ABSTRACT. A quantitative understanding of radiation effects essential in both radiation therapy and radiation protection. Various approaches of effect modelling attempt to describe and predict radiation effects upon exposure and may reflect mechanistic insights on various levels, e.g. on the conversion of DNA damage into cellular or tissue effects. In the presentation, general strategies of effect models will be reviewed in particular for models with a low number of free parameters, which largely ignore the complexity of biological processes by taking into account effectively the most essential processes. Here, two examples will be given: First, the Local Effect Model (LEM) is used to predict the relative biological effectiveness of high LET radiation. Its capabilities and limitations have been investigated using the Particle Irradiation Data Ensemble (PIDE) which is a data collection of in-vitro experiments after ion vs photon irradiation [1, 2]. To go beyond model tests against experimental data of one type, consistency of the model assumptions were challenged by applying the LEM concept to different endpoints with the same set of model parameters. As a second example, a model for the anti-tumor effects of the combination of radiation and immunotherapy is introduced [3]. The model reflects the radiation mediated immune activation upon the administration of immune checkpoint blockers and succeeds to describe tumor growth curves for a broad set of in-vivo data. It is used to predict suitable exposure conditions for establishing an abscopal effect, which is experimentally testable. In summary, parameter poor models can allow robust effect predictions and associated uncertainty estimates.

ABSTRACT. Purpose: Since the early years, particle therapy treatments have been associated with concerns for late toxicities, especially secondary cancer risk (SCR). Nowadays, this concern is related especially to patients for whom long-term survival is expected (e.g. breast cancer, paediatrics). We present a modelling analysis aiming at improving our understanding of the RBE for mutation induction (RBEM) for different particle species. Methods: We built a database collecting all available RBE data for mutation induction (in vitro HPRT mutation assay) from literature (105 entries, distributed among 4 cell lines and 14 particle species). Statistical and modelling analysis were applied to the data. The latter was performed by applying the microdosimetric kinetic model (MKM) to describe the mutagenesis in analogy to the lethal lesion induction. Exploiting the formal similarity between mutation induction and tumor induction, the new MKM formalism has been used in an updated version of the Schneider approach to evaluate the excess absolute risk (EAR) of secondary cancer in case of particle therapy. Using this new framework, a case of lymphoma has been simulated using the Topas Monte Carlo code, to evaluate the EAR following a treatment with protons. Results: A correlation analysis between RBE for survival (RBES) and RBEM reveals significant correlation between these two quantities. The correlation is stronger when looking at a subset of data based on e.g. cell line and particle species. We also show that the MKM can be successfully employed to describe RBEM, obtaining comparably good agreement with the experimental data. The EAR for the organs at risk has been also evaluated within the MKM-based framework, showing the impact of variable LET and the intra-fraction correlation between survival and tumor induction. Conclusions: We show that RBES and RBEM are strongly related. Together with the successful application of the MKM, in analogy to the RBES, RBEM and EAR can be readily implemented into TPS evaluations. This might contribute to a more accurate estimation of secondary cancer risk in particle therapy.

ABSTRACT. Carbon ion (C-ion) radiotherapy has physical and biological advantages over conventional radiotherapy due to high energy deposits at the end of their course (Bragg peak). Photon irradiation induces uniform production of reactive oxygen species (ROS) in cells while C-ions exhibit a huge ionization density in individual tracks causing a localized ROS production at the nanometric scale. Based on our experimental data and Monte-Carlo simulations, we proposed the paradigm of the “stealth-bomber” to conceptualize two opposing processes responsible for the biological superiority of C-ions over photons. The "bomber" effect encompasses most of the deleterious properties of C-ions on cancer cells at the molecular and cellular levels. It is triggered when the biological targets, such as DNA or organelles, are on the trajectories of C-ions. In this case, the ROS clustered in the tracks are responsible for: complex and irreparable DNA damage; increased levels of oxidized proteins; the absence of dependency on telomere length, and the absence of dependency on intracellular oxygen concentration for the induction of cell death. The consequence, at an equivalent physical dose of photons, is a higher cell killing, specifically on cancer stem cells, by a p53-independent and ceramide-dependent mechanism. The “stealth” effect symbolizes the property of C-ions to deceive the cellular defenses. Indeed, the absence of significant ROS production outside the C-ion tracks does not allow the achievement of a decisive ROS threshold necessary to activate survival pathways and defense mechanisms. This is objectified by: the decrease in the detection of DNA lesions and the activation of their repair; the non-activation of proliferative and invasive pathways; the absence of stabilization of the HIF-1α transcription factor and the non-activation of its numerous targets; the specific regulation of key effectors of the proteostasis network. Altogether, our results led us to propose this new paradigm, setting ROS spatial distribution at the nanometric scale as a highly relevant point, to explain the differential cellular responses to C-ion and X-ray irradiations. It also strongly suggests that hadrontherapy with C-ions will always display a much better efficacy relative to the most advanced conventional radiotherapy technology.

ABSTRACT. In the next future, due to development studies on deuterium-tritium fusion reactors as well as to the decommissioning of old nuclear power facilities, the tritium release in the environment is expected to increase. New impact mitigation strategies, combined to a better understanding of tritium impact on health and environment, are therefore needed. In this context, the recently-concluded European multidisciplinary project TRANSAT [1] (Transversal Action for Tritium, 2017-2022) has contributed to improve the knowledge on tritium management in fission and fusion facilities and on the impact of specific relevant tritiated products, such as micrometric steel and cement particles, on environment and human health.

In this framework, we aimed at investigating the possible biological damage induced by radiation emitted by such tritiated particles at the cellular and subcellular level. Modeling is essential for the reconstruction of dose levels associated to common toxicological indicators of contamination with radioactive particles, as particle concentration. We thus created a software replica of the setup used for in-vitro experiments, with the bronchial epithelium BEAS-2B cell line exposed to tritiated steel and cement particles. We characterized the radiation field associated to the particle presence in the proximity of cells, to estimate, in particular, the dose absorbed by cell nuclei, the main critical target for radiation action. The dosimetric reconstruction has allowed us to make predictions on the radiation-induced DNA damage, as a well-recognized indicator of the biological effectiveness of these tritiated products. Dosimetric results will be presented and discussed seeking for a better interpretation of the outcome of experimental in-vitro genotoxicity assays, to advance in informing dose-response curves and help in the challenge of building the bridge between radiobiological damage and risk for relevant specific exposure scenarios.

[1] https://transat-h2020.eu/ and Liger K, Grisolia C, Cristescu I, Moreno C, Malard V, Coombs D, et al. Overview of the TRANSAT (TRANSversal Actions for Tritium) project. Nucl Eng Des/Fusion 2018; 136:168–72.

ABSTRACT. Cells of different origin often differ in their response to radiation. Cells of the same type isolated from individuals also differ in radiation response but the underlying mechanisms are not known. Genetic factors are commonly thought to be a substantial contributor but the inheritance of variably responsive phenotypes among a population of healthy individuals does not follow a classical Mendelian inheritance pattern suggesting that it is not based on mutations or polymorphisms in single genes. Rather, a radiosensitive phenotype can be considered to be a multi-factorial, complex trait, that is based on the inheritance of an unknown number of low-penetrance risk alleles. Apart from the genetic component, the reaction of cells and individuals to radiation is influenced by environmental factors and by a third component - chance. Chance is understood as stochastic molecular variation that can influence on phenotypic variation. The interesting question is what is the impact of the three components on the response of a cell or an individual to radiation. We have investigated the response of peripheral blood lymphocytes of two donors to radiations of different qualities during 3 seasons of one year. At each season, 3 weekly replicates were carried out. The analysed endpoints were mRNA levels of selected genes and the frequency of chromosomal aberrations. The aim was to assess the interindividual and seasonal variability of response. Could a pattern of response be identified when mean values were calculated from all repeats? We also compared the intra-, and inter-cellular variability of radiation-induced 53BP1 foci in U2OS cells. Finally, we tested if patients who undergo radiotherapy two times for cancers of different locations develop similar levels of normal tissue toxicities that would reflect intrinsic sensitivity traits. The results of the experiments will be presented and discussed in the light of prospects of finding biomarkers of individual response to radiation.

ABSTRACT. Presently, carbon ions represent the ions with the optimal combination of physical and radiobiological features for achieving the best results in terms of local control for radioresistant and hypoxic tumours while sparing of the surrounding normal tissues. By the end of 2021, more than 40.000 patients have been treated with C-ions with very encouraging outcomes, according to the Particle therapy co-operative Group (PTCOG) and several new clinical facilities are also being built or are planned for construction worldwide (https://www.ptcog.ch/). Concurrently with the growth of the clinical activity, we are witnessing a significant rise in research studies that are focusing on a deeper understanding of the molecular mechanisms underlying cellular and tissue responses to the targeted and non-targeted effects of carbon ions in order to better exploit their radiobiological features and identify the tumors that can benefit from this treatment the most. In this talk, the current knowledge of the cellular and molecular mechanisms of action of carbon ions for the treatment of cancer will be compared to that of X-rays, with a focus on the newly emerging topics of heavy ion radiobiology such as signalling pathway activation, microenvironment response, and immunomodulatory properties. Finally, a brief digression on experimental models that are not conventional for radiobiology will be critically presented.

ABSTRACT. The effects of chronic irradiation of aquatic biota in water bodies within the Chernobyl exclusion zone (CEZ) during 1998-2021 were studied. The absorbed dose rate for hydrobionts from water bodies of the CEZ registered in a range from 0.2 to 430 μGy/h and in the reference lakes - up to 0.08 μGy/h. It is determined that the rate of chromosomal aberrations in the root meristem tissues of aquatic plants in the most contaminated lakes on average in 2-3 times, and in cells of the pond snail embryos in 4-6 times exceeding the spontaneous mutagenesis level, inherent to aquatic organisms. During the period of studies, a tendency to decrease of chromosomal aberration level in mollusks from all lakes of the exclusion zone was registered. The probabilistic prediction of the chromosomal aberration rate for gastropod snails in lakes of the CEZ have shown that spontaneous mutagenesis level (2.0-2.5%) can be reach in the most contaminated lakes in 2060-s-2070-s. Analysis of leukograms of fish peripheral blood showed the decrease of lymphocyte cells, as well as the increase in the number of granulocytic cells with increase of radiation dose rate. Along with changes in leukograms an increased level of morphological damages of erythrocytes (structural and proliferation abnormalities) was determined, which is generally for pray fish in 4-12 times and for predatory fish in 7-15 times higher than in fish from reference lakes. High number of erythrocytes with structural and proliferation abnormalities in blood of fish from lakes with high levels of radioactive contamination allows us to assume that the qualitative indexes of red cells in blood of fish are more sensitive to chronic radiation impact in comparison with the elements of white blood. A variety of forms of pathological changes in the structure of blood cells, mainly erythrocytes, may indicate low resistance of cytogenetic apparatus of fish in the face of considerable mutagenicity and genotoxicity of environment. In this situation the ionizing radiation causes damage to the lipid structures of biological membranes (e. g. lysosomes) and violation of their barrier functions that ensure compartmentalization in the cell. This leads to disruption of spatial isolation of enzymes to their substrates and release enzymes to further destruction of macromolecules and intracellular structures. As a result, there are changes not only in the cytoskeleton, but also in functioning of all the organelles in the cell.

ABSTRACT. Radiation therapy has made significant advancements in the past years and is considered as an indispensable tool in cancer treatment. Its main challenge is to destroy cancer cells without the depletion of healthy tissues. This is achieved by understanding the radioresistance mechanisms of cancer cells as well as normal stem cells upon ionizing radiation exposure. Stem cells possess the ability to regenerate themselves and differentiate into specialized cells. These two mechanisms are more or less regulated in order to limit abnormal expansion and lineage imbalance. Recently promising radiation therapy techniques such as hadrontherapy are increasing worldwide. A great concern is the late effects of these radiation qualities since accelerated particles produce complex DNA damage not only in the tumor but also touch the surrounding healthy tissue exposed to low dose levels. Furthermore, examining the biological plausibility of low-dose irradiation is an essential factor to follow up oxidative stress response that may impact proliferation and differentiation processes of normal stem cells. For this reason, we have carried out a study that compares different radiation qualities (X-ray, proton, and carbon ion) on adipose-derived stem cells (ADSC) in presence and absence of nuclear factor erythroid 2-related factor 2 (Nrf2), the cryoprotective controller against oxidative stress. Our results suggested an enhanced cell killing effect when these cells were subjected to carbon-ion irradiation in absence of Nrf2, and to a lesser degree when exposed to X-ray and proton.

We are now analyzing the stemness properties of the exposed cells before and after irradiation as well as their adipogenic and osteogenic abilities by FACS. This study conducts a mechanistic understanding of stem cell response to irradiation which leads to improving knowledge about the overall radiosensitivity of human stem cells.

ABSTRACT. PURPOSE: The increasing risk of acute large-scale radiological/nuclear exposures of population underlines the necessity of developing rapid and high-throughput biodosimetric tools for estimation of received dose and initial triage. As ionizing radiation triggers complex response on genome and proteome level, both were already reported as suitable indicators of radiation-induced damage in vitro or in animal models. Our goal is to identify and quantify radiation-responsive plasma proteins in TBI patients using mass spectrometry.

MATERIAL AND METHODS: Peripheral blood was taken before and 24 hours after TBI (2 x 2.0 Gy). Plasma samples of leukaemic patients (n=24) were immuno-depleted, reduced, alkylated, and digested. Healthy donors (n=15) of corresponding sex and age were sampled in parallel to reduce bias caused by oncological condition and temporal effects. Both “label-free” and iTRAQ relative quantification approaches were applied using RP-nanoLC-ESI-MS/MS system with Q-Exactive mass spectrometer (Thermo). Proteins were identified using Proteome Discoverer v.2.2 platform (Thermo). Subsequent analysis was carried out using Metascape freeware (http://metascape.org).

DISCUSSION: We acquired a list of plasmatic proteins with statistically significant up-regulation (ratio ≥ 1.2) or down-regulation (ratio ≤ 0.83) 24 hours after irradiation (p ≤ 0.01). We ruled out proteins deregulated in non-irradiated patients when compared to healthy donors as possibly associated with the disease and those changed in healthy donors within 24 hours as the timely unstable ones. Finally, we obtained a list of candidate proteins and assessed their biological function concerning radiation.

CONCLUSION: Finally, we obtained a list of candidate biomarkers and an overview of identified proteins will be presented. We assessed their radiobiological relevance and selected the top candidates for verification. As this is an ongoing project, the validation is still in the process. The human plasma proteome of oncological TBI patients represents an uncovered area, thus the obtained data might have future implications to biological dosimetry.

