Molecular Investigation on a Triple Negative Breast Cancer Xenograft Model Exposed to Proton Beams.

Specific breast cancer (BC) subtypes are associated with bad prognoses due to the absence of successful treatment plans. The triple-negative breast cancer (TNBC) subtype, with estrogen (ER), progesterone (PR) and human epidermal growth factor-2 (HER2) negative receptor status, is a clinical challenge for oncologists, because of its aggressiveness and the absence of effective therapies. In addition, proton therapy (PT) represents an effective treatment against both inaccessible area located or conventional radiotherapy (RT)-resistant cancers, becoming a promising therapeutic choice for TNBC. Our study aimed to analyze the in vivo molecular response to PT and its efficacy in a MDA-MB-231 TNBC xenograft model. TNBC xenograft models were irradiated with 2, 6 and 9 Gy of PT. Gene expression profile (GEP) analyses and immunohistochemical assay (IHC) were performed to highlight specific pathways and key molecules involved in cell response to the radiation. GEP analysis revealed in depth the molecular response to PT, showing a considerable immune response, cell cycle and stem cell process regulation. Only the dose of 9 Gy shifted the balance toward pro-death signaling as a dose escalation which can be easily performed using proton beams, which permit targeting tumors while avoiding damage to the surrounding healthy tissue.

Proton Therapy and Src Family Kinase Inhibitor Combined Treatments on U87 Human Glioblastoma Multiforme Cell Line.

Glioblastoma Multiforme (GBM) is the most common of malignant gliomas in adults with an exiguous life expectancy. Standard treatments are not curative and the resistance to both chemotherapy and conventional radiotherapy (RT) plans is the main cause of GBM care failures. Proton therapy (PT) shows a ballistic precision and a higher dose conformity than conventional RT. In this study we investigated the radiosensitive effects of a new targeted compound, SRC inhibitor, named Si306, in combination with PT on the U87 glioblastoma cell line. Clonogenic survival assay, dose modifying factor calculation and linear-quadratic model were performed to evaluate radiosensitizing effects mediated by combination of the Si306 with PT. Gene expression profiling by microarray was also conducted after PT treatments alone or combined, to identify gene signatures as biomarkers of response to treatments. Our results indicate that the Si306 compound exhibits a radiosensitizing action on the U87 cells causing a synergic cytotoxic effect with PT. In addition, microarray data confirm the SRC role as the main Si306 target and highlights new genes modulated by the combined action of Si306 and PT. We suggest, the Si306 as a new candidate to treat GBM in combination with PT, overcoming resistance to conventional treatments.

SiCILIA-Silicon Carbide Detectors for Intense Luminosity Investigations and Applications.

Silicon carbide (SiC) is a compound semiconductor, which is considered as a possible alternative to silicon for particles and photons detection. Its characteristics make it very promising for the next generation of nuclear and particle physics experiments at high beam luminosity. Silicon Carbide detectors for Intense Luminosity Investigations and Applications (SiCILIA) is a project starting as a collaboration between the Italian National Institute of Nuclear Physics (INFN) and IMM-CNR, aiming at the realization of innovative detection systems based on SiC. In this paper, we discuss the main features of silicon carbide as a material and its potential application in the field of particles and photons detectors, the project structure and the strategies used for the prototype realization, and the first results concerning prototype production and their performance.

Computational approaches in the estimation of radiobiological damage for human-malignant cells irradiated with clinical proton and carbon beams.

