RT) requires precise positioning, particularly with high single doses. Fiducial markers in combination with onboard imaging are excellent tools to support this. The purpose of this study is to establish a pancreatic cancer mouse model for high-precision image-guided RT (IGRT) using the liquid fiducial marker BioXmark (Nanovi, Kongens Lyngby, Denmark)., Methods: In an animal-based cancer model, different volumes of BioXmark (10-50 µl), application forms, and imaging modalities-cone-beam computer tomography (CBCT) incorporated in either the Small Animal Radiation Research Platform (SARRP) or the small-animal micro-CT Scanner (SkyScan; Bruker, Brussels, Belgium)-as well as subsequent RT with the SARRP system were analyzed to derive recommendations for BioXmark., Results: Even small volumes (10 µl) of BioXmark could be detected by CBCT (SARRP and Skyscan). Larger volumes (50 µl) led to hardening artefacts. The position of BioXmark was monitored at least weekly by CBCT and was stable over 4 months. BioXmark was shown to be well tolerated; no changes in physical condition or toxic side effects were observed in comparison to control mice. BioXmark enabled an exact fusion with the original treatment plan with less hardening artefacts, and minimized the application of contrast agent for fractionated RT., Conclusion: An orthotopic pancreatic tumor mouse model was established for high-precision IGRT using a fiducial marker. BioXmark was successfully tested and provides the perfect basis for improved imaging in high-precision RT. BioXmark enables a unique application method and optimal targeted precision in fractionated RT. Therefore, preclinical trials evaluating novel fractionation regimens and/or combination treatment with high-end RT can be performed.

Background: Chemoradiation of locally advanced non-metastatic pancreatic cancer can lead to secondary operability by tumor mass reduction. Here, we analyzed radiomodulating effects of oridonin and ponicidin in pancreatic cancer in vitro . Both agents are ent-kaurane diterpenoids, extracted from Isodon rubescens , a plant that is well known in Traditional Chinese Medicine. Cytotoxic effects have recently been shown in different tumor entities for both agents., Materials and Methods: Pancreatic cancer cell lines AsPC-1, BxPC-3, Panc-1 and MIA PaCa-2 were pretreated with oridonin or ponicidin and irradiated with 2 Gy to 6 Gy. Long-term survival was determined by clonogenic assay. Cell cycle effects and intensity of γH2AX as indicator for DNA double-strand breaks were investigated by flow cytometry. Western blotting was used to study the DNA double-strand break repair proteins Ku70, Ku80 and XRCC4., Results: Oridonin and ponicidin lead to a dose-dependent reduction of clonogenic survival and an increase in γH2AX. Combined with irradiation we observed additive effects and a prolonged G2/M-arrest. No relevant changes in the levels of the DNA double-strand break repair proteins were detected., Conclusions: Pretreatment with oridonin or ponicidin followed by irradiation lead to an additional reduction in survival of pancreatic cancer cells in vitro , presumably explained by an induced prolonged G2/M-arrest. Both agents seem to induce DNA double-strand breaks but do not interact with the non-homologous end joining (NHEJ) pathway., Competing Interests: Disclosure: No potential conflicts of interest were disclosed.

X-ray microbeam radiotherapy can potentially widen the therapeutic window due to a geometrical redistribution of the dose. However, high requirements on photon flux, beam collimation, and system stability restrict its application mainly to large-scale, cost-intensive synchrotron facilities. With a unique laser-based Compact Light Source using inverse Compton scattering, we investigated the translation of this promising radiotherapy technique to a machine of future clinical relevance. We performed in vitro colony-forming assays and chromosome aberration tests in normal tissue cells after microbeam irradiation compared to homogeneous irradiation at the same mean dose using 25 keV X-rays. The microplanar pattern was achieved with a tungsten slit array of 50 μm slit size and a spacing of 350 μm. Applying microbeams significantly increased cell survival for a mean dose above 2 Gy, which indicates fewer normal tissue complications. The observation of significantly less chromosome aberrations suggests a lower risk of second cancer development. Our findings provide valuable insight into the mechanisms of microbeam radiotherapy and prove its applicability at a compact synchrotron, which contributes to its future clinical translation.

Radiation biology is a highly interdisciplinary field at the interface of biology, physics, and medicine. It is characterized by rapid advances in biological and technical knowledge. The potential for using these advances to optimize medical care, radiation protection, and related fields can be exploited only with complementary activities to support the education of young academics. A small number of academic institutions have committed resources into radiation-related courses and curricula; however, few offer a comprehensive interdepartmental research and training program. At the Technical University of Munich (TUM), a full Master of Science (MSc) course in radiation biology has been established. This article describes the TUM MSc radiation biology program, discusses the scope of the field, the teaching goals, and the interdisciplinary curriculum. Detailed information on the full MSc program can be found continuously updated at www.radonc.med.tum.de/masterradiationbiology.