ABSTRACT. Radiation therapy damages tumors and surrounding normal tissue, probably in part through the recruitment of immune cells. Endothelial high-mannose N-glycans are, in particular, involved in monocyte-endothelium interactions. Trimmed by the class I -mannosidases, these structures are quite rare in normal conditions. Here, we show that the expression of the endothelial -mannosidase MAN1C1 protein decreases after irradiation. We modeled two crucial steps in monocyte recruitment after irradiation by developing in vitro real-time imaging models. Inhibition of MAN1C1 expression by siRNA gene silencing increases the abundance of high-mannose N-glycans and improves the adhesion rate of monocytes on endothelial cells in flow conditions. In contrast, inhibition of MAN1C1 expression decreases radiation-induced transendothelial migration of monocytes. Consistently, overexpression of MAN1C1 in endothelial cells using lentiviral vectors decreases monocyte adhesion and enhances transendothelial migration. Hence, we propose a role for endothelial MAN1C1 in the recruitment of monocytes through endothelial high-mannose N-glycan interactions, particularly in the adhesion step to the endothelium.

ABSTRACT. Unresectable locally advanced non-small cell lung cancer (NSCLC) is a frequent cancer with a prognosis that remains poor despite several treatments for radiotherapy, chemotherapy and immunotherapy. Mechanisms of resistance to treatments have been highlighted such as KRAS mutations. A new therapy type tyrosine kinase inhibitor targeting the KRAS G12C mutation has enabled efficacy in metastatic NSCLC and could have an interest in the treatment by radiotherapy locally advanced forms. However, the radiosensitizing effect of such molecules targeting KRAS remains unknown. KRAS resistance mechanisms could be related to hypoxia and the proliferation of tumor stem cells and irradiation by carbon ions and protons compared to photon irradiation might also have future interest in the treatment of these patients. In this study, we propose to use in vitro models of KRAS G12C mutated cell lines (vs. non-mutated). These cells are treated with a combination of KRAS G12C inhibitors and irradiations (X-rays, carbon ions and protons). Following a complete characterization of the cell lines and drug toxicity tests, the clonogenic survival of the cells is then analysed. Our preliminary results show a differential response of the cells, depending on the cell lines, irradiation doses and quality and drug combinations.

ABSTRACT. Cancer is intrinsically complex, comprising both heterogeneous cellular composition and extracellular matrix. In vitro cancer research models have been widely used in the past to model and study cancer. Although two-dimensional (2D) cell culture models have been the traditional hallmark of cancer research, they have many limitations, such as the disturbance of interactions between cellular and extracellular environments and changes in cell morphology, polarity, division mechanism, differentiation and cell motion. This implies that 2D tumor models are ineffective to accurately recapitulate complex aspects of tumor cells growth, as well as their drug and radiation responses. Over the past decade there has been significant uptake of three-dimensional (3D) in vitro models by cancer researchers, highlighting a complementary model for studies of radiation effects on tumors, especially in conjunction with chemotherapy. In this work we present two alternative 3D in vitro models obtained by the use of: 1) 3D bioprinting techniques and 2) VITVO cell culture bioreactors containing a fiber-based matrix, to be used for radiobiological purposes. Preliminary results show clearly different spatial cell organization in the 3D compared to 2D environment, leading to different responses of medulloblastoma cells to radiations, also in terms of cell survival. Our findings can shed new light on understanding the features of the 3D cell model and its application in basic research into clinical radiotherapy and medicine. Furthermore, positioning itself halfway between 2D cell culture and animal models, 3D systems could affect modeling of radiation-tissue interactions, thus opening up new possibilities in the study of radiation response mechanisms of healthy and tumor tissues.

ABSTRACT. Increasing the dose within the tumour mass and simultaneously reducing the dose to healthy tissues is still a major challenge in radiotherapy, although several strategies have been proposed. At present, nanotechnologies play an important role in biomedicine, and different types of nanoparticles have received significant attention in this field, such as gold nanorods (AuNRs) [1]. Low energy electrons (Auger electrons) are produced by the interaction of photons with the gold in the NRs. These electrons are similar to those emitted by the decay of 99mTc, a radioactive nuclide widely used for diagnostic purposes in nuclear medicine. At the cellular level, these short-range charged particles lead to a dense deposition of ionising energy associated with increased radiobiological efficiency. Auger and internal conversion (IC) electron emitters appropriately targeted to the DNA of tumour cells may therefore represent an interesting new radiotherapy system: 99mTc could indeed be used as a theragnostic radiopharmaceutical once loaded on AuNRs and delivered to the tumour site. This work aims to be a proof of concept to evaluate in vitro the toxicity of AuNRs in T98G cells and their efficacy in inducing radio-induced damage at cellular and/or molecular level following photons irradiation, thus mimicking the labeled 99mTc used in clinical settings. Preliminary data will be presented on the chemical characterization of AuNRs (with aspect ratio 3.2 and Surface Plasmon Resonance bands at 520 and 680 nm) and the loading of 99mTc on their surface. Spectroscopic characterization, such as Uv-vis, Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS), will be performed to investigate the drug-AuNRs interaction. Finally, preliminary radiobiological data on cell killing and/or chromosomal damage will be shown.

[1] Appl. Sci. 2019, 9(16), 3232; https://doi.org/10.3390/app9163232

ABSTRACT. Heavy ion particle therapy is a rapidly growing and potentially the most effective and precise radiotherapy technique. However, range uncertainties jeopardize the benefits of the sharp Bragg peak and force to use wide margins extending in the normal tissue. The use of radioactive ion beams (RIBs) for simultaneous treatment and online range verification using positron emission tomography (PET) could help to overcome this limitation, showing an increased signal/noise ratio, a reduced biological washout (thanks to the shorter measurement time required to collect the signal) and alignment of the activity peak with the Bragg peak compared to PET imaging of fragments produced by primary stable ion beams1. In this context, the BARB (Biomedical Applications of Radioactive ion Beams) project was initiated at GSI with the goal to assess the technical feasibility and investigate possible advantages of RIBs in preclinical studies2,3. During the first year of experiments within this project, radioactive Carbon and Oxygen beams (10,11C and 15O) were produced by isotopic separation with the fragment separator (FRS) and transported to the medical vault of GSI. Thanks to the upgrade of the SIS-18 in the FAIR in Darmstadt, it is possible to achieve RIB intensities sufficient to treat a small animal tumor.

ABSTRACT. Conventional treatments for cancer patients are based on two main approaches: radiotherapy and chemotherapy. Radiotherapy makes use of ionizing radiation, inducing damage to the DNA of target tissues, and it is often used as an adjuvant to surgical resections. Chemotherapy employs drugs that block specific targets involved in proliferative activity to inhibit the cell cycle progression. The combined administration of such treatments has proven to be more effective than treatments with individual therapies. More generally, oncological research has recently seen a very rapid progress towards the so-called personalized medicine approaches, which allows patients allocation and stratification into treatment schemes based on the differential analysis and subtyping of tumors, including consideration of tumour specific resistance to treatments and interactions with the immune system.

In the context of investigating radiation resistance, such as that observed in vitro in the Caco-2 cell line (a cell line derived from colorectal adenocarcinoma) (1,2), we wanted to test a possible synergistic effect between ionizing radiation (0.5-5.0 Gy from a conventional radiotherapy X-ray accelerator) and shortage or absence of metabolites induced by Asparaginase, a bacterial amidohydrolase used for many decades in the treatment of Leukemia (3). Preliminary data from our Radiation Biophysics and Radiobiology Lab obtained with different radiobiological techniques, including cell clonogenic inactivation assay and cell-cycle characterization, show that the combination of X-ray exposure and deprivation of specific nutrients (asparagine and glutamine) seem to exacerbate the cell stress response, mostly at low doses, and that the two stressors have a synergistic action.

ABSTRACT. Boron- Neutron Capture Therapy (BNCT) is a type of radiotherapy which is based on the ability of non-radioactive boron-10 isotope to capture thermal neutrons. In the experiment, triple-negative types (MDA-MB-231, MDA-MB-468) and luminal type (MCF-7) of breast cancer cells were used. The main aim of the research was to analyze the influence of the breast cancer cells sensitization by gold nanoparticles on their cell cycle distribution and their survivability rate. MDA-MB-231, MDA-MB-468 and MCF-7 were exposed on average to 2,5 Gy of neutron radiation from MARIA research reactor located in National Center for Nuclear Research in Świerk in Poland. Before irradiating cells, solutions of boron-10 compound (BPA) and gold nanoparticles were added and nextly incubated. Cell cycle distribution was measured by flow cytometry method (BD LSR II) within 24h. In the case of the clonogenic assay, cells were incubated for 14 days and later colonies were counted. Preliminary results show that there is difference in the cell cycle distribution and the survivability rate between cells after using BNCT treatment method with sensitization by gold nanoparticles and without usage of gold nanoparticles. The obtained results will be presented during the conference.

ABSTRACT. Microbeam Radiation Therapy (MRT) is an innovative preclinical concept in radiotherapy that collimates X-ray radiation in micrometer-wide, planar beams. Previous research has shown that MRT substantially spares normal tissue, while being equally effective in tumor ablation. In order to validate doses measured with radiochromic film dosimetry, biological dosimetry using the cytokineses blocked micronuclei (CBMN) assay was applied on a cellular level.

CBMN assay was performed using CHO cells after homogeneous irradiation to establish a dose response curve, and afterwards with either sham or microbeam. At least 1000 binucleated cells were analyzed with Metafer (Metasystems, Germany). Microbeam radiation was performed at the XenX irradiation device (XStrahl, UK), equipped with a special microbeam collimator. Planar microbeams with a peak-width of 50 μm and a center-to-center distance of 400 μm were used.

The dose-response curve was fitted with the linear equation (y=0.44x + 0.13). Irradiations with a physically calculated peak dose of 2 Gy and a valley dose of 0.05 Gy resulted in 2.05 +/- 0.12 Gy and 0.02 +/- 0.05 Gy respectively using biological dosimetry. However, after irradiations with a calculated peak dose of 82 Gy and a valley dose of 2 Gy, the micronuclei which were counted in the valleys equaled to 2.29 +/- 0.02 Gy.

This is the first study determining precisely the absorbed doses in the peak and valley regions of MRT by film and biological dosimetry. Moreover, the slightly higher measured doses than the physically planned doses in the valleys indicate effects on a cellular level, which could be due to bystander effects and/or enhanced cell migration.

ABSTRACT. Introduction: Radiotherapy, as one of the most important prostate cancer treatment modalities can modify systemic immune responses but little is known on how long immune dysfunction persists in cancer survivors. Our aim was to perform a detailed analysis of the systemic immune status of prostate cancer patients treated with various radiotherapy protocols. Materials and methods: Blood samples were collected from 21 patients treated with low-dose rate (LDR) and 18 patients treated with high-dose rate (HDR) brachytherapy before and at 5 time points after therapy. Cellular and soluble changes were analysed in peripheral blood. Results: In LDR patients before therapy both the adaptive (decreased CD4+ effector T cells) and the innate immune system (decreased NK cell pools) were changed. While the innate immune response recovered as the tumour was cured, a mild long-term deficit in the adaptive immune response persisted even 3 years later. Similar, but stronger changes were detected in HDR patients. Before therapy the level of B cells, memory T cells decreased, senescent T cells level and degranulating NK cell levels strongly increased compared to control group, and these alterations persisted up to 36 months. Levels of PDGF-AA, ENA-78 and RANTES increased in both patient groups before therapy, however they did not normalize in HDR patients potentially indicating an increased predisposition for the development of long-lasting side effects such as radiation-induced late fibrosis. Conclusion: Significant differences were noted in systemic immune parameters of both prostate cancer patient groups highlighting the importance of radiotherapy in modulating systemic immune responses. Funding: This work was supported by a Hungarian research grant of the National Research, Development, and Innovation Office (NKFI-124879) and by the H2020 Framework Program (grant agreement number 899995, EURAMED rocc-n-roll).

ABSTRACT. Background & aim Several human syndromes with a genetic defect in one of the DNA double strand break (DSB) recognition and repair proteins have been described. These patients are defined by their immunological defects, cancer susceptibility, neurological abnormalities and sensitivity to ionizing radiation (IR). Malfunction of the patient’s immune system may be the most overt clinical feature and initially present with a primary immunodeficiency disease (PID). As DNA damaging agents are often applied in diagnostic and therapeutic procedures, identification of radiosensitive individuals in this patient population is essential to optimize their clinical care. As an alternative for the well-established lymphocyte-based assays, which are not feasible for certain PID patients, reliable fibroblast-based radiosensitivity (RS) analysis is proposed in this study.

Materials & methods Fibroblasts derived from PID patients with a confirmed or suspected DNA repair defect (mutations in ATM, Artemis, XLF, LIGIV, NBS1, RAG1/2) were irradiated with X-rays (0,5 and 1 Gy) in the G0 phase of the cell cycle. RS was assessed with the γ-H2AX foci test and an optimized cytokinesis-block micronucleus (MN) assay. Using fibroblasts from healthy individuals, the cut-off value for RS was determined.

Results & conclusion Patients with an Artemis and RAG1/2 mutation could respectively be identified as radiosensitive and not radiosensitive with both the γ-H2AX foci and G0 MN assay. The MN test was not feasible for XLF and LIG4 mutated fibroblasts, but their high γ-H2AX foci levels post-irradiation clearly indicated a radiosensitive phenotype. For these patients, the observed in vitro RS correlated with the expected clinical response to IR. However, ATM defective fibroblasts, known for their extreme RS, show high MN yields following irradiation, while these cells were not considered radiosensitive with the γ-H2AX foci assay. Also for NBS1 mutated cells, residual foci levels did not reach higher than the RS cut-off value. Although limitations in the use of a single RS assay were observed, the combination of the fibroblast MN and γ-H2AX foci test clearly improved the RS assessment for PID patients.

ABSTRACT. Cellular response to ionizing radiation-induced DNA damage may vary depending on genetic and environmental factors. The interesting question is how far environmental factors influence the radiation response of cells in culture. To investigate whether there is a difference between intra- and intercellular variation in sensitivity to radiation-induced DNA damage we compared frequencies of 53BP1 foci in nuclei of binucleated cells and in nuclei of neighboring cells. Frequencies of foci in micronuclei and mononucleated cells were also assessed, and nucleus areas were examined to see if the density of foci per unit nucleus/micronucleus area is different in nuclei in mononucleates vs binucleates and in micronuclei. Experiment was carried out with U2OS cells which were stably transfected with a plasmid coding for 53BP1-GFP. Cells were seeded on glass coverslips, placed in Petri dishes, and exposed to 2 Gy of gamma radiation. Cytochalasin B was added immediately after irradiation. After 24 h the cells were irradiated again with 2 Gy of gamma radiation. Kinetics of foci formation was analyzed after 30, 60, 120 and 180 minutes of repair time. Foci were analyzed using a confocal microscope and scored manually on images. The analysis has not been completed at the time of abstract submission. The preliminary results show that the intercellular variability in focus frequency is significantly higher than the intracellular variability suggesting that environmental factors have an impact on the response of cells in cell culture. A difference in focus frequency was also observed between nuclei of mononucleates and binucleates.