PURPOSE: The use of Monte Carlo (MC) simulations capable of reproducing radiobiological effects of ionising radiation on human cell lines is of great importance, especially for cases involving protons and heavier ion beams. In the latter, huge uncertainties can arise mainly related to the effects of the secondary particles produced in the beam-tissue interaction. This paper reports on a detailed MC study performed using Geant4-based approach on three cancer cell lines, the HTB-177, CRL-5876 and MCF-7, that were previously irradiated with therapeutic proton and carbon ion beams. METHODS: A Geant4-based approach used jointly with analytical calculations has been developed to provide a more realistic estimation of the radiobiological damage produced by proton and carbon beams in tissues, reproducing available data obtained from in vitro cell irradiations. The MC "Hadrontherapy" Geant4 application and the Local Effect Model: LEM I, LEM II and LEM III coupled with the different numerical approaches: RapidRusso (RR) and RapidScholz (RS) were used in the study. RESULTS: Experimental survival curves are compared with those evaluated using the highlighted Geant4 MC-based approach via chi-square statistical analysis, for the combinations of radiobiological models and numerical approaches, as outlined above. CONCLUSION: This study has presented a comparison of the survival data from MC simulations to experimental survival data for three cancer cell lines. An overall best level of agreement was obtained for the HTB-177 cells.

Impact on the Transcriptome of Proton Beam Irradiation Targeted at Healthy Cardiac Tissue of Mice.

Proton beam therapy is considered a step forward with respect to electromagnetic radiation, thanks to the reduction in the dose delivered. Among unwanted effects to healthy tissue, cardiovascular complications are a known long-term radiotherapy complication. The transcriptional response of cardiac tissue from xenografted BALB/c nude mice obtained at 3 and 10 days after proton irradiation covering both the tumor region and the underlying healthy tissue was analyzed as a function of dose and time. Three doses were used: 2 Gy, 6 Gy, and 9 Gy. The intermediate dose had caused the greatest impact at 3 days after irradiation: at 2 Gy, 219 genes were differently expressed, many of them represented by zinc finger proteins; at 6 Gy, there were 1109, with a predominance of genes involved in energy metabolism and responses to stimuli; and at 9 Gy, there were 105, mainly represented by zinc finger proteins and molecules involved in the regulation of cardiac function. After 10 days, no significant effects were detected, suggesting that cellular repair mechanisms had defused the potential alterations in gene expression. The nonlinear dose-response curve indicates a need to update the models built on photons to improve accuracy in health risk prediction. Our data also suggest a possible role for zinc finger protein genes as markers of proton therapy efficacy.

Characterization of a flexible a-Si:H detector for in vivo dosimetry in therapeutic x-ray beams.

BACKGROUND: The increasing use of complex and high dose-rate treatments in radiation therapy necessitates advanced detectors to provide accurate dosimetry. Rather than relying on pre-treatment quality assurance (QA) measurements alone, many countries are now mandating the use of in vivo dosimetry, whereby a dosimeter is placed on the surface of the patient during treatment. Ideally, in vivo detectors should be flexible to conform to a patient's irregular surfaces. PURPOSE: This study aims to characterize a novel hydrogenated amorphous silicon (a-Si:H) radiation detector for the dosimetry of therapeutic x-ray beams. The detectors are flexible as they are fabricated directly on a flexible polyimide (Kapton) substrate. METHODS: The potential of this technology for application as a real-time flexible detector is investigated through a combined dosimetric and flexibility study. Measurements of fundamental dosimetric quantities were obtained including output factor (OF), dose rate dependence (DPP), energy dependence, percentage depth dose (PDD), and angular dependence. The response of the a-Si:H detectors investigated in this study are benchmarked directly against commercially available ionization chambers and solid-state diodes currently employed for QA practices. RESULTS: The a-Si:H detectors exhibit remarkable dose linearities in the direct detection of kV and MV therapeutic x-rays, with calibrated sensitivities ranging from (0.580 ± 0.002) pC/cGy to (19.36 ± 0.10) pC/cGy as a function of detector thickness, area, and applied bias. Regarding dosimetry, the a-Si:H detectors accurately obtained OF measurements that parallel commercially available detector solutions. The PDD response closely matched the expected profile as predicted via Geant4 simulations, a PTW Farmer ionization chamber and a PTW ROOS chamber. The most significant variation in the PDD performance was 5.67%, observed at a depth of 3 mm for detectors operated unbiased. With an external bias, the discrepancy in PDD response from reference data was confined to ± 2.92% for all depths (surface to 250 mm) in water-equivalent plastic. Very little angular dependence is displayed between irradiations at angles of 0° and 180°, with the most significant variation being a 7.71% decrease in collected charge at a 110° relative angle of incidence. Energy dependence and dose per pulse dependence are also reported, with results in agreement with the literature. Most notably, the flexibility of a-Si:H detectors was quantified for sample bending up to a radius of curvature of 7.98 mm, where the recorded photosensitivity degraded by (-4.9 ± 0.6)% of the initial device response when flat. It is essential to mention that this small bending radius is unlikely during in vivo patient dosimetry. In a more realistic scenario, with a bending radius of 15-20 mm, the variation in detector response remained within ± 4%. After substantial bending, the detector's photosensitivity when returned to a flat condition was (99.1 ± 0.5)% of the original response. CONCLUSIONS: This work successfully characterizes a flexible detector based on thin-film a-Si:H deposited on a Kapton substrate for applications in therapeutic x-ray dosimetry. The detectors exhibit dosimetric performances that parallel commercially available dosimeters, while also demonstrating excellent flexibility results.

