BACKGROUND AND PURPOSE: This study presents a multi-center comparison of in vitro cell survival measurements and RBE calculations following proton irradiations conducted under harmonized experimental conditions across six European institutions participating in the INSPIRE framework. MATERIALS AND METHODS: V79-4 cells were irradiated using spread-out Bragg peak (SOBP) proton fields of two configurations delivering 6 and 8 Gy with widths of 6 and 4 cm, respectively. Each center adhered to a standardized protocol, utilizing the same phantom design to minimize uncertainties related to sample positioning. X-ray reference irradiations were also performed to assess cell radiosensitivity across the participating centers. RESULTS: Despite the consistent protocol, significant inter-institutional variability was observed in the survival measurements. For both treatment plans, the largest variation was detected in the most distal points of the SOBP (coefficients of variation of 43 % and 60 % for the 6 Gy and 8 Gy plans, respectively). Kruskal-Wallis statistical test confirmed the significant differences between the centers for each of the measured position in the proton field for both SOBP configurations. Discrepancies were observed in calculated RBE data as well, albeit preserving the expected trend for the values to slightly increase towards the distal edge of the SOBP (up to 1.5 and 1.3 for the 6 Gy and 8 Gy plans, respectively). CONCLUSION: The results of the study highlight the minimal biological variation one could expect performing proton RBE measurements in well-aligned experimental conditions and challenges in conducting large-scale, multi-center radiobiological experiments and inter-comparisons between literature data sets.

• Keywords
• Cell survival, Proton therapy, Relative biological effectiveness,
• Publication type
• Journal Article MeSH

This paper has been prepared to commemorate the 70th anniversary of the Institute of Biophysics of the Czech Academy of Sciences (IBP CAS), which has a long-standing tradition in researching the biological effects of ionizing radiation (IR). Radiobiology has recently gained renewed importance due to several compelling factors. The demand for a better understanding of the biological effects of both low and high doses of various types of ionizing radiation, along with improved radiation protection, is increasing-particularly in the context of critical ongoing human activities such as medical diagnostics, radiotherapy, and the operation of nuclear power plants. This demand also extends to newly emerging scenarios, including the development of hadron and FLASH radiotherapy, as well as mixed radiation field exposures related to planned manned missions to Mars. Unfortunately, there is also an urgent need to address the heightened risk of nuclear materials and weapons misuse by terrorists or even rogue states. Additionally, nuclear energy is currently the only viable alternative that can provide efficient, sustainable, and ecological coverage for the dramatically increasing current and future energy demands. Understanding the risks of IR exposure necessitates exploring how different types of IR interact with living organisms at the most fundamental level of complexity, specifically at the level of molecules and their complexes. The rising interest in radiobiology is, therefore, also driven by new experimental opportunities that enable research at previously unimaginable levels of detail and complexity. In this manuscript, we will address the important questions in radiobiology, focusing specifically on the mechanisms of radiation-induced DNA damage and repair within the context of chromatin architecture. We will emphasize the differing effects of photon and high-LET particle radiation on chromatin and DNA. Both forms of IR are encountered on Earth but are particularly significant in space.

• Keywords
• Biological effects of ionizing radiation, Chromatin architecture at micro- and nano-scale, DNA damage and repair, Densely ionizing (high-LET) particle radiation, Institute of biophysics of the Czech academy of sciences, Microscopy, Photon radiation, Radiobiological research, Single molecule localization microscopy (SMLM),
• Publication type
• Journal Article MeSH
• Review MeSH

Boron has been suggested to enhance the biological effectiveness of proton beams in the Bragg peak region via the p + 11B → 3α nuclear capture reaction. However, a number of groups have observed no such enhancement in vitro or questioned its proposed mechanism recently. To help elucidate this phenomenon, we irradiated DU145 prostate cancer or U-87 MG glioblastoma cells by clinical 190 MeV proton beams in plateau or Bragg peak regions with or without 10B or 11B isotopes added as sodium mercaptododecaborate (BSH). The results demonstrate that 11B but not 10B or other components of the BSH molecule enhance cell killing by proton beams. The enhancement occurs selectively in the Bragg peak region, is present for boron concentrations as low as 40 ppm, and is not due to secondary neutrons. The enhancement is likely initiated by proton-boron capture reactions producing three alpha particles, which are rare events occurring in a few cells only, and their effects are amplified by intercellular communication to a population-level response. The observed up to 2-3-fold reductions in survival levels upon the presence of boron for the studied prostate cancer or glioblastoma cells suggest promising clinical applications for these tumour types.

• Keywords
• Biological effectiveness, Cell survival, Proton radiotherapy, Proton-boron capture therapy, Sodium mercaptododecaborate (BSH),
• MeSH
• Boron chemistry   MeSH
• Glioblastoma radiotherapy   drug therapy   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Prostatic Neoplasms radiotherapy   drug therapy   MeSH
• Proton Therapy * methods   MeSH
• Protons MeSH
• Boron Neutron Capture Therapy * methods   MeSH
• Cell Survival drug effects   radiation effects   MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Boron MeSH
• Protons MeSH

DNA double-strand breaks (DSBs), marked by ionizing radiation-induced (repair) foci (IRIFs), are the most serious DNA lesions and are dangerous to human health. IRIF quantification based on confocal microscopy represents the most sensitive and gold-standard method in radiation biodosimetry and allows research on DSB induction and repair at the molecular and single-cell levels. In this study, we introduce DeepFoci - a deep learning-based fully automatic method for IRIF counting and morphometric analysis. DeepFoci is designed to work with 3D multichannel data (trained for 53BP1 and γH2AX) and uses U-Net for nucleus segmentation and IRIF detection, together with maximally stable extremal region-based IRIF segmentation. The proposed method was trained and tested on challenging datasets consisting of mixtures of nonirradiated and irradiated cells of different types and IRIF characteristics - permanent cell lines (NHDFs, U-87) and primary cell cultures prepared from tumors and adjacent normal tissues of head and neck cancer patients. The cells were dosed with 0.5-8 Gy γ-rays and fixed at multiple (0-24 h) postirradiation times. Under all circumstances, DeepFoci quantified the number of IRIFs with the highest accuracy among current advanced algorithms. Moreover, while the detection error of DeepFoci remained comparable to the variability between two experienced experts, the software maintained its sensitivity and fidelity across dramatically different IRIF counts per nucleus. In addition, information was extracted on IRIF 3D morphometric features and repair protein colocalization within IRIFs. This approach allowed multiparameter IRIF categorization of single- or multichannel data, thereby refining the analysis of DSB repair processes and classification of patient tumors, with the potential to identify specific cell subclones. The developed software improves IRIF quantification for various practical applications (radiotherapy monitoring, biodosimetry, etc.) and opens the door to advanced DSB focus analysis and, in turn, a better understanding of (radiation-induced) DNA damage and repair.

• Keywords
• 53BP1, P53-binding protein 1, Biodosimetry, CNN, convolutional neural network, Confocal Microscopy, Convolutional Neural Network, DNA Damage and Repair, DSB, DNA double-strand break, Deep Learning, FOV, field of view, GUI, graphical user interface, IRIF, ionizing radiation-induced (repair) foci, Image Analysis, Ionizing Radiation-Induced Foci (IRIFs), MSER, maximally stable extremal region (algorithm), Morphometry, NHDFs, normal human dermal fibroblasts, RAD51, DNA repair protein RAD51 homolog 1, U-87, U-87 glioblastoma cell line, γH2AX, histone H2AX phosphorylated at serine 139,
• Publication type
• Journal Article MeSH