ABSTRACT. One of the limitations of carbon ion therapy (CIT) is the range uncertainty, which is usually mitigated by adding wider margins to the CTV to ensure its dose coverage. While PET imaging is the beam range verification method with most clinical applications in CIT, for 12C ion beams its applicability remains limited by the low signal-to-noise ratio and the shift between the dose and activity peaks. Instead, direct application of β+-emitting radioactive beams (RIBs), such as 11C, for treatment could increase the signal magnitude and improve its correlation with the dose distribution, allowing to reduce the tumor margins and, consequently, the dose to surrounding healthy tissues. The experimental campaign of the BARB project1, ongoing at GSI, aims at the full physical and radiobiological characterization of RIBs for therapy, with the final goal of treating the tumor in a small animal. This study aims to estimate the potential clinical benefits of tumor margin reduction granted by RIB treatments. We have developed a FLUKA beam model for radioactive 11C ions to be used within the TRiP98 treatment planning system and established the workflow to predict the resulting activity maps. We demonstrate that in 11C plans, the activity fall-off strictly follows the dose fall-off and could be theoretically used to trace the sub-millimeter dose distribution shifts, allowing to practically eliminate the range margins. Following that, we compared the robustly optimized 11C and 12C treatment plans for head-and-neck and liver tumors. Reduced margins in 11C plans lead to a substantial decrease of dose to the bystander critical structures. The results of the NTCP evaluation, additionally performed to estimate the toxicity for critical organs in the target proximity, suggest a better toxicity profile compared to 12C beams in selected patients. Work supported by ERC Advanced Grant 2020 n. 883425 (BARB, Biomedical Applications of Radioactive ion Beams) to Marco Durante.

1 Boscolo D, et al. Radioactive Beams for Image-Guided Particle Therapy: The BARB Experiment at GSI. Front. Oncol. 11:737050. doi: 10.3389/fonc.2021.737050

ABSTRACT. The cellular response to ionising radiation, either naturally occurring or otherwise, involves an intricate and coordinated chain of events. These events encompass activation of DNA damage response and DNA repair pathways via post-translational modification, regulation of transcription and translation, defining the health relevant outcomes such as apoptosis, genomic instability or proliferation. The precise nature and interactions within the cellular response to ionising radiation largely depend on the damage incurred. Although the biological response and effects of high doses of ionising radiation are well documented, less is known about responses to low-dose exposure. Given the potential implications for human health, we wished to investigate the regulation and coordination of transcriptional and translational events and how they relate to DNA double-strand break repair after exposure to low-dose ionising radiation using biologically relevant in vitro models, namely human-derived fibroblast cell lines. Following a combination of molecular and bioinformatic lines of interrogation including RNA-seq, mRNA stability analysis, ribopuromycylation and γH2AX foci analysis, we present observed changes as a function of time in coordinated transcriptional, post-transcriptional, translational and DNA double-strand break repair pathway responses to low doses (20 mGy and 100 mGy) of γ-rays. Taken together, these data help to decipher early molecular responses to low-dose radiation within the context of delayed health related outcomes, such as genomic instability, and represent the first steps to building an integrated low-dose response model.

ABSTRACT. Despite the improvements in cancer treatment over the past decades, tumor recurrence and metastases are still the main concern for the therapy's success1. Tumors are composed of a heterogeneous population of cells. Besides those cells, Cancer Stem Cells (CSCs) are the most aggressive and resistant subpopulation2. Circulating Cancer Stem Cells are particularly resistant to radiation and present specific cell surface markers needed to resist in the bloodstream3. An increased metastasis formation ability characterizes these cells. These cells are challenging to identify, and are present in a few number in the bloodstream. The possibility of culturing them in vitro and characterizing them would significantly increase our knowledge about the mechanisms responsible for metastasis formation. An essential role in forming these cells is due to the tumor microenvironment and, in particular, hypoxia. In this study, we select cells with CTCs (Circulating Tumor Cells)-like phenotypes for further characterization using stressors such as hypoxia and radiation. We cultivate the cells in hypoxia for one week (acute hypoxia) and two weeks (chronic hypoxia), while irradiation is performed with a target dose of 4 Gy of X-rays. Our results evidence a subpopulation of cells with an increased sphere formation capacity and migration under hypoxia and radiation. Further, WB analysis shows an increase in CD133 expression, a cancer stem cell marker, after treatment. This study will shed light on the mechanisms responsible for the CTCs formation.

ABSTRACT. The relationship between radiation dose, dose rate and cancer risk is uncertain. Accordingly, the justification for a dose and dose rate effectiveness factor (DDREF) to adjust cancer risk estimates at low doses and low dose rates (LDLDR) remains controversial. This study aims at investigating short- and long-term radiation-induced biological effects of low doses at different low dose rates as compared to a single acute dose rate of 137Cs gamma radiation on AHH-1 lymphoblasts. Cells exposed to 0, 25, 50 or 100 mGy delivered either chronically at 1.6, 8 or 12 mGy/h or acutely at 0.35 Gy/min are monitored over 30 days for molecular, cellular and cytogenetic endpoints. Short-term effects to be analysed include cell viability at 1-, 3-, and 6-days post-exposure by the MTT assay, and cell reproductive death by the colony formation assay. Additionally, cell growth is continuously followed up, and cell pellets, collected on selected days, will become available for transcriptional and post-transcriptional analyses to investigate immediate and long-term changes at these levels by RNA sequencing and/or quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot, respectively. Finally, stable chromosomal aberration analyses will be performed by fluorescence in situ hybridization (FISH) 30 days after exposure to examine the potential detrimental effects of low doses and dose rates on AHH-1 cells. Analyses of the first two experiments are ongoing. Preliminary data do not show an impaired cell growth of irradiated cells as compared to control up to day 19, but there is a trend in the first available replicate for a reduced cell viability of exposed cells relative to control at 3- and 7- days post-exposure. Our study will contribute to defining the shape of the dose response curve after low doses delivered at different dose rates, providing insight on whether the experimental data supports a DDREF for the analysed endpoints and conditions.

ABSTRACT. Cells exposed to both scattered low linear energy transfer (LET) and densely ionising high LET radiation delivered at the same time react more strongly than expected based on additivity. Here we studied the relationship between DNA double strand break (DSB) location inside the nucleus and chromatin structure. High-resolution transmission electron microscopy (TEM) was used to assay MDA-MB-231 cells 30 min after 5 Gy of gamma and/or alpha irradiation. DSB marker γH2AX immunolabelling was detected using nanosized gold beads. Additionally, cellular response to single (1 x 1.5 Gy) versus fractionated dose delivery (5 x 0.3 Gy) to low and/or high LET radiation was assessed in two cancer cell lines (MDA-MB-231 and U2OS). The highest total number of gold beads as well as foci were found in cells irradiated with alpha radiation just prior to gamma radiation (called mixed beam, 50% dose of each), followed by alpha, then gamma radiation. DSB induced by mixed beams tended to be surrounded by open chromatin (lighter regions in TEM), yet foci containing the highest number of beads, i.e. larger foci representing complex damage, remained in the heterochromatic areas. The γH2AX focus area was also greater in mixed beam-treated cells when analysed by immunofluorescence. The strongest reduction in cell viability and colony formation was demonstrated in MDA-MB-231 and U2OS cells after mixed beams, in comparison to each radiation quality delivered separately, after fractionated exposure. This may partially be explained by a delay in recompaction of chromatin in MDA-MB-231 cells, where the heterochromatin marker H3K9me3 was low after fractionated mixed beam as well as alpha radiation exposure. In conclusion, we provide information on induction and location of damage with higher complexity when cells are irradiated with a mixed field. The stronger cell kill induced by fractionated exposure to mixed beams in two cancer cell lines indicates a therapeutic potential of combined high and low LET irradiation.

ABSTRACT. Around 400 000 children and adolescents develop cancer each year worldwide, where Hodgkin lymphoma (HL) and non-Hodgkin lymphoma represent around 10% of all cancer cases 1,2. The treatment regimen of HL consists of chemotherapy and radiotherapy. With this, 10 years overall survival is achieved in over 90% of patients. However, one severe complication is represented by the late toxic effects of the therapy, where radiotherapy is considered as the major contributing factor. There is an increasing occurrence of therapy-related second cancers (SCs), where breast cancer is the most common type among HL childhood survivors3–7. Even though the late consequences of radiation exposure are known, predisposing factors and adverse molecular pathways involved in development of SCs remain unclear. Recent advances in genomics bring new approaches for finding biomarkers of susceptibility to SC. Genome-wide association studies of childhood HL survivors revealed the single nucleotide polymorphism located on chromosome 6q21 (gene PRDM1) as significantly associated with elevated risk of SC development. The presence of this risk allele resulted in lower basal and radiation-induced expression of PRDM18. The aim of this study is to investigate the role of the gene PRDM1 in the radiation response of healthy epithelial breast cells. We will present preliminary data from knockdown of PRDM1 in MCF10A cells, where we see small, but consistent differences in response at the level of cell cycle arrest and apoptosis genes, cell viability and PRDM1 itself.

1. World Health Organization, 2021 [Accessed 2022 April 08]; Available from: https://apps.who.int/iris/handle/10665/347370 2. Bloxham, N. and Burke, G.A.A., Paediatr Child Health, 2021. 31: p. 410-414 3. Buglione, M. et al., Crit Rev Oncol/Hematol, 2021. 167: 103437 4. Wei, C., Stevens, M. and Crowne, E., Curr Opin Endocr Metab Res, 2020. 14: 112-116 5. Schaapveld, M. et al., NEJM, 2015. 373: 2499-2511 6. Bakkach, J. et al., Crit Rev Oncol/Hematol, 2021. 157: 103175 7. Toltz, A. et al., J Appl Clin Med Phys, 2015. 16: 167-178 8. Best, T. et al., Nat Med, 2011. 17: 941-943

ABSTRACT. Glioblastoma multiforme is the most common malignant brain tumor with a very poor prognosis. High infiltration rates, uncontrolled cell growth, and the strong ability to develop therapy resistance are components of the aggressive nature of this type of cancer. Despite multimodal treatment, the tumor often recurs in the vicinity of 1-2 cm to the primary tumor. In this study, the migration behavior of U87 glioblastoma after low- and high-LET irradiation is analyzed to figure out whether the migration is enhanced or also influenced in other ways by radiation exposure. For the migration assay, the cells were seeded in Ibidi Culture-Inserts 2 Well to generate a cell-free gap of about 500 µm between two cell populations. Targeted irradiation was performed with 55 MeV carbon ions and 20 MeV protons at the ion microprobe SNAKE (superconducting nanoprobe for nuclear physics experiments) located at the 14 MV tandem accelerator in Garching (GER). The closure of the gap after irradiation was observed by phase-contrast microscopy (10x, PH1) under live-cell conditions. There are differences in the migration behavior of cells regarding their velocity and directness visible. Cells irradiated with high-LET carbons tend to be faster, but have lost all orientation, therefore the gap is closing the slowest. Irradiation with low-LET protons cause also a loss of orientation but has nearly no effect on the velocity of the cells. When only one cell population on one side of the gap was irradiated, the migration behavior changes again. Here, the cells are better oriented regardless of the type of irradiation. However, the one-sided proton irradiation leads to a deceleration of the cells. Our results show that with different irradiation conditions the migration behavior changes. Especially, the behavior changes when only a part of the cells is irradiated. These findings indicate that the close presence of non-irradiated cells has a strong effect on the migration behavior of the whole population.

ABSTRACT. It is well known that radiotherapy of head and neck (H&N) cancer causes both early and late effects that may lead to unplanned interruptions in treatment and a reduced quality of life1. Previous animal studies have used treatment regimens and fields that are not clinically compatible. The aim of this study was to establish a preclinical model with relevant endpoints, optimized radiation field and delivery setup to study radiation-induced normal tissue responses in H&N cancer. C57BL/6J mice were irradiated with X-rays to total doses ranging from 30 to 85 Gy in 10 fractions over 5 days. The radiation field in the H&N area covered the skin of the neck including lip, oral cavity and salivary glands and was controlled by an X-ray imaging system. The maximal follow-up time was 100 days post irradiation (p.i.). Early radiation-induced effects were monitored by macroscopic examinations of the oral cavity. Structural changes were examined through histopathological analysis of the lip, oral mucosa tissues and salivary glands. Blood and saliva sampling was performed at baseline and p.i. X-ray irradiation with total doses above 30 Gy induced dose-dependent radiation dermatitis of the lower lip, while oral mucositis was observed for doses above 75 Gy. The peak of acute effects was observed around 15-20 days p.i for all doses, while the severity level and time of first appearance demonstrated a dose dependency. Histopathological examinations showed radiation damages in the tongue, lip and parotid glands. Significant reduction of saliva volume was observed in mice exposed to 75 Gy. The optimal dose for the current model was found to be 75 Gy, while 85 Gy was not tolerable for this mouse strain. A preclinical model to study radiation-induced normal tissue changes in the H&N area was established. The model allows investigating several clinically relevant tissue responses simultaneously, with the opportunity to study systemic effects through e.g. biochemical analysis of body fluids.

1 Sroussi, H.Y., et al., Common oral complications of head and neck cancer radiation therapy: mucositis, infections, saliva change, fibrosis, sensory dysfunctions, dental caries, periodontal disease, and osteoradionecrosis. Cancer Med, 2017. 6(12): p. 2918-2931

ABSTRACT. The lung and prostate cancers are the most common men's cancers, which are treated in clinical practice with different methods, such as: surgery, radiotherapy, chemotherapy, immunotherapy [1]. However, new methods of cancer treatment are still being investigated therefore the interest in exosomes studies is increasing in the last few years. Exosomes are spherical membrane nanovesicles with a diameter of 30 to 150 nm. Exosomes are the only extracellular vesicles formed by exocytosis [2]. They are released by most eukaryotic cells, both healthy and cancerous. Due to the fact that exosomes are present in human body fluids such as urine, blood or saliva, they can be used for noninvasive diagnostic tests. Exosomes play an important role in tumor immune response, metastasis, angiogenesis, and survival [3]. The studies on exosomes isolated from cells exposed to photon radiation used commonly in conventional radiotherapy show the influence of this radiation quality on exosome release and its features. There is no research done on densely ionizing particles such as protons and alpha radiation, thus we have evaluated the cellular response of human prostate cancer cells exposed to 0, 2, and 6 Gy of alpha radiation emitted from the Am-241 source. The irradiated PC3 and DU145 cell lines characterized by different radiosensitivity were studied with apoptosis, lactate dehydrogenase (LDH), and interleukin-6 (IL-6) assays. Additionally, the corresponding concentration and size of isolated exosomes were measured with nanoparticle tracking analysis (NTA). We found that exposure to ionizing radiation resulted in gross changes in viability and cell damage. There were increased amounts of apoptotic or necrotic cells as a function of doses. We demonstrated that irradiated PC3 cells secrete higher quantities of exosomes compared to DU145 cells. We also discussed the diameter of isolated exosomes and no statistically significant differences were found between control and irradiated cells.