Proton Bragg curve and energy reconstruction using an online scintillator stack detector.

Real-time measurement and characterization of laser-driven proton beams have become crucial with the advent of high-repetition-rate laser acceleration. Common passive diagnostics such as radiochromic film (RCF) are not suitable for real-time operation due to time-consuming post-processing; therefore, a novel approach is needed. Various scintillator-based detectors have recently gained interest as real-time substitutes to RCF-thanks to their fast response for a wide range of dose deposition rates. This work introduces a compact, scalable, and cost-effective scintillator-based device for proton beam measurements in real-time suitable for the laser-plasma environment. An advanced signal processing technique was implemented based on detailed Monte Carlo simulations, enabling an accurate unfolding of the proton energy and the depth-dose deposition curve. The quenching effect was accounted for based on Birks' law with the help of the Monte Carlo simulations. The detector was tested in a proof-of-principle experiment at a conventional cyclotron accelerating protons up to 35 MeV of energy. The signal comparison with a standard RCF stack was also performed during the test of the device, showing an excellent agreement between the two diagnostics. Such devices would be suitable for both conventional and laser-driven proton beam characterization.

Proton boron capture therapy (PBCT) induces cell death and mitophagy in a heterotopic glioblastoma model.

Despite aggressive therapeutic regimens, glioblastoma (GBM) represents a deadly brain tumor with significant aggressiveness, radioresistance and chemoresistance, leading to dismal prognosis. Hypoxic microenvironment, which characterizes GBM, is associated with reduced therapeutic effectiveness. Moreover, current irradiation approaches are limited by uncertain tumor delineation and severe side effects that comprehensively lead to unsuccessful treatment and to a worsening of the quality of life of GBM patients. Proton beam offers the opportunity of reduced side effects and a depth-dose profile, which, unfortunately, are coupled with low relative biological effectiveness (RBE). The use of radiosensitizing agents, such as boron-containing molecules, enhances proton RBE and increases the effectiveness on proton beam-hit targets. We report a first preclinical evaluation of proton boron capture therapy (PBCT) in a preclinical model of GBM analyzed via µ-positron emission tomography/computed tomography (µPET-CT) assisted live imaging, finding a significant increased therapeutic effectiveness of PBCT versus proton coupled with an increased cell death and mitophagy. Our work supports PBCT and radiosensitizing agents as a scalable strategy to treat GBM exploiting ballistic advances of proton beam and increasing therapeutic effectiveness and quality of life in GBM patients.

Hydrogenated amorphous silicon high flux x-ray detectors for synchrotron microbeam radiation therapy.