Background: Radiotherapy (RT) is an established treatment for patients with primary and recurrent prostate cancer. Herein, the effects of definitive and salvage RT on the composition of lymphocyte subpopulations were investigated in patients with prostate cancer to study potential immune effects., Patients and Methods: A total of 33 prostate cancer patients were treated with definitive (n = 10) or salvage RT (n = 23) after biochemical relapse. The absolute number of lymphocytes and the distribution of lymphocyte subpopulations were analyzed by multiparameter flow cytometry before RT, at the end of RT, and in the follow-up period., Results: Absolute lymphocyte counts decreased significantly after RT in both patient groups and a significant drop was observed in the percentage of B cells directly after RT from 10.1 ± 1.3 to 6.0 ± 0.7% in patients with definitive RT and from 9.2 ± 0.8 to 5.8 ± 0.7% in patients with salvage RT. In contrast, the percentages of T and natural killer (NK) cells remained unaltered directly after RT in both patient groups. However, 1 year after RT, the percentage of CD3 + T cells was significantly lower in patients with definitive and salvage RT. The percentage of regulatory T cells was slightly upregulated in primary prostate cancer patients after definitive RT, but not after salvage RT., Conclusion: Definitive and salvage RT exert similar effects on the composition of lymphocyte subpopulations in prostate cancer patients. Total lymphocyte counts are lower in both patient groups compared to healthy controls and further decreased after RT. B cells are more sensitive to definitive and salvage RT than T and NK cells.

Background: Sulforaphane (SFN), an herbal isothiocyanate enriched in cruciferous vegetables like broccoli and cauliflower, has gained popularity for its antitumor effects in cell lines such as pancreatic cancer. Antiproliferative as well as radiosensitizing properties were reported for head and neck cancer but little is known about its effects in pancreatic cancer cells in combination with irradiation (RT)., Methods: In four established pancreatic cancer cell lines we investigated clonogenic survival, analyzed cell cycle distribution and compared DNA damage via flow cytometry and western blot after treatment with SFN and RT., Results: Both SFN and RT show a strong and dose dependent survival reduction in clonogenic assays, an induction of a G2/M cell cycle arrest and an increase in γH2AX protein level indicating DNA damage. Effects were more pronounced in combined treatment and both cell cycle perturbation and DNA damage persisted for a longer period than after SFN or RT alone. Moreover, SFN induced a loss of DNA repair proteins Ku 70, Ku 80 and XRCC4., Conclusion: Our results suggest that combination of SFN and RT exerts a more distinct DNA damage and growth inhibition than each treatment alone. SFN seems to be a viable option to improve treatment efficacy of chemoradiation with hopefully higher rates of secondary resectability after neoadjuvant treatment for pancreatic cancer.

The first stereocontrolled Cu-catalyzed sequential 1,6/1,4-asymmetric conjugate addition (ACA) of C-metalated hard nucleophiles to cyclic dienones is reported. The use of DiPPAM (diphenylphosphinoazomethinylate) followed by a phosphoramidite as the stereoinducing ligands facilitated both high ee values for the 1,6-ACA and high de values for the 1,4-ACA reaction components, which thus gave enantioenriched 1,3-dialkylated moieties. The absolute configurations were determined by using vibrational circular dichroism (VCD) and optical rotatory dispersion (ORD) spectroscopy, in combination with DFT calculations and X-ray analysis. Interestingly, DFT calculations for the mechanism of enantioselective 1,6-addition by using an unprecedented Cu-Zn bimetallic catalytic system confirmed this attribution. Lastly, exploring intramolecular cyclization avenues for enantioenriched 1,3-dialkylated products provided access to the challenging drimane skeleton., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report here a unique example of an in situ generated aluminum initiator stabilized by a C 2 -symmetric salen ligand which shows a hitherto unknown high activity for the ROP of rac-lactide at room temperature. Using a simple and robust catalyst system, which is prepared from a salen complex and an onium salt, this convenient route employs readily available reagents that afford polylactide in good yields with narrow polydispersity indices, without the need for time-consuming and expensive processes that are typically required for catalyst preparation and purification. In line with the experimental evidence, DFT studies reveal that initiation and propagation proceed via an external alkoxide attack on the coordinated monomer.