ABSTRACT. The reaction of tissue to irradiation is based on the radiation effects to single cells. The most notable reactions of cells to irradiation are cell cycle arrest, proliferation and cell death. Here, we present a new approach to investigate these biological endpoints in one experiment: The use of long-term live-cell phase-contrast videos analyzed by a deep-learning-based algorithm called CeCILE (Cell Classification and In-vitro Lifecycle Evaluation). With this method, we can observe and analyze the behavior and the health conditions of single cells over several days after treatment. After irradiation up to six sample dishes containing the cells can be mounted in parallel on a microscope inside a top-stage incubator and are imaged for up to 5 days. The created videos can then be analyzed by CeCILE, which is based on a faster RCNN-based algorithm for object detection and is trained on a hand-labelled dataset of microscopic videos. In these videos all cells were assigned to one of four classes, which defines the cells’ state in the cell cycle. Then, a tracking algorithm assigns an individual ID to every cell in the video. The tracking is conducted employing hidden Markov models to simulate the cell movements and states and an embedded recursive Bayesian filter to predict their behaviors via inference. It additionally provides an improved tracking with stable cell identification in complex scenes as well as the possibility to predict future states and fill previous gaps in a trajectory caused by detection failures. In conclusion, we are able to investigate the behavior of irradiated cells in one simple experiment. The first version of of CeCILE was able to achieve similar results compared to state-of-the-art assays. The upgrade with the Markov-model-based tracking allows to go further by inspecting the behavior of every single cell by evaluating the cell-cycle, the lineages and the circumstances of cell death.

ABSTRACT. Objectives: Translational cancer radiotherapy main goal is to deliver high doses at the tumor site, in order to inhibit the growth of resistant cancer cells, and simultaneous reduction of adverse effects in surrounding healthy tissues. For this purpose, targeted nanoparticle therapies have been proposed as a solution. Here, we propose a method based on iron oxide nanoparticles (IONP) for the intracellular delivery of doxorubicin in order to enhance the cytotoxic effects of ionizing radiation. Materials and methods: Iron oxide nanoparticles functionalized with polyethylene glycol were synthesized in order to be used as drug delivery systems for doxorubicin. The physico-chemical characterization of the nano-systems was done using relevant methods. The biological effects of the IONP were assessed on both 2D and 3D cell cultures of human cervical adenocarcinoma cells, squamous cell carcinoma and normal keratinocytes. IONP uptake and retention was assessed through optical microscopy, while clonogenic survival was used to measure the radio-sensitization effect of the nanoparticles at different doses X-Rays (150 kV) in both 2D and 3D cell models. Data was presented as mean ± SEM. Results: Efficient internalization of IONP occurred in cancer cells, with the nanoparticles accumulating in the perinuclear area. In 2D cell cultures, IONPDOX enhanced the cytotoxicity of 150 kV X-rays in 2D HeLa cell cultures with a DMFSF= 0.1= 1.29 ± 0.02. Efficient penetration of the IONP was obtained after 48h of exposure in 3D spheroids and exposure to 150 kV lead to a DMFSF0.1=1.07 ± 0.07 for HeLa cells. Conclusions: The IONP are good candidates for the controlled delivery of DOX to enhance the cytotoxic effects of ionizing radiation. Acknowledgement: This work was supported by the Austrian Agency for International Cooperation in Education and Research OeAD grant No. ICM-2018-10056, by Romanian Ministry of Education and Research grants no. 543 PED/2020 and Norway-Romania Grants: RO-NO-2019-0510, CTR 41/2020.

ABSTRACT. Introduction: In radiology, low X-ray energies (<120 keV) are used to obtain an optimal image while in radiotherapy, higher X-ray energies (MeV) are being used to eradicate tumor tissue. Within the energy range of 0.1- 6 MeV, the radiobiological effectiveness (RBE) has been stated as equal to 1. However, the energy deposition of X-rays shows differences in function of their energy spectrum, which might lead to changes in biological responses. Therefore, in this study we compare the DNA damage response (DDR) in peripheral blood lymphocytes (PBLs) exposed to different X-ray beam qualities and quantities. Methods: The DDR was evaluated by the γ-H2AX foci assay, the cytokinesis-block micronucleus assay and a SYTOX-based cell death assay in peripheral blood lymphocytes exposed to X-rays. Cells were irradiated in T25 flasks with a 220 kV X-ray research cabinet (SARRP, X-Strahl) or a 6 MV X-ray Linear accelerator (Elekta Synergy). Three main physical parameters were investigated: beam quality (V), mean photon energy (eV) and dose-rate (Gy/min). The addition of Cu filtration influenced the mean photon energy at the SARRP while dose-rates were varied by adjusting tube current for 220 kV X-rays (0,33-3 Gy/min) and by adjusting water-phantom depth in the 6 MV set-up (3-6 Gy/min). Results: The RBE of 220 kV X-rays compared to 6 MV X-rays was higher than 1. When an identical beam quality was filtered to increase the mean photon energy, the RBE decreased. Within the tested dose rate ranges no specific effects were observed. Conclusion: The DDR is influenced by the beam quality and mean photon energies. This study demonstrates that it is crucial to consider and report these physical parameters in radiobiological experiments.

ABSTRACT. Introduction The formation and decay of γH2AX foci is measured to assess the response of cells to ionizing radiation. Foci can be measured microscopically or with the help of flow cytometry (FACS). The aim of the study was to compare the kinetics of foci formation and decay using both methods in U2OS cells and peripheral blood lymphocytes (PBL). Foci in U2OS cells were analysed after 2 Gy of gamma radiation at 0, 15, 30, 60, 120, 180 min and 24 h of repair time. In PBL foci were analysed after 2 Gy and 60, 120, 180 min and 24 h of repair time. Results and conclusion The results demonstrate that the kinetics of foci formation and decay in U2OS cells and PBL counted manually are more expressive and dynamic than results obtained by FACS. Fluorescence intensities measured by FACS are more spread-out than foci frequencies measured microscopically, resulting in smaller signal differences between selected repair times than in the manual method. This is probably due to the differences in the nature of measured signals: cell fluorescence intensity vs focus frequency. The advantage of FACS analysis is that the measured signal level is less dependent on the precision of sampling time post irradiation. This is an asset when the γH2AX focus assay is used to measure differences in individual response to radiation. This project received funding from the European Union´s Horizon 2020 R&I program under grant agreement No 945196. References 1. Marková et al.l International Journal of Radiation Biology 2007 2. Lisowska et al. International Journal of Radiation Biology 2013 3. Plodowska et al. Scientific Reports, submission date 04.01.2022

ABSTRACT. Exposure to radon often takes place in combination with chemical stressors such as cigarette smoke and epidemiological studies show that radon and cigarette smoke interact in inducing lung cancer. The mechanisms of this interaction are not understood. A component of cigarettes is nicotine, the intake of which improves physical and mental ability due to the release of catecholamines into the bloodstream, being the main reason for smoking and chewing tobacco. The interesting question is if nicotine modulates the DNA damaging potential of alpha particles emitted by radon. Bronchial epithelial BEAS-2B cells were pretreated with 2 µM nicotine and given 1 or 2 Gy of alpha particles. γH2AX formation and decay were analyzed to measure the direct effects on DNA damage response. Alpha exposure lead to a biphasic response with peaks at 1 h and 6 h, whereas nicotine alone induced no foci. The combined exposure produced a delayed, flattened response, where more foci remained unrepaired after 24 hours indicating delayed DNA repair. Also, fewer foci were detected at the time of the alpha peak (1h) in the combined treatment group. Preliminary data of comet assay showed a reduction in DNA tail moment at 1 h post combined exposure. During a 13-day time period post-exposure both the cell viability kinetics and viable cell counts showed a faster recovery in irradiated cells with nicotine treatment. These results are consistent with results from colony formation assay and suggest that nicotine interacts with alpha radiation at the DNA damage and cellular level. This project has received funding from the Euratom research and training program 2014-2018 under grant agreement No 900009.

ABSTRACT. Brain cancers are among the most difficult tumours to treat and remain a leading cause of death in Europe. Current treatments based on surgical removal followed by chemotherapy and radiotherapy (RT) can have devastating neurological consequences. An innovative approach to improve RT efficacy is to use radiosensitizers to increase the radiation effectiveness on tumours, sparing the healthy surrounding tissue and preventing the long-term cognitive side effects by reducing the dose. In particular, nanoparticles provide unique chemical and physical properties and following irradiation can intensify the production of secondary electrons and free radicals, like reactive oxygen species (ROS), which can cause indirect damage to the cells and in turn enhance RT effects (1). In this contest, nanodiamonds (NDs) are attracting increasing interest due to their appealing properties, including inertness, fluorescence, biocompatibility and the possibility of surface functionalization (2). In the hydrogenated form (H-NDs), obtained by adding hydrogen moieties as surface terminations, it has been proved that they exhibit a negative electron affinity together with a positive charge in aqueous solutions, which ensures their high reactivity, locally enhancing the damage caused by radiation and the dose deposit in their surrounding area (3). In this work, NDs were processed using thermal treatments carried out in a controlled atmosphere and their surface was modified to achieve the desired specific characteristics to optimize their radiosensitizing properties (4). Differently surface-terminated NDs undergone on both physical and chemical analysis and were employed in different concentration to study their possible application in RT treatments. In vitro Raman/photoluminescence microscopy was performed to assess NDs cellular uptake and localization in human medulloblastoma cell cultures and NDs toxicity and their effect following X-ray irradiation was evaluated by cell vitality assays.

1 Retif P., et al. Theranostics 2015, 5(9):1030-1044. 2 Mochalin VN., et al. Nat Nanotechnol. 2011, 7(1):11-23. 3 Grall R., et al. Biomaterials 2015, 61:290-8. 4 Aprà P., et al. Nanomaterials 2021, 11, 2740.

ABSTRACT. DNA double‐strand breaks (DSBs) produced by ionizing radiation (IR) induce the complex process of DNA repair signalised by histon phosphorylation (γH2AX)1. Quantitative analysis of γH2AX foci is a standard method in radiobiology; however, applying direct stochastic optical‐reconstruction microscopy (dSTORM) enables deeper insight at nanoscale into the DNA repair process2. Our aim was to reveal the dynamics of γH2AX formation after X-ray irradiation at different dose levels in a time-dependent manner, using dSTORM over diffraction limited 3D confocal microscopy. Glioblastoma cells (U251) were irradiated with 250 KeV X-ray at 0, 2, 5 Gy dose levels and were fixed at 30 min, 24 and 72 hours after irradiation for γH2AX immunofluorescense staining. Increased γH2AX foci, and cluster density was detected at both dose levels 30 minutes after irradiation, but both returned to the control level at 24 hours. At the same time, the highest volume of foci and clusters was measured at 24 hours. dSTORM‐based analysis of γH2AX revealed that micron-sized foci are composed of distinct, smaller parts with a diameter of few tens of nanometers (nanoclusters). We analysed the epitope/cluster, area/cluster ratios, cluster densities, and spatial nanofoci distribution with dSTORM superresolution microscopy. The density of these nanoclusters and the number of epitopes showed correlation with the delivered dose in the first post-irradiation time point and with the dynamics of the loss of γH2AX nanofoci. dSTORM superresolution microscopy provided higher accuracy over 3D confocal microscopy in unrevealing the molecular structure and organisation of radiation induced γH2AX foci and in the study of molecular rearrangements during the repair process. The superresolution imaging opens a novel perspective for radiation biology research.

ABSTRACT. Radiotherapy is the most widely used solid cancer treatment. It is based on the exposure of tumors to Ionizing Radiation (IR), by using photons or particles radiation such as carbon ions or protons. IR interacts with the molecules in a cell, which may cause DNA damage and oxidative stress and may lead to cellular damage and cell death. Here we focus on the effect of different Linear Energy Transfer (LET) on cells at the level of DNA damage formation. The increase in LET results in an increase in the ionization density along the particle track. This promotes an increase in the amount and complexity of DNA damages. Our aim is to compare the effect of ionizing radiation with different LETs on cell survival and DNA damage formation. We irradiated wild-type TK6 cells with x-rays, protons and two different LETs of carbon ions. Thereafter, the survival and the formation of micronuclei (CBMN assay), an indicator of genotoxic lesions, are being investigated. The analysis is ongoing and the results obtained until know show that carbon ions radiation has stronger DNA damaging and cell killing effects than x-ray. The results also indicate that irradiation of cells by LET 28 keV/µm c-ions produces more MN than LET 73 keV/µm C-ions which might be due to the fact the 28 keV/µm carbon ions induce more close and complex DNA damages due to release of energy in shorter distance than 73 keV/µm. Another explanation is that 73 keV/µm carbon ions has higher killing effects than 28 keV/µm thus the exposed cells are eliminated from being prepared for MN formation assay. The experiments on proton beam irradiation are planned and the results will be presented and discussed in my poster.

ABSTRACT. Heavy-ion cancer therapy requires modelling of ion-beam biological effects in tumors and normal tissues. The Relative Biological Effectiveness (RBE) of heavy ions is a complex quantity, characterized by significant variations along the beam path and by the dependence on several factors including radiation quality, dose, and considered cell type and endpoint. Therefore, it requires a precise modelling, especially for the pencil-beam scanning technique. Two radiobiological models, LEM I (Local Effect Model) and MKM (Microdosimetric Kinetic Model), are currently in use for heavy ions in clinical facilities, although other models are available including BIANCA (BIophysical ANalysis of Cell death and chromosome Aberrations), which has shown good agreement with in vitro and in vivo carbon-ion experimental data [1, 2]. In this work we present the first application of BIANCA to carbon-ion treatment planning scenarios. Following an interface with the FLUKA Particle Therapy Tool [3], BIANCA was applied to re-calculate the RBE-weighted dose distribution for three patient cases (chordoma, head-and-neck and prostate) previously irradiated at CNAO (Centro Nazionale di Adroterapia Oncologica) in Pavia, where the radiobiological optimization was based on LEM I. The predictions obtained by BIANCA were based either on chordoma cell survival (RBEsurv), considered as representative of the effectiveness in the tumor, and on dicentric aberrations in peripheral blood lymphocytes (RBEab), which are indicators of normal tissue damage, including secondary tumors. In the target and in the entrance channel the values predicted by BIANCA were lower than those obtained by LEM I, whereas the two models provided very similar results in the Organs At Risk. The observed differences between RBEsurv and RBEab suggest that, in normal tissues, the information on cell survival should be integrated by information more closely related to the induction of late damage, such as chromosome aberrations. This peculiar ability of the BIANCA model may represent an improvement for treatment plan optimization in ion-beam therapy. 1. M.P. Carante et al., Phys. Med. Biol. (2019), 64, 215008 2. M.P. Carante et al, Int. J. Mol. Sci. (2020), 21, 3973 3. W. Kozlowska et al., Phys. Med. Bio. (2019), 64, 075012

ABSTRACT. Different types of ionizing radiation (IR) interact differently with the cellular genome. Densely interacting particles with high linear energy transfer (LET) form concentrated double-strand breaks (DSBs) along particle tracks. Sparsely IR with low LET induces dispersed simple DSBs that are easier to repair. It is of interest whether the sequential order of high and low LET IR affects the DNA damage response and DSB repair focus formation. To investigate the DNA damage response after high and low LET IR and mixed beams (MB) thereof, we irradiated U2OS cells expressing a GFP-tagged version of the DSB sensor protein NBS1 with alpha particles, gamma radiation, and an alternating consecutive combination of both. NBS1 foci were recorded in live cells using an inverted fluorescence microscope during 5 h after irradiation. Time-lapse movies were analyzed for different parameters and the temporal dynamics of NBS1 foci occurrence and decay. The results of our analyses align with the prediction that high LET IR causes fewer microscopic DSB foci in a small area of the cell nucleus as compared to low LET IR. In the case of MB, the sequence of the IR types applied showed significant differences. The cells irradiated first with alpha particles showed a greater number of foci that repaired more slowly. When low LET gamma radiation was applied first, the decrease in the large number of NBS1 foci over time was rapid. In both cases, there was a significant increase in the foci size after the 2nd hour of observation, especially in the case of irradiation with alpha first. The current results suggest that the presence of high LET IR damage delays the sensing and repair of successive low LET-induced DSB damage. It can also be considered that high LET IR leads to more excessive chromatin damage which may include oxidative damage. An increase in the size of the foci may indicate the amassing of damaged DNA segments that are difficult to repair or that are congregated to facilitate repair.