Objective. Microbeam radiation therapy (MRT) is an alternative emerging radiotherapy treatment modality which has demonstrated effective radioresistant tumour control while sparing surrounding healthy tissue in preclinical trials. This apparent selectivity is achieved through MRT combining ultra-high dose rates with micron-scale spatial fractionation of the delivered x-ray treatment field. Quality assurance dosimetry for MRT must therefore overcome a significant challenge, as detectors require both a high dynamic range and a high spatial resolution to perform accurately.Approach. In this work, a series of radiation hard a-Si:H diodes, with different thicknesses and carrier selective contact configurations, have been characterised for x-ray dosimetry and real-time beam monitoring applications in extremely high flux beamlines utilised for MRT at the Australian Synchrotron.Results. These devices displayed superior radiation hardness under constant high dose-rate irradiations on the order of 6000 Gy s-1, with a variation in response of 10% over a delivered dose range of approximately 600 kGy. Dose linearity of each detector to x-rays with a peak energy of 117 keV is reported, with sensitivities ranging from (2.74 ± 0.02) nC/Gy to (4.96 ± 0.02) nC/Gy. For detectors with 0.8µm thick active a-Si:H layer, their operation in an edge-on orientation allows for the reconstruction of micron-size beam profiles (microbeams). The microbeams, with a nominal full-width-half-max of 50µm and a peak-to-peak separation of 400µm, were reconstructed with extreme accuracy. The full-width-half-max was observed as 55 ± 1µm. Evaluation of the peak-to-valley dose ratio and dose-rate dependence of the devices, as well as an x-ray induced charge (XBIC) map of a single pixel is also reported.Significance. These devices based on novel a-Si:H technology possess a unique combination of accurate dosimetric performance and radiation resistance, making them an ideal candidate for x-ray dosimetry in high dose-rate environments such as FLASH and MRT.

Development of a portable hypoxia chamber for ultra-high dose rate laser-driven proton radiobiology applications.

BACKGROUND: There is currently significant interest in assessing the role of oxygen in the radiobiological effects at ultra-high dose rates. Oxygen modulation is postulated to play a role in the enhanced sparing effect observed in FLASH radiotherapy, where particles are delivered at 40-1000 Gy/s. Furthermore, the development of laser-driven accelerators now enables radiobiology experiments in extreme regimes where dose rates can exceed 109 Gy/s, and predicted oxygen depletion effects on cellular response can be tested. Access to appropriate experimental enviroments, allowing measurements under controlled oxygenation conditions, is a key requirement for these studies. We report on the development and application of a bespoke portable hypoxia chamber specifically designed for experiments employing laser-driven sources, but also suitable for comparator studies under FLASH and conventional irradiation conditions. MATERIALS AND METHODS: We used oxygen concentration measurements to test the induction of hypoxia and the maintenance capacity of the chambers. Cellular hypoxia induction was verified using hypoxia inducible factor-1α immunostaining. Calibrated radiochromic films and GEANT-4 simulations verified the dosimetry variations inside and outside the chambers. We irradiated hypoxic human skin fibroblasts (AG01522B) cells with laser-driven protons, conventional protons and reference 225 kVp X-rays to quantify DNA DSB damage and repair under hypoxia. We further measured the oxygen enhancement ratio for cell survival after X-ray exposure in normal fibroblast and radioresistant patient- derived GBM stem cells. RESULTS: Oxygen measurements showed that our chambers maintained a radiobiological hypoxic environment for at least 45 min and pathological hypoxia for up to 24 h after disconnecting the chambers from the gas supply. We observed a significant reduction in the 53BP1 foci induced by laser-driven protons, conventional protons and X-rays in the hypoxic cells compared to normoxic cells at 30 min post-irradiation. Under hypoxic irradiations, the Laser-driven protons induced significant residual DNA DSB damage in hypoxic AG01522B cells compared to the conventional dose rate protons suggesting an important impact of these extremely high dose-rate exposures. We obtained an oxygen enhancement ratio (OER) of 2.1 ± 0.1 and 2.5 ± 0.1 respectively for the AG01522B and patient-derived GBM stem cells for X-ray irradiation using our hypoxia chambers. CONCLUSION: We demonstrated the design and application of portable hypoxia chambers for studying cellular radiobiological endpoints after exposure to laser-driven protons at ultra-high dose, conventional protons and X-rays. Suitable levels of reduced oxygen concentration could be maintained in the absence of external gassing to quantify hypoxic effects. The data obtained provided indication of an enhanced residual DNA DSB damage under hypoxic conditions at ultra-high dose rate compared to the conventional protons or X-rays.