The reliance of all cell types on the mitochondrial function for survival makes mitochondria an interesting target when trying to understand their role in the cellular response to ionizing radiation. By harnessing highly focused carbon ions and protons using microbeams, we have performed in situ live cell imaging of the targeted irradiation of individual mitochondria stained with Tetramethyl rhodamine ethyl ester (TMRE), a cationic fluorophore which accumulates electrophoretically in polarized mitochondria. Targeted irradiation with both carbon ions and protons down to beam spots of <1 μm induced a near instant loss of mitochondrial TMRE fluorescence signal in the targeted area. The loss of TMRE after targeted irradiation represents a radiation induced change in mitochondrial membrane potential. This is the first time such mitochondrial responses have been documented in situ after targeted microbeam irradiation. The methods developed and the results obtained have the ability to shed new light on not just mitochondria's response to radiation but to further elucidate a putative mechanism of radiation induced depolarization and mitochondrial response.

Background: This work investigates on putative cytotoxic effects in four different hepatocellular carcinoma (HCC) cell lines after irradiation with photons or carbon ions in combination with new targeted molecular therapy using either Temsirolimus (TEM) or Gemcitabine (GEM)., Methods and Materials: The HCC cell lines HepG2, Hep3B, HuH7, and PLC were cultured and irradiated with photons or carbon ions at the Heidelberg Ion Beam Therapy Center using the raster-scanning method. For combination experiments, cell lines were first treated with Temsirolimus or GEM before irradiation. Cytotoxicity was measured by a clonogenic survival assay. The evaluation of the experiments and the obtained survival curves were based on the concept of additivity defined by Steel and Peckham., Results: The results for the combination of carbon ions and both tested systemic substances TEM and GEM showed independent toxicities in all four cell lines. Supra-additive effects were observed in PLC cells for photon irradiation combined either with TEM or GEM and in HuH7 cells for the combination of photons with TEM., Conclusion: Addition of targeted therapy substances Temsirolimus and GEM to photon irradiation showed additive cytotoxicity in HCC cell lines, whereas independent toxicities where reached by the combination of carbon ions to these substances. It can be assumed that combining 12C with systemic substances only has independent effects because heavy ions cause direct damage because of their high-LET character resulting in complex and clustered double-strand breaks. Nonetheless, further investigations are warranted in order to determine whether addition of systemic therapy allows a reduction of radiation doses in combination therapy. This could possibly lead to better responses and tolerances in patients with HCC.

The relative biological effectiveness (RBE) based on the induction of dicentrics in any cell type is principally an important information for the increasing application of high-LET radiation in cancer therapy. Since the standard system of human lymphocytes for measuring dicentrics are not compatible with our microbeam irradiation setup where attaching cells are essential, we used human-hamster hybrid A L cells which do attach on foils and fulfil the special experimental requirement for microbeam irradiations. In this work, the dose-response of A L cells to photons of different energy, 70 and 200 kV X-rays and 60 Co γ-rays, is characterized and compared to human lymphocytes. The total number of induced dicentrics in A L cells is approximately one order of magnitude smaller. Despite the smaller α and β parameters of the measured linear-quadratic dose-response relationship, the α/β-ratio versus photon energy dependence is identical within the accuracy of measurement for A L cells and human lymphocytes. Thus, the influence of the reference radiation used for RBE determination is the same. For therapy relevant doses of 2 Gy ( 60 Co equivalent), the difference in RBE is around 20% only. These findings indicate that the biological effectiveness in A L cells can give important information for human cells, especially for studies where attaching cells are essential.

Preclinical mouse models that allow the evaluation of novel techniques in radiotherapy using charged particles like protons or heavier ions are discussed here. Focused beams of protons or carbon ions offer distinct physical characteristics that might contribute to an overall improved risk-benefit profile in radiotherapy. In the last decade, novel concepts in radiotherapy aimed to optimize the delivery of high radiation doses to the tumor while sparing the surrounding healthy tissue. 2D in vitro tumor cell cultures provide experimental models to determine the radiosensitivity of isolated cells to different radiation qualities. However, these cell culture models are not capable of mimicking the complexity and heterogeneity of 3-dimensionally growing clinical tumors with their individual tumor microenvironment. Therefore, small animal tumor models are urgently needed to study radiation responses after particle radiotherapy in vivo. Here we describe the recent developments and the accurate testing of novel techniques in particle radiotherapy using up-to-date preclinical animal models., (© 2018 S. Karger AG, Basel.)