ABSTRACT. Despite all improvements in radiotherapy, many tumors resist to this treatment and require high doses causing inacceptable damage to healthy tissues. Noble metal nanoparticles (MP) with high atomic number (high Z) have been shown to preferentially accumulate in proliferating tumor tissues and amplify radiation dose on the microscale. Hence, MN can serve as radiosensitizers, enhancing both radiotherapy efficiency and specificity. Simultaneously, MN allow tumor imaging (theranostics). Despite these advantages, biological processes provoked by MN in cells prior to and after irradiation (IR) remain obscure. We combined 3D confocal microscopy, single molecule localization microscopy (SMLM), flow cytometry and other biophysical/biological methods in combination with advanced software approaches to shed a new light, at the multiscale, on contradictory results presented in the literature on IR-mediated DNA double strand break (DSB) induction and repair in presence of MN. While we observed more DSBs in tumor cells irradiated in presence of MN (MN+), compared to MN(-) controls, this phenomenon reflected increased proportions of G2 cells in MN(+) cell populations rather than MN-mediated augmentation of direct DSB induction by IR. This is supported by micro- and nano-morphology of γH2AX/53BP1 foci, which slightly differed for MN(+) and MN(-) cells. At the nanoscale, Ripley´s distance frequency analysis of SMLM signal coordinate matrices revealed relaxation of heterochromatin (H3K9me3) clusters upon IR, which was more prominent in MN(+) cells. The expansion of radiosensitive G2 cells correlated with slightly decreased post-IR (PI) survival of MN(+) cells. Interestingly, low MN concentrations accelerated DSB repair but increased presence of unrepaired γH2AX/53BP1 foci at 24 h PI. MN thus exert multiple but mostly indirect effects on chromatin. Cytoplasmic effects, e.g., lysosome damage, cannot be excluded although they are not sufficiently supported by our preliminary results.

ABSTRACT. Carbon ion irradiation (C-ions) present a high biological efficacy and an excellent ballistic precision compared with photons. Indeed, a body of arguments supports their superiority related to the spatial distribution of Reactive Oxygen Species (ROS) at the nanometric scale, condensed in their tracks whereas diffused in the cells exposed to photons1. The ROS induced by both radiation lead to misfolded and oxidized proteins addressed to the ubiquitin-proteasome system or the autophagy pathway to maintain cellular homeostasis. Understanding the fate of oxidized proteins and the role played by the proteasome could help decipher the molecular advantages of carbon ions. The activity of the proteasomal catalytic 20S subunit, the levels of oxidized proteins through Oxiblot®, and their proteasomal addressing through the K48 ubiquitin expression, were studied in response to C-ion and photon irradiation for two Head and Neck Squamous Cell Carcinomas (HNSCC) cell lines and their radioresistant sub-population of Cancer Stem Cells (CSCs). The activation of the autophagy was investigated by flow-cytometry (Muse®). First, we showed a decrease in the 20S proteasomal activity after C-ion exposure compared with photons in HNSCC cell lines and their CSCs sub-populations. We also observed lower proteasomal addressing of the abnormal proteins traduced by a decreased K48 ubiquitin expression. However, the accumulation of oxidized proteins differs with enhanced oxidation after C-ions in parental cell lines, not observed in CSCs. These results, supported by preliminary data recently obtained at the French Heavy Ions Accelerator (GANIL), suggest a different fate of the abnormal proteins in CSCs after C-ion exposure, potentially addressed to the autophagy pathway. Besides, the condensation of ROS along the particle tracks, as evidenced by Monte-Carlo simulations, may lead to heavily oxidized protein difficult to repair and, therefore, toxic for the cells. Altogether, these data confirm the central role of the proteasome in the biological specificities of C-ions, especially on CSCs, and support the relevance of the proteasome as a therapeutic target.

1 Wozny AS, Vares G, Alphonse G, Lauret A, Monini C, Magné N, Cuerq C, Fujimori A, Monboisse J, Beuve M, Nakajima T, Rodriguez-Lafrasse C. ROS Production and Distribution: A New Paradigm to Explain the Differential Effects of X-ray and C-Ion Irradiation on Cancer Stem Cell Migration and Invasion Cancers 2019. Apr3;11(4):468

ABSTRACT. In the era of personalized therapy, radiotherapy (RT) could be used in combination with radiation modifying agents both to improve the therapeutic index and to personalize cancer treatment plans [1]. In this scenario zebrafish embryo represents a potential model in the radiobiology field, considering that embryogenesis is the most radiosensitive stage in the vertebrate life cycle and that the aqueous environment in which embryos develop favours homogeneity in the radiation dose distribution [2]. In order to suggest innovative RT treatments, we describe the characterization of the biological effects induced in zebrafish embryos exposed to X-ray beams, alone or in combination with curcumin, already known for anti-oxidant and anti-tumor properties [3]. Distinct batches of 24 hours post fecondation (hpf) embryos were exposed to 0-15 Gy X-rays. For the combined treatment, embryos were pre-treated for 18 hours with 0-10 μM curcumin, and subsequently irradiated using the above mentioned dose range. Sister batches of 6 hpf embryos were either used as untreated controls or subjected to single treatment with curcumin following the same experimental setting used in the combined treatment. Treated and control embryos were carefully examined by daily steromicroscope observation until 120 hpf, to estimate the mortality rate as well as developmental delay and alterations. In addition, behavioural analysis was performed to assess alteration in swimming capacity or delay in response to induced physical stimuli, as well as any possible variation in the heart rate values at 48 and 72 hpf. We found that the X-ray single exposure in a dose-dependent manner, as well as the treatment with curcumin alone at concentrations greater than 5 μM, inflicted gross malformations (including pericardial and yolk-sac edema, skeleton defects, cardiac dysfunctions and variation in the pigmentation degree), behavioural defects and lethality. In striking contrast, the occurrence of these phenotypic alterations was markedly reduced, at different extents, in embryos exposed to the combined treatment, strongly suggesting that the adverse effects induced by RT were mitigated in these embryos by 0-5 µM curcumin pre-treatment. Additional experiments are planned to accomplish the characterization at a molecular level of the observed effects.

ABSTRACT. In breast cancer (BC) care, radiotherapy (RT) is considered an efficient treatment, both for controlling localized tumors or as a therapeutic option in case of inoperable or recurrent tumors. However, the choice of a unique treatment plan for all BC patients may not be the best option. As known, BC is a heterogeneous disease at both clinical and molecular levels, with distinct subtypes also linked to the hormone receptor (HR) status (i.e. estrogen – ER, progesterone – PR) [1-2]. Thus, radiobiological research is needed to understand molecular differences that affect the radiosensitivity of different BC subtypes to obtain more successful treatments plans. For this purpose, the aim of this study was to analyze gene expression profiles (GEPs) induced by high ionizing radiation (IR) doses (9 and 23 Gy) in primary radioresistant BC cells. Firstly, we selected, collected (72h and 1-week post-RT), and expanded the IR radioresistant cell fractions of two primary BC cells isolated from surgically removed breast tumors, with opposite HR expression status: BCpc7 (ER+/PR+) and BCpcEMT (ER-/PR-): the negative expression of HRS is often described in radioresistant BC and associated with a bad prognosis. Secondly, differential gene expression analyses revealed that a conspicuous number of genes had significantly altered expression levels in 72h and 1-week post-RT radioresistant fractions compared to the early irradiated cells (24h) used as reference. Pathway analyses revealed that GEPs of the two radioresistant cell fractions, were strictly involved in the regulation of the cell cycle/cell death balance, hypoxia, inflammation as well as in viability, and in cell communication. Further investigation is now in progress to identify pathway signatures linked to HR status. We trust in the idea that genes may function as biomarkers of disease providing the rationale for the development of molecularly based signatures to predict the RT responses in cancers, including BC [3-4].

ABSTRACT. Today, the technological development of radiation therapy (RT) has led to more performing and innovative technologies which can deliver, with high precision, increasing doses of ionizing radiation (IR) saving the organ at risk. This property of the proton beam is due to its typical curve of energy deposition through the matter (Bragg peak), which represents a better conformational option with respect to conventional photon beams [1]. Additionally, in Proton Boron Capture Therapy (PBCT), the reaction between protons and boron particles enhance the Relative Biological Effectiveness (RBE) of protons, representing a chance for radio-resistant and hypoxic malignancies for which there is no effective therapy [2-3]. Here, we investigate the molecular responses induced by PBCT in Glioblastoma (GBM) U87 xenograft mice models, which belongs to the category of the foremost radio-resistant and hypoxic cancers, by using the RNA-Sequencing approach, to study the biological processes activated by PT with or without boron administration. Comparative differential gene expression analyses revealed that a conspicuous number of genes had significantly altered expression levels compared to the reference mock-irradiated cells. Pathway analyses revealed that differentially expressed genes (DEGs) compared to the control, were strictly involved in the regulation of actin cytoskeleton, axon guidance, and the binding and communication with other cells through adherent, gap, and Focal junctions. Moreover, we reported the deregulation of the cell cycle /cell death balance, probably driven by Notch signalling, known to be a key regulator of neuronal cell growth and homeostasis in particular down-regulation of cells survival, growth, and viability and, on the other hand, an up-regulation of cell death (apoptosis, necrosis, and others), were described in PBCT GBM cells. Taking together these data, we encourage clarifying the biological response and therapy efficacy induced by PBCT to enhance cancer therapies' success rate, especially for the foremost radio-resistant and hypoxic cancers [4-5]. In addition, we trust that these gene expression data could be useful to select potential new biomarkers against which specific targeted therapies could be directed, in order to suggest a combined treatment approach with Proton Therapy

ABSTRACT. Tumor microenvironment (TME) consists in a complex interplay of cells and soluble factors holding a critical role in neoplastic development. Glioblastoma (GBM), a WHO grade IV glioma, is a malignant primary brain tumor for which combination of surgery, chemotherapy and radiotherapy is the first-line approach despite severe adverse effects. Significant pathophysiological changes have been found in GBM TME, such as oxidative stress, neuroinflammation and glia activation. This has been reported to occur spontaneously and upon severe therapeutic regimens, resulting in dismal prognosis and recurrences. Microglia, is among the most important players in favouring GBM growth and proliferation, representing target cells of immune escape mechanisms. Our study aims at analysing radiation-induced effects in modulating intercellular communication and how the plethora of molecules secreted in TME determines protective mechanisms in naïve GBM cells through mitochondrial activation and metabolic rearrangement. We first evaluated irradiated microglia and microglia-to-GBM cells interactions mediated by both paracrine and autocrine signalling. Conditioned media (CM) from human irradiated microglia were collected and GBM cell lines (i.e. U-87 MG and U-251 MG) were exposed to either mock irradiated or 2 Gy/15 Gy irradiated microglia-derived CM. We observed not significant changes in apoptosis, promotion of proliferation and colony formation and mitochondrial fitness modulation. In particular, we found that mitochondrial metabolism was affected by direct irradiation and such an effect was not observed in GBM cultures exposed to 2 Gy or 15 Gy irradiated microglia-derived CM. Our results suggest that irradiation direct damages on either GBM cells or microglia are not transferred to naïve cells and that off-target radiotherapy modulates microglia to support GBM proliferation and metabolism.

ABSTRACT. Radiotherapy plays a key role in the management of cancer patients. Technical and physical innovations have helped to enhance accuracy of radiotherapy dose delivery. Furthermore, multimodal combinations with molecularly tailored drugs or immunotherapy yield promising survival benefits in selected patient subgroups. Yet high loco-regional failure-rates and frequent development of metastases still limit patient outcome in relevant cancer subtypes. We will present our most recent findings and concepts on the role of cancer metabolism in the radiation response and radioresistance. We used a systematic collection of metabolic and radiobiological data from irradiated cancer cell lines including syngeneic cell pairs for mathematical modeling of metabolic adaptative processes, as well as genetic and pharmacologic approaches to gain more insight into the interplay between metabolic adaptation to radiotherapy-induced cell stress, clinically relevant oncogenic drivers, the cellular radiation response, and cancer cell radiosensitivity. Our data demonstrate that in addition to DNA damage and oxidative stress exposure to ionizing radiation induces a severe energy stress. Mutant KRAS or the activation associated protein kinase B mutant protein AKT-E17K enhanced the capacity of cancer cells to adapt their metabolism to cope with these radiotherapy-induced stresses with impact on energy metabolism, antioxidant defense, and repair of DNA damage damage. These metabolic adaptive processes were associated with specific metabolic dependencies allowing for a targeted radiosensitization [1, 2]. Understanding metabolic phenotypes of radioresistance and associated metabolic bottlenecks in a cancer cell-specific context offers largely unexploited avenues for a future biological individualization and optimization of clinical radiotherapy, e.g., by targeting critical metabolic nodes of oncogene-dependent metabolic reprogramming.

1 Krysztofiak A et al., iScience Oct 28;24(11):103366. 2 Götting I, Matschke J, Jendrossek V, unpublished

Acknowledgement: Supported by grants of the DFG (GRK1739 to VJ; MA8970/1-1 to JM), and the European Commission under the Horizon 2020 Marie Skłodowska-Curie Innovative Training Program THERADNET (MSCA ITN (ETN) THERADNET (Grant Agreements No. 860245) to VJ and JM.

ABSTRACT. Zahradnicek, O.1, Jelinek Michaelidesova, A 1,2, Danilova, I.1,2, Pachnerova Brabcova, K.1, Vachelova, J.1, Kundrat, P.1, Vilimovsky, J.3, David, M. 2,4, Vondracek, V.3,4, Davidkova, M. 1 1 Nuclear Physics Institute of the Czech Academy of Sciences, Husinec - Řež 130, 250 68 Řež, Czech Republic; zahradnicek@ujf.cas.cz, michaelidesova@ujf.cas.cz, danilova@ujf.cas.cz, brabcova@ujf.cas.cz, vachelova@ujf.cas.cz, kundrat@ujf.cas.cz, davidkova@ujf.cas.cz 2 Czech Technical University in Prague, Faculty of Nuclear Sciences and Physical Engineering, Břehova 78/7, 13 115 19 Prague, Czech Republic 3 Proton Therapy Center Czech, Prague, Budínova 2437/1a, 180 00 Prague, Czech Republic; jan.vilimovsky@ptc.cz, vladimir.vondracek@ptc.cz 4 Thomayer University Hospital, Vídeňská 800, 140 59 Prague, Czech Republic; miroslav.david@ftn.cz

The killing efficiency of proton beams in the presence of boron increases as was previously observed in in vitro cultivated cancer cells. The number of tested cell lines was low and the mechanism of the increased cell killing in the presence of boron is not understood. The insight and understanding of what happened to and within cells doped by boron compounds after protons irradiation could open new perspectives for streamlining proton therapy of aggressive tumors. Here we tested the glioblastoma U-251 MG and U-87 MG cell lines which were adherently cultivated. Adherent U-251 MG and U-87 MG cells were doped by boron compounds with different isotopic ratios 24 hours before irradiation. Cell samples were irradiated by a 190.6 MeV proton beam in plateau and Bragg peak positions. Control boron compound free cell samples were also included in the experiments. To evaluate the effect of proton therapy in the presence/absence of boron compounds the clonogenic cell survival test was used as a basal method. To examine the intercellular damage in relation to cell death or decreasing cellular proliferation, we analyzed the number of DNA double-strand breaks marked by the expression of gammaH2AX and the number of lysosomes in which an increasing amount of number is related to damage to organelles.

ABSTRACT. While current technology in radiation therapy permits precise delivery of radiation dose to the tumor, with a decreased risk of side effects to the healthy surrounding tissue, still the mechanisms underlying DNA damage response (DDR) that lead to tumor resistance, are not yet clearly understood. After irradiation, signaling pathways are activated, enabling cell cycle arrest for DNA repair via the DDR-related kinases and their downstream targets. ATM, ATR, and Chk1 kinases play an important role in DDR activation and enhance resistance to radiotherapy. In our previous study, the use of ATM, ATR and Chk1 inhibitors have been studied as potential tools for radiosensitivity estimation. More specifically, these inhibitors, including the ATR inhibitor VE-821, have been used as alternatives to caffeine in order to modify the classical G2-chromosomal radiosensitivity assay [1], and this modified assay was performed in exponentially growing RPE and 82-6 hTERT human cells lines, proposing the efficacy of the above inhibitors, especially of VE-821, in radiosensitization [2]. For this reason, in this study, the classical G2 assay, using caffeine, as well as the modified G2-assay, using VE-821, have been performed in several cancer cell lines, such as epidermoid carcinoma (A431), lung cancer (A549) and prostate adenocarcinoma (PC3). Cells were irradiated during the G2/M-phase under the presence or absence of caffeine (for the classical G2-assay) and VE-821 (for the modified G2-assay). The induced chromatid breaks were recorded and used in order to evaluate the radiosensitivity of these cancer cell lines and their potency for radiosensitization. The comparison between the classical and the modified G2 assay shows the potential use of VE-821 for the evaluation of cancer cell radiosensitivity and validates this enhanced modified G2 assay. In addition, the results strongly support the concept that ATM and ATR inhibitors, can possibly act as attractive anticancer agents in radiation oncology.

ABSTRACT. In order to better describe the biological effects of ionizing radiation, understanding the mechanisms of radiolysis at the molecular scale is a key step. Proteins are by far the most abundant biomolecules in the cell, yet very few studies describe their radiolysis by accelerated ions. The aim of our team is therefore to develop a systematic study of these effects on protein biomolecules, from amino acids to whole proteins, and with various ions, energies and dose-rates.

In this work, myoglobin, a small heme-protein, was irradiated by accelerated ions of a few MeV energies, in highly concentrated native gels, 20 % w/w, similar to protein content in the cell. The impact of ions on its secondary structure was followed by mean of infrared spectroscopy, showing reproducible and organized change in its conformation, from alpha helices to mostly beta-structures (cf. Figure 1). UV-Visible spectra were also recorded under irradiation, and the combination of the data allowed identifying the formation of a significant quantity of carbon monoxide under irradiation. The results obtained with low–energy protons will be presented and compared to helium and carbon ions, and the possible source for carbon monoxide will be discussed.

ABSTRACT. Radiation therapy is the most-effective cytotoxic therapy available to treat localized solid cancers. With the introduction of charged particle radiotherapy (proton therapy), the area of irradiated healthy tissue surrounding the tumor was further decreased. The aim of this study is to investigate the role of the p53 pathway in response to both X-rays and proton therapy treatments. p53 is a transcription factor with a key role in the stress-depended regulation of DNA repair, cell cycle arrest, and apoptosis. As a model, we used 3 isogenic derivatives of the colon cancer-derived cells HCT116: parental, TP53-/-, and CDKN1A-/- (coding for p21, the main p53 target involved in cell cycle arrest). We analyzed cellular responses to irradiation, focusing on DNA damage, p53 targets activation, apoptosis induction, and 3D culture disaggregation. As expected, X-rays and proton irradiation caused DNA damage an hour after treatment in all cellular systems, detected by the formation of γ-H2AX foci. Interestingly, despite their different genetic background, all three cell lines retained a similar ability to repair the DNA damage. Surprisingly, the p53 and p21 null cells showed a higher apoptotic rate, indicating that the two cell lines could be more radiosensitive than the parental cells. Moreover, to better mimic the shrinkage effect of radiation therapy on solid cancers, 3D spheroids were also used. HCT116 parental, p53-/-, and p21-/- cells spontaneously formed spheroids in ultra-low attachment plates. Notably, while parental spheroids showed a reduction in diameter 10 days after X-rays and proton irradiation but still maintained a proper 3D organization, the p53-/- and notably p21-/- spheroids completely disaggregated. Furthermore, the viability of the p21-/- spheroids drastically dropped in response to X-rays and proton irradiation, and the analysis of PARP cleavage and activation of Caspase 3 highlighted an increase in apoptosis, particularly in p21 null cells. Taken collectively, these data suggest that the absence of p53-dependent responses through p21 enhances the sensitivity to irradiation. This study revealed a dichotomy in p21 role: in addition to its canonical tumor-suppressive role, it seems to hold a radioprotective function in these cancer cells that, when depleted for p21, are considerably more prone to apoptosis. These findings could set the stage for future studies based on therapies targeting p21 in combination with charged-particles radiotherapy.

ABSTRACT. Globally, head and neck cancer (HNC) is the sixth most common cancer. Developed countries have seen an increase in the number of HNC cases, and this can be explained by HPV being a primary etiological source of HNC. Radiation therapy is a common treatment for HNC, and HNC patients can experience severe late radiation toxicity such as dry mouth and dysphagia that can significantly impact their quality of life. HPV-positive HNC patients are usually younger than HPV-negative HNC patients and with a better prognosis, HPV-positive HNC patients will have to deal with the long-term effects of radiation for longer. It is not known why some patients develop toxicity, and currently, it is difficult to predict before radiotherapy which patients will experience these long-term and sometimes irreversible late toxicities. Optical spectroscopic methods, such as Raman spectroscopy, can provide a rapid, label-free, and non-destructive measurement of the biochemical content of cells and biofluids. This project aims to identify Raman spectral biomarkers from blood plasma to predict the development of radiation-induced toxicity prior to treatment commencement. Baseline plasma samples (n=40) were collected from HPV-positive HNC patients who were enrolled in the De-ESCALATE clinical trial. Raman spectra from plasma of patients who experienced late toxicity grade 0-2 and grade 3+ were analysed using principal component analysis (PCA) for the exploration and visualisation of trends in the data, then partial least squares discriminant analysis (PLS-DA) was used for the development of classification models. Identification of predictive spectral biomarkers would allow the stratification of HNC patients according to the risk of developing radiation toxicity and could guide the selection of treatment modalities to reduce this risk in high-risk patients or allow dose escalation in low-risk patients to improve tumour control.

ABSTRACT. Breast cancer is the most common malignancy accounting for 29.2% of all cancers in women1 and it has been one of the first models treated with radiotherapy (RT) by stages I (tumor size up to 2 cm and no affected lymph nodes) to stage III (spread of the tumor to the lymph knots or tissue near the breast) to reduce the risk of recurrence after surgery. However, increasingly, the treatment plan requires the addition of chemotherapy drugs that often cause resistance. Nanotechnology proposes the use of nanoparticles (NPs) as novel gateways to enhance the therapeutic efficacy of anticancer agents at the target site of action due to their tumor-targeting abilities, which can limit the undesired systemic effects of chemotherapy agents and also reduce drug resistance2,3. In particular, hyaluronic acid (HA) coated NPs (HA-NPs) represent a very promising candidate for ligand-targeted therapy, considering the high expression of surface receptor CD44, that specifically binds with HA, in breast cancer cells4. This work aims at investigating the effects of ionizing radiation on cell's ability to internalize HA-NPs. Specifically, fluorescent (rhodamine B) HA- coated poly lactic co glycolic acid (PLGA) NPs (HA-PLGA-NPs) were formulated and placed in contact for 5h with healthy breast cell line (MCF10A) and its triple-negative cancerous counterpart (MDA-MB-231) irradiated with two doses of X-rays: 2 and 10 Gy. To quantify the cellular internalization capability of HA-PLGA-NPs, the samples were observed with a confocal microscope and the fluorescent signals was analyzed using ImageJ Fiji software. The results demonstrate an amplifying effect of the internalization process of HA-coated NPs by irradiated tumor cells, representing the background to continue the internalization study related to the response of cells to irradiation to finally establish the most suitable time to administration of NPs - basic therapy after radiotherapy.

ABSTRACT. Despite the enormous advances that have been made in medical technology and techniques using ionising radiation, the challenges associated with these rapid developments are still relevant. The biological outcomes assessment is subject to uncertainty due to the use of averaged, macroscopic physical quantities. That is why we notice the importance of the choice of physical values best describing the quality and quantity of radiation used. Since the initiation of radiation-induced damage is dominated by interactions occurring in the DNA or within its environs, the distribution of such interactions is crucial to properly assess the biological effects. The first results of the attempt to connect fundamental nanodosimetric concepts with radiobiological parameters characterising the survival of a well-established cell line irradiated with an ion beam will be presented. To obtain a mathematical formula, we have analysed radiobiological data for V79 cells derived from the PIDE database1 and correlated the parameters describing biological endpoints with nanodosimetric characteristics of the radiation used in these experiments. The nanodosimetric quantities were determined retrospectively based on available information about irradiation conditions using Monte Carlo simulation for the JetCounter device with the Geant4-DNA physics option 4. Since larger clusters are more efficient with delivering lethal damage, a simple sum of probabilities associated with cluster size from 2 to infinity, namely F2 parameter, cannot be a good predictor of radiobiological outcome, although it is often considered a valuable candidate2,3. Instead, we use a different quantity that takes into account both the cluster-size probabilities and the probability of damage due to ionisation in a minimal possible way. For now, the proposed model based on this quantity does not include any intermediate steps but provides good data consistency.

1Friedrich T, Scholz U, Elsässer T, et al. Systematic analysis of RBE and related quantities using a database of cell survival experiments with ion beam irradiation. J Radiat Res, 54 (3), pp. 494-514 (2013). doi:10.1093/jrr/rrs114 2Grosswendt B. Nanodosimetry, from radiation physics to radiation biology. Radiat Prot Dosimetry, 115(1-4), pp. 1-9 (2005). doi: 10.1093/rpd/nci152 3Conte V, Selva A, Colautti P, et al. NANODOSIMETRY: TOWARDS A NEW CONCEPT OF RADIATION QUALITY. Radiat Prot Dosimetry, 180(1-4), pp. 150-156 (2018). doi: 10.1093/rpd/ncx175

ABSTRACT. The surviving fraction of cells decreases exponentially with the increase of the absorbed dose at high doses1. At low doses, however, experiments show that surviving fraction differ from this due to the effects of hyper-radiosensitivity and induced radioresistance in many different cell lines2. The result is a function that starts steeper and after a local minimum starts to increase to a local maximum as the dose increases before following the exponential decrease at higher doses. The aim of this study was to test if the hypothesis of minimum mutation load3 can describe both hyper-radiosensitivity and induced radioresistance at low doses. In this case the principle means that the most damaged cells in a vicinity use apoptosis to reduce the mutation rate in the tissue. For this purpose, a python code was created to simulate the surviving fraction on different doses, with the following presumptions: • the number of cells is in equilibrium in the tissue with random placement in a given radius circle, • the radiation induced mutations follow Poisson distribution, • and the cells are able to communicate the condition of their DNA with signals whose concentration distribution follow a normal distribution centered on the given cell. The cells whose DNA damage are above a threshold (compared to the average damage of the vicinity) go into apoptosis and the neighboring cells divide to uphold the equilibrium. Thus, the overall rate of mutagenic damage decreases in the tissue. In order to test the model, results were compared to a large volume of experimental data4 The initial parameters were calculated from the experimental data and the results show that this model gives comparable result to the commonly used phenomenological induced repair model, which can be further improved with additional fit of the parameters. While the exact procedure of communication between the cells is not known, this model gives a possible general explanation to the hypersensitivity in the tissue due to any kind of mutagenic effect not just ionizing radiation.

ABSTRACT. Background: Radionuclide therapy using 177Lu-octreotate in patients with spread neuroendocrine tumours show promising results, but could be optimised. A previous study showed slower tumour regrowth and prolonged overall survival for mice that received hyper-fractionated versus single injection with the same total dose of 177Lu-octreotate, but the reasons for these differences are not known. Aim: The purpose of this study was to assess differences in biodistribution in tumour and normal tissues after single administration and hyper-fractionated treatment regime. Methods: Fifteen female BALB/c nude mice were treated with a total of 120 MBq, injected as 1x120 or 3x40 MBq, of 177Lu-octreotate (n=3/group). The mice were euthanized after 1, 3 or 7 days after treatment start and tumour and normal tissues were excised, weighed and measured for 177Lu activity to determine activity concentration. From the activity concentration, tumour-to-normal-tissue activity concentration values (T/N) was determined. Results: Overall, the activity concentrations in the various tissues were higher after hyper-fractionation, compared to single injection. The mean 177Lu activity concentration in tumours were 4 times higher 7 days after treatment start, for the group that received hyper-fractionated treatment compared to single administration. T/N values increased by approximately a factor of 2-3 at day 7 for normal tissues, including kidneys and bone marrow, after hyper-fractionation compared to single administration. Conclusion: Hyper-fractionation results in higher uptake of 177Lu-octreotate in tumours and higher T/N values, and could be beneficial in the treatment of tumours with overexpressed somatostatin receptors.

ABSTRACT. Estimation of cancer risks following ionising radiation exposure are mostly based on epidemiological studies of the Japanese atomic bomb survivors who received acute high-dose exposures to external radiation (gamma rays and neutrons). Animal studies have been used to provide additional information on the effects of different dose rates on tumorigenesis, however, most of these studies have used acute high-dose rate radiation and there is less information about the effects from chronic exposure to low dose-rate radiation.

This study examines the quantitative effects of exposure to chronic low dose-rate gamma radiation compared with acute high dose-rate gamma irradiation in the ApcMin/+ mouse model. F1 C57BL/6 x CBA/Ca ApcMin/+ and Apc+/+ mice were bred at UKHSA and shipped to Norway for exposure to chronic low-dose gamma radiation (2.1 mGy h-1, total doses 0, 1.7 and 3 Gy) in the FIGARO facility at the Norwegian University of Life Sciences, Aas, or exposed to high-dose rate gamma irradiation (0.3 Gy min-1, total doses 0, 1.5 and 3 Gy) at the Medical Research Council Co-60 gamma irradiation facility (Harwell Campus, Didcot, Oxfordshire, UK). Following the completion of the irradiations the mice were returned to UKHSA and killed 200 days after the end of their exposure period for tumour evaluation.

Intestinal tumour numbers in the different exposure groups will be presented and compared. The results will contribute to knowledge of low-dose rate radiation intestinal tumorigenesis and therefore inform judgements on Dose-Rate Effectiveness Factor values for low dose-rate vs. high dose-rate gamma exposure.

Funding for the project was received from the European Atomic Energy Community Seventh Framework Programme FP7/2007-2011 under grant agreement No 249689 via the Network of Excellence (NoE) Low Dose Research towards Multidisciplinary Integration (DoReMi) and the Research Council of Norway through its Centres of Excellence funding scheme, project # 223268/F50 CERAD.

ABSTRACT. Strategies to overcome tumor radioresistance are urgently needed to increase radiotherapy effectiveness, reducing the overall treatment time and spare as much as possible normal tissues. In this regard, telomere targeting has been recently proposed as a radiosensitizing strategy. Molecules known as G4-ligands interact with telomeres stabilizing secondary structures known as G-quadruplex (G4s), inducing telomere uncapping and dysfunction, increased chromosomal instability, decreased cell proliferation and radiosensitization. RHPS4 is one of the most effective and studied telomeric G4-ligand, and our laboratory provided evidence of its in vitro and in vivo radiosensitizing effect on glioblastoma cells suggesting the potential of telomeric G4-ligands for therapeutic applications in radiotherapy. Unfortunately, RHPS4 route to clinical testing was halted by off-target cardiotoxicity and analogues were developed to overcome this problem. In the present work, we investigated the capability of RHPS4 and one of its most promising analogs named 190 to impair proliferation and to induce telomere dysfunction and radiosensitization in breast cancer cells (MCF7, MCF7Y537S, HCC1937, MDA-MB-231) and in a non-tumorigenic mammary epithelial cell line (MCF10A) as normal control. Here we provided the evidence of strong antiproliferative action of RHPS4 and 190 compounds, as well as the ability to induce genomic and telomeric replicative stress, which is turned into genomic and telomeric damage. However, despite the RHPS4 and 190 capability to increase IR effects in glioma cells (used as reference) we did not observed radiosensitization in breast cancer cell lines that displayed higher resistance to telomeric DNA damage induced by the ligand.

ABSTRACT. The phenomenon of low-dose hyper-radiosensitivity (HRS) is an effect in which cells die from excessive sensitivity to low doses (< 0.5 Gy) of ionizing radiation but become more resistant (induced radioresistance, IRR) to larger doses. One possibility to benefit in the clinic from the HRS effect is by using low-dose fractionated radiation (LDFR) as an enhancer of systemic chemotherapy. The fact that our National Research Institute of Oncology, as the first in Poland, has started a phase II clinical trial using LDFR (0.5 Gy fractions) combined with induction chemotherapy (ChT) in patients with locally advanced squamous cell carcinoma of head and neck (SCCHN) gave us a unique opportunity to recognize potential predictors and mechanism underlying the clinical response to such treatment. The aim of the study is to compare in vitro the effects (assessed by clonogenic, pATM and γH2AX foci assays) of LDFR (4 x 0.5 Gy) versus a single dose of 2 Gy on carboplatin and paclitaxel in normal fibroblasts derived from patients with SCCHN enrolled in the clinical trial (LDFR+ChT) and to answer the question whether the chemopotentiating effects of LDFR apply to normal cells and depend on HRS status. To date, skin fibroblasts from 10 SCCHN patients have been obtained. Preliminary results on fibroblasts of two patients showed that carboplatin- and paclitaxel-potentiating effects were two-fold greater with LDFR 4 x 0.5 Gy than those with a single dose of 2 Gy. Research is ongoing and all results will be presented at the conference. This work is supported by the National Science Centre, Poland, grant no 2020/39/O/NZ5/02625.

ABSTRACT. Chondrosarcoma is a primary tumor of bone and cartilage. It is known for its resistance to conventional treatments such as radiotherapy, as well as its high metastatic potential for high-grade tumors. A possibility that could explain this resistance, is the presence of cancer stem cells (CSCs) described as having increased DNA repair systems. In order to overcome this problem, certain therapeutic strategies are being explored, such as hadrontherapy (proton and carbon ions therapy) and radiosensitizing molecules such as PARP inhibitors whose effects have been previously described in the context of chondrosarcoma . In this study, we observed the effect of different irradiation qualities (X-rays, protons and carbon ions) coupled with a PARP inhibitor: olaparib, on a chondrosarcoma cell line presenting a high stem cell potential: the JJ012 cell line. The impact of combination of different irradiation qualities coupled with olaparib treatment, was analyzed by clonogenic assays. The effect of treatments on the cancer stem cells proportion was studied using an ALDH activity test by flow cytometry, sphere formation assay and RT-qPCR. Preliminary results indicate that carbon ions are more effective than protons, which are themselves more effective than X-rays on JJ012 chondrosarcoma cells. It also appears that olaparib increases the radiosensitivity of cells to different treatments (X-rays, protons, C-ions). This radiosensitivity seems higher with X-rays and protons than with carbon ions treatment. According to the first results on CSCs, it seems that carbon ions are a good alternative against this subpopulation, while the effects of olaparib has not been proven yet.

Vares et al., « A Multimodal Treatment of Carbon Ions Irradiation, MiRNA-34 and MTOR Inhibitor Specifically Control High-Grade Chondrosarcoma Cancer Stem Cells ». Césaire et al., « Sensitization of Chondrosarcoma Cells with PARP Inhibitor and High-LET Radiation ».

ABSTRACT. Epidemiological studies demonstrated an elevated cardiovascular disease (CVD) risk associated with high local doses of ionizing radiation to the heart, as reported in patients undergoing thoracic radiotherapy for malignant diseases such as breast cancer, Hodgkin's disease, or childhood cancers. Radiation-induced CVD is characterized by the metabolic remodelling in the heart mainly due to the inactivation of the transcription factor peroxisome proliferator-activated receptor alpha (PPAR alpha) thereby inhibiting lipid metabolic enzymes. The goal of the present study was to investigate the potential protective effect of fenofibrate, a known agonist of PPAR alpha on radiation-induced cardiac toxicity. To this end, we compared for the first time, the cardiac proteome of fenofibrate- and placebo-treated mice 20 weeks after local heart irradiation (16 Gy) using label-free proteomics. The observations were further validated using immunoblotting, enzyme activity assays, and ELISA [1]. The analysis showed that fenofibrate restored the signalling pathways negatively affected by irradiation such as lipid metabolism, mitochondrial respiratory chain, extracellular matrix hemostasis, redox response, endothelial NO signalling and inflammatory status. These findings emphasize the role of PPAR alpha related metabolic pathway in cardiac injury after irradiation and suggest a molecular target for potential diagnosis and prognosis of radiation-induced CVD risk to select individuals for optimal prevention and therapeutic interventions.

1. Azimzadeh, O., et al., Activation of PPARα by Fenofibrate Attenuates the Effect of Local Heart High Dose Irradiation on the Mouse Cardiac Proteome. Biomedicines, 2021. 9(12).

ABSTRACT. In this work, we shed new light on the highly debated issue of chromatin fragmentation in cryopreserved cells. We describe replicating cell-specific DNA damage and higher-order chromatin alterations after freezing and thawing. We identified DNA structural changes associated with the freeze-thaw process and correlated them with the viability of frozen and thawed cells. And simultaneously evaluated DNA defects and the higher-order chromatin structure of frozen and thawed cells with and without cryoprotectant treatment. We found that in replicating (S phase) cells, DNA was preferentially damaged by replication fork collapse, potentially leading to DNA double strand breaks (DSBs), which represent an important source of both genome instability and defects in epigenome maintenance. This induction of DNA defects by the freeze-thaw process was not prevented by any cryoprotectant studied. Both in replicating and non-replicating cells, freezing and thawing altered the chromatin structure in a cryoprotectant-dependent manner. Freezing and thawing effects are tested to radio-sensitize the tumor cells.

ABSTRACT. Studying fundamental chemical mechanisms of the effects of ionizing radiations on biomolecules is crucial to have a better understanding of their radiobiological effects. These ionizing radiations can be used in a therapeutical context in radiotherapy to treat cancers by damaging tumoral tissues. FLASH radiotherapy, using very high dose rates (>40Gy/s) could have a preserving effect towards healthy tissues, prompting a lot of interest recently [1]. At this moment, molecular mechanisms of the FLASH effect are still far from being completely understood.

Dose-rate effects at the chemical stage, on water radiolysis and radiolysis of biomolecules, could be an essential element of the FLASH effect observed on biological systems. Water radiolysis produces several reactive species, among which hydroxyl radical is the most potent one towards biomolecules. We have quantified and reconstructed the kinetics of formation of hydroxyl radical HO•, using several scavenging probes. Experiments were conducted at several dose-rates, from 0.1 to more than 2000 Gy/s, under irradiation by 1MeV electrons. The data obtained show a significant dose-rate effect on the yields of HO•. Radiolysis of amino acids and of a small peptide, aspartame, was also studied in solution, the very same conditions. Even taking into account the dose-rate effect on HO•, a clear effect could be observed on the yields of radiolysis products of both biomolecules. Similar results were obtained with phenylalanine and aspartame, showing that the radiolysis mechanisms remain similar when the amino acid is included in a peptide. These results could be very interesting to better understand FLASH effect.

ABSTRACT. Despite more sophisticated delivery technology, modern radiotherapy is faced with many challenges.1 Clinical results have shown that, apart from well-known advantageous effects of hadrons, less conventional beams might provide additional benefits. 2 Due to their favorable physical and biophysical characteristics, helium ions (4He) arose as promising therapeutic modality.2,3 They provide minimal lateral scattering compared to protons, and reduced fragmentation tail compared to carbon ions.2 Since innovation in radiation therapy requires understanding of the complex biological effects of ions, more experimental data about the effects of 4He ions on cancerous cells is needed.4 To get deeper insight into their biological effects, 4He ions are used on two types of human cancer cell lines of different origin, i.e. MCF-7 breast adenocarcinoma and HTB140 melanoma cells. Previous experiments with gamma rays as well as with protons and carbon ions, have demonstrated high level of radioresistance of these cells.5 Changes in cell cycle as well as the induction of apoptosis are used as endpoints to evaluate biological response of selected cell lines to 4He irradiations. Irradiation dose was adjusted to the cell line and previously determined level of radiosensitivity.6 Cell cycle distribution and induction of apoptosis were monitored in equal time intervals, up to 72 h after irradiations. According to the results, for MCF-7 cell line significant changes in terms of reduction in the number of S-phase cells was pronounced at 24 and 48 h time points, while in HTB140 cells changes in cell cycle were induced somewhat later and were still detectable 72 h after irradiations. Analysis of apoptosis has shown that 4He ions make MCF-7 cells more prone to apoptotic cell death than HTB140 cells. This was confirmed by both changes in the number of SubG1 cells and changes in Bax/Bcl-2 ratio. The obtained results point to resistance to apoptosis as a possible mechanism underlying high level of radioresistance of HTB140 cells. Further experiments will be conducted to determine which modes of cell death contribute to biological response of this cell line to irradiation with 4He ions.

ABSTRACT. Radiation-induced DNA damage is considered as the most important cellular damage as it could lead to the loss of clonogenic capacity and cell death.1,2 Cell inactivation caused by irradiation results both from direct as well as from indirect actions mediated by free radicals.3 To discriminate the direct from indirect radiation actions is important because it provides essential information about the mechanisms by which radiation ultimately affects cells.4 Free radical scavengers, such as DMSO, can be used to reduce effects of indirectly induced DNA damages without affecting the direct effects of irradiation.4,5 The aim of this study is to estimate the contribution of each of these effects on breast adenocarcinoma cells irradiated with three types of irradiations, i.e. γ-rays, protons and carbon ions. The MCF-7 cells were pre-treated with DMSO and then irradiated with 60Co γ-rays, protons or carbon ions with doses ranging from 1-5 Gy. Cells were exposed to 62 MeV/u protons and carbon ions. Radiation position for protons was in the middle of the spread-out Bragg peak, while irradiations with carbon ions were carried out within slightly broadened Bragg peak to obtain LET with the highest biological effectiveness. Degree of protection (DP) was calculated for each dose and plotted as a function of the DMSO concentration. The contribution of indirect action in cell killing was obtained from the maximum DP provided by DMSO.4,5,6 According to the results, the DP of DMSO increased in all irradiated cells, in a concentration dependent manner. After γ-irradiations, the estimated direct effects were around 35%, while the 65% of total irradiation effects could be attributed to indirect actions. In cells irradiated with protons and carbon ions, higher contribution of direct effects was observed, being around 44% and 48%, respectively. Although contribution of direct effects was higher in proton and carbon ion irradiated cells, a substantial fraction of indirect actions was found, being around 56% for proton and 52% for carbon ions. This points to important role of free radical actions in high LET irradiation induced cell death. The calculated relative biological effectiveness at 10% survival (RBED10 values) also show that in high LET radiations, direct actions had a stronger impact on cellular growth inhibition than indirect effects.

ABSTRACT. In our previous study (1), we found the unique enhancing effects of low-dose fractionated radiation (LDFR) on cisplatin in two cervical cancer cell lines SiHa and CaSki (both cell lines with Human Papilloma Virus (HPV) presence and low-dose hyper-radiosensitivity (HRS) phenomenon absence). The aim of the present study is to identify molecular mechanisms underlying the chemopotentiating effects of LDFR, especially the role of radiation-induced ATM nucleoshuttling (RIANS), in four human head and neck cancer cell lines (with different status of HRS and with or without HPV infection). The presence of HRS phenomenon in the cell lines is estimated by flow cytometry-based clonogenic survival assay. The potentiating effects of LDFR 4 x 0.5 Gy versus a single dose of 2 Gy on carboplatin and paclitaxel are compared using clonogenic survival, pATM and gammaH2AX foci assays. For combined experiments, the cells are treated with cytostatic drug and irradiated 24 hours later with 6 MV X-ray beam. To date, HRS status was assessed for two cell lines. FaDu cells appeared to be HRS-positive (with extremely pronounced HRS region) and SCC-25 cells HRS-negative. In case of FaDu cells, preliminary results showed that LDFR 4 x 0.5 Gy enhanced the effects of carboplatin and paclitaxel (assessed by clonogenic survival assay) at the same level as a single dose of 2 Gy that suggests no sparing effect of fractionation when low radiation doses (0,5 Gy) are combined with these cytostatics. Research is ongoing and results for four cell lines will be presented at the conference.

This work is supported by the National Science Centre, Poland, grant no 2019/35/O/NZ3/03039.

1. Słonina D, Kabat D, Biesaga B, Janecka-Widła A, Szatkowski W. Chemopotentiating effects of low-dose fractionated radiation on cisplatin and paclitaxel in cervix cancer cell lines and normal fibroblasts from patients with cervix cancer. DNA Repair 103, 103113 (2021) doi: 10.1016/j.dnarep.2021.103113.

ABSTRACT. The three-dimensional architecture of genomes on the micro-, meso- and nano-scale acts in combination with epigenetic modifications as an important player of gene reg¬ulation and, consequently, fundamental biological processes such as DNA damage response and repair. So far only little is known about the impact of chromatin archi¬tecture and its geometry and topology on DNA double strand break (DSB) induction and repair pathway selection and progression at individual damage sites. How does a cell nucleus as system as a whole, process DSBs and re-organize the chromatin towards functionally intact repair units? And how, on the other hand, are repair cen¬tres oriented towards specific repair mechanisms formed at individual DSB sites? We present investigations of spatial and topological parameters of chromatin domains and DNA repair foci during a time period of repair to glimpse key aspects related to these questions. Nano-probing of chromatin damage sites and the recruited DNA repair proteins in combination with super-resolution Single Molecule Localization Mi¬croscopy (SMLM) are powerful methods for geometric and topological analyses of meso- and nano-structures in single cells and at single DSB sites and, thus, to study mechanisms of their formation and repair pathway regulation. We used variable technological tools based on image-free SMLM, nano-scaled molecule distribution analyses, appropriate metrics following Ripley´s distance frequencies and cluster formation analyses, as well as topological quantifications employing persistence ho¬mology. Comparing the topology of repair foci suggests general similarities in repair cluster formation, indicating a non-random, molecule topology at given time points during repair. The data reveal a specific architecture of DNA damage foci for a given chromatin domain and cell type. Characteristics of chromatin architecture around complex damage sites, repair focus nano-architecture or spatial arrangements of re¬pair proteins may contribute to control repair process. Our studies contribute to the understanding of whole system cellular radiation response.

ABSTRACT. Monte Carlo simulations of electron and proton irradiations in an oxygenated water with different oxygen concentrations were carried out using GEANT4 and TRAX-Chem toolkits by different groups. In this paper, the accuracy and efficiency of these codes are compared in order to evaluate the effect of oxygen in FLASH (ultra-high dose rate) irradiation. The recent studies on animal models indicated that FLASH radiation treatment, remarkably increases the radio resistance of normal tissues, while the tumor control remains similar to conventional treatment. Also, observations show that oxygen plays a significant role in this kind of irradiation, but the mechanism of the oxygenation effect in FLASH irradiation is not completely clarified. Simulated oxygen consumption, interaction of water radicals induced by particle radiation in the pre-chemical and chemical stages, radiolytic reactive oxygen species production and the calculated temporal yield (G-values in the chemical evolution time) were compared for electron and proton irradiations at various energies. A general close agreement was observed in all cases analyzed by these codes separately. It was concluded that both toolkits are suited for this class of simulation, although further improvements are needed in both toolkits.

ABSTRACT. Microtubules (MTs) are one of the three components of cell cytoskeleton composed by 13 protofilament laterally tied together and assembled around a hollow core, defined as a rod or strand, with a diameter about 25nm. MTs have some similarities with DNA, the preferred macromolecule target of ionizing radiation damage, but also many differences. Among the common points we can mention the dimensions and involvement of acetylation and methylation process. On the other hand, microtubules are uniformly distributed over the cell volume and are characterised by completely different repair/reorganisation mechanisms than DNA. A Previous work has shown effects from ionizing radiation on MT of non-tumor breast cells1. In this work MTs rearrangement and distribution of breast cancer cells after irradiation with proton in the dose range between 2 and 9 Gy, will be presented

ABSTRACT. Key to the DNA damage induced by radiation is the presence of oxygen. In conditions of low oxygen (radiobiological hypoxia) significantly less DNA damage is induced by radiation leading to therapy resistance and disease progression. Regions of hypoxia occur in most solid tumours and, although the degree of hypoxia (how little oxygen) varies, these include areas of radiobiological hypoxia. The focus of my work is the mechanistic investigation of the response to radiobiological hypoxia with a view to identifying therapeutic strategies to improve radiotherapy response. In particular, we have focused on the DNA damage response induced in radiobiological hypoxia which is unusual as it is dependent on replication stress and occurs in the absence of detectable DNA damage. Recently, we have implicated the unfolded protein response as part of the response to hypoxia-induced replication stress. An overview of these studies will be presented.

ABSTRACT. FLASH radiotherapy (RT) is attracting a significant interest since the first investigations carried out in 2014, demonstrated by the increasing number of related publications [1]. Several preclinical studies worldwide have demonstrated that ultra-high dose rate (UHDR) beams produce an improvement of normal tissue sparing while maintaining high tumor control probability compared to conventional dose-rate RT (FLASH effect). However, to fully understand the mechanisms and the biological processes, reliable beam monitoring and dosimetry technologies must be developed and new protocols are needed [2]. This is crucial to support the first clinical trials and for the clinical translation of FLASH RT. Currently used detectors saturate at these extreme regimes, therefore the optimization of already established technologies as well as the investigation of novel radiation detection and dosimetry methods are required [3]. The main challenges coming from the peculiar beam parameters characterizing UHDR beams for FLASH RT will be discussed. A status of the current technology will be provided, including recent developments for established detectors and novel approaches currently under investigation with a view to predict future directions in terms of dosimetry approaches and practical procedures for the clinical translation of FLASH RT.

1. V. Favaudon et al., Science Translational Medicine, 6(245), 245ra93 (2014). 2. F. Romano et al., Medical Physics, in-press, doi: 1002/mp.15649 3. M. McManus M., SCIENTIFIC REPORTS, vol. 10, ISSN: 2045-2322 (2020).

ABSTRACT. Introduction Compared to 2D cell cultures, ECM-derived bioscaffolds retain specific growth factors that facilitate cell adhesion, tissue integration, remodeling and differentiation. Additionally, these scaffolds are porous allowing the transport of oxygen and nutrients to the seeded cells and ensuring the waste metabolites’ physiological output. Gynecological mucosal melanomas (MMs) and pancreatic cancer (PaC) are aggressive tumors with a dismal prognosis. Preclinical and clinical data showed that both tumors display intrinsic resistance to conventional radiotherapy (RT), while C-ion RT could be valuable for their treatment. Materials and methods MM HMV-II and PaC PANC-1 cells were seeded in decellularized biological scaffolds and irradiated with 2 Gy and 4 Gy of photons (X-ray) or C-ions. Afterwards, every seven days for four weeks, control and irradiated scaffolds were formalin-fixed, embedded in paraffin and sectioned. Their histological specimens were stained with Hematoxylin and Eosin, Periodic acid-Schiff, Masson's trichrome, Alcian blue and Picrosirius red, and revised independently by two anatomopathological experts. Results MM and PaC cell lines repopulated the scaffolds in a histological-coherent way and were able to grow into depth, without dedifferentiation. Images of the sections indicate significant different morphological features, according to the type of RT and the characteristics of each cell line, starting from >7 days’ time point. In particular, C-ions exhibited more severe effects on PANC-1 cells in terms of early molecular alteration (binucleated cells, increased nuclei size, cytoplasm disaggregation, diapedesis). Melanin production of HMV-II cells was premature in the scaffolds treated with X-ray compared to C-ions. Conclusions Hepatic scaffolds provide a fertile 3D environment for tumor cell growth and proliferation and represent a promising experimental approach for more comprehensive radiobiological studies, even in the case of long-term studies.

ABSTRACT. Experimental synchrotron X-ray-generated microbeam radiation therapy (MRT) is an innovative model of cancer radiotherapy with an excellent therapeutic ratio, but optimization of the irradiation protocols, as well as assessment of metastatic spread, are needed to go ahead toward clinical implementation. Here, we demonstrated that: (i) two 396-Gy peak-dose fractions of MRT are more effective than one in attenuating tumor growth in the B16-F10 melanoma mouse model; (ii) both single dose MRT and broad beam irradiation accelerated the formation of metastasis in superficial cervical lymph nodes but remarkably the second MRT fraction triggered a very pronounced regression of locoregional metastasis that lasted for 5 weeks. This observed reduction cannot be explained by direct exposure of cervical lymph nodes to low-dose scattered radiation, therefore we hypothesized the presence of abscopal effects. In search for factors that generated this anti-tumor/anti-metastatic response, we measured plasma concentrations of 34 cytokines in cohorts of mice that received either one or two MRT fractions. Neutrophil and T cell-attracting chemokines CXCL5, CXCL12 and CCL22 were significantly increased two days after the second MRT irradiation, indicating that delayed melanoma growth and metastasis progression in animals treated with two MRT fractions could be a consequence of increased recruitment of anti-tumor neutrophils and T cells. Indeed, we demonstrated elevated infiltration of neutrophils and activated T-cells in the tumors following the second MRT. Our study indicates the approach for an optimal MRT regimen that promotes local and locoregional tumor control with the potential to manage distant metastasis, a most common cause of death even after successful treatment of the primary melanoma.

ABSTRACT. Our understanding of the impact of dose-rate in radiobiology and radiotherapy have been well defined for many years, particularly when considering the effect of reducing dose-rate relative to standard therapeutic dose-rates of around 1-2 Gy/min. The recent observation of FLASH radiation effects has re-focused interest in the role of high dose-rate exposures typically of ~ 100 Gy/s. Overall, the potential benefits of FLASH are related to the protection of normal tissues within the body. Although already the subject of clinical trials, the mechanisms underpinning FLASH responses are not yet fully defined and there are significant gaps in our understanding1. With this, there is a resurgence of interest in much higher dose-rates than the current FLASH regime to test whether novel biological responses will be apparent. A major focus is developing the potential of laser-based sources for the production of electrons, X-rays and charged particles. With rapid technological developments, dose-rates for protons, at near clinical energies (60-100 MeV), of 109Gy/s have been achieved for radiobiology studies2 and with electrons and gamma-rays there is the potential to reach extreme dose-rates > 1013Gy/s3. In these regimes, there are major gaps in our understanding, not only of biological response, but of the physical and chemical processes that are likely to be involved. Ultimately, the concept of FLASH and dose-rate effects are likely to be highly influenced by the spatial distribution of dose, at the tissue level, an expanding area of research. Defining both temporal and spatial effects will lead to a greater understanding of how cells, tissues and organism integrate radiation dose and open new opportunities for optimized radiation-based therapies.

1 FRIEDL, A., et al., 2022, Radiobiology of the FLASH Effect. Medical Physics, 49, 1993-2013 2 CHAUDHARY, P., et al., 2022, Development of a portable hypoxia chamber for ultra-high dose rate laser-driven proton radiobiology applications. BMC Radiation Oncology 17, 77 3 McANESPIE, C., et al., 2022, High-dose femtosecond-scale gamma-ray beams for radiobiological applications. Physics in Medicine and Biology 67, 085010

ABSTRACT. The current interest in ultra-high dose rate irradiation stems mainly from its well documented radiobiological sparing effect, compared to conventional dose rates, which has been thoroughly established in many cultured cancer cell lines in vitro, as well as in several different normal tissues in vivo. Although the size of the effect seems to depend on various conditions, for instance, including dose-rate parameters and environmental factors, there is also recent evidence for a differential sparing of normal tissue, as compared to the anti-tumour effect, which in theory may be used to widen the therapeutic window in radiotherapy. While encouraging data continue to accumulate, one of the present challenges in FLASH radiotherapy concerns how this widened window may best be exploited in clinical applications, alongside other differentially sparing treatment techniques that are already available, such as target conformity and dose fractionation. In this lecture, an overview of the FLASH project at Skåne University Hospital and Lund University will be presented, including preclinical in vitro and in vivo experiments, veterinary clinical trials, as well as the planned route towards a future clinical implementation, including novel technical solutions for ion chamber-based dosimetry, treatment planning, and motion management.

ABSTRACT. Compared to conventional dose-rate irradiation, ultra-high dose-rate irradiation can substantially widen the radiation therapy window, allowing the radiotherapist to spare the healthy tissues while controlling the tumor with the same efficacy. Nowadays, this effect is known as the FLASH effect, and it is a breakthrough in radiotherapy. Although the normal tissue sparing at ultra high dose-rate has been demonstrated with electrons, photons, and protons, so far, evidence with heavy ions is limited to in vitro cell experiments. We present the first in vivo results with high-energy 12C- ions delivered at an ultra-high dose rate.

In our results, irradiation with an ultra-high dose rate of carbon ions was able to control murine osteosarcoma in the posterior limb of C3H/He mice the same as with conventional dose-rate irradiation.

Moreover, FLASH irradiation decreases normal tissue toxicity and significantly reduces lung metastasis compared to conventional dose-rate irradiation and sham-irradiated animals.

ABSTRACT. Purpose: FLASH radiotherapy (FLASH-RT) is a new technique, involving treatment of tumours at ultra-high dose rates, which has been shown to reduce normal tissue from radiation-induced toxicity, whilst equalling the anti-tumour effect of conventional dose rate radiotherapy (CONV-RT). Here, we performed a dose-response study in mice comparing the effect of FLASH-RT versus CONV-RT on skin and lung toxicity as well as tumour response in a lung cancer xenograft model.

Experimental Design: Two human tumours xenografted in CD1-nude mice and one syngeneic tumour xenografted in C57BL/6J mice were used for the comparative determination of the antitumor response. Mice were treated using a 6 MeV electron linear accelerator once xenografts reached 80 mm3 with FLASH-RT or CONV-RT, both at a single dose of 20 Gy or at a fractionated dose of 30 Gy (3 x 10 Gy). Acute and late radiation effects were quantified in healthy C57BL/6J mice by skin toxicity scoring, lung CT-scan imaging and histopathological analysis, after hemithorax irradiation in the dose range of 10 to 30 Gy in a single fraction.

Results: We found that FLASH-RT and CONV-RT showed similar efficacy with regards to growth delay/control of lung cancer cells transplanted into immunocompromised and immunocompetent mice. No differences were observed between the treatments with single dose of 20 Gy and fractionated dose of 30 Gy, for both, FLASH and CONV irradiations. No macroscopic signs of cutaneous lesions were observed after 30 Gy hemithorax FLASH-RT, although we observed hair depigmentation restricted to the irradiated area. In contrast, mice exposed to 20 or 25 Gy CONV-RT developed severe cutaneous lesions and earlier hair depigmentation. Both, lung CT-scan imaging and histopathological analysis, demonstrated lower inflammation after FLASH-RT compared to CONV-RT.

Conclusions: In this study, the results showed that FLASH-RT reduces radiation-induced skin and lung toxicity, while showing equivalent tumour response as CONV-RT.