High-Resolution Photoemission Study of Neutron-Induced Defects in Amorphous Hydrogenated Silicon Devices.

In this paper, by means of high-resolution photoemission, soft X-ray absorption and atomic force microscopy, we investigate, for the first time, the mechanisms of damaging, induced by neutron source, and recovering (after annealing) of p-i-n detector devices based on hydrogenated amorphous silicon (a-Si:H). This investigation will be performed by mean of high-resolution photoemission, soft X-Ray absorption and atomic force microscopy. Due to dangling bonds, the amorphous silicon is a highly defective material. However, by hydrogenation it is possible to reduce the density of the defect by several orders of magnitude, using hydrogenation and this will allow its usage in radiation detector devices. The investigation of the damage induced by exposure to high energy irradiation and its microscopic origin is fundamental since the amount of defects determine the electronic properties of the a-Si:H. The comparison of the spectroscopic results on bare and irradiated samples shows an increased degree of disorder and a strong reduction of the Si-H bonds after irradiation. After annealing we observe a partial recovering of the Si-H bonds, reducing the disorder in the Si (possibly due to the lowering of the radiation-induced dangling bonds). Moreover, effects in the uppermost coating are also observed by spectroscopies.

DNA double-strand breaks in cancer cells as a function of proton linear energy transfer and its variation in time.

PURPOSE: The complex relationship between linear energy transfer (LET) and cellular response to radiation is not yet fully elucidated. To better characterize DNA damage after irradiations with therapeutic protons, we monitored formation and disappearance of DNA double-strand breaks (DNA DSB) as a function of LET and time. Comparisons with conventional γ-rays and high LET carbon ions were also performed. MATERIALS AND METHODS: In the present work, we performed immunofluorescence-based assay to determine the amount of DNA DSB induced by different LET values along the 62 MeV therapeutic proton Spread out Bragg peak (SOBP) in three cancer cell lines, i.e. HTB140 melanoma, MCF-7 breast adenocarcinoma and HTB177 non-small lung cancer cells. Time dependence of foci formation was followed as well. To determine irradiation positions, corresponding to the desired LET values, numerical simulations were carried out using Geant4 toolkit. We compared γ-H2AX foci persistence after irradiations with protons to that of γ-rays and carbon ions. RESULTS: With the rise of LET values along the therapeutic proton SOBP, the increase of γ-H2AX foci number is detected in the three cell lines up to the distal end of the SOBP, while there is a decrease on its distal fall-off part. With the prolonged incubation time, the number of foci gradually drops tending to attain the residual level. For the maximum number of DNA DSB, irradiation with protons attain higher level than that of γ-rays. Carbon ions produce more DNA DSB than protons but not substantially. The number of residual foci produced by γ-rays is significantly lower than that of protons and particularly carbon ions. Carbon ions do not produce considerably higher number of foci than protons, as it could be expected due to their physical properties. CONCLUSIONS: In situ visualization of γ-H2AX foci reveal creation of more lesions in the three cell lines by clinically relevant proton SOBP than γ-rays. The lack of significant differences in the number of γ-H2AX foci between the proton and carbon ion-irradiated samples suggests an increased complexity of DNA lesions and slower repair kinetics after carbon ions compared to protons. For all three irradiation types, there is no major difference between the three cell lines shortly after irradiations, while later on, the formation of residual foci starts to express the inherent nature of tested cells, therefore increasing discrepancy between them.

Biological Impact of Target Fragments on Proton Treatment Plans: An Analysis Based on the Current Cross-Section Data and a Full Mixed Field Approach.

Clinical routine in proton therapy currently neglects the radiobiological impact of nuclear target fragments generated by proton beams. This is partially due to the difficult characterization of the irradiation field. The detection of low energetic fragments, secondary protons and fragments, is in fact challenging due to their very short range. However, considering their low residual energy and therefore high LET, the possible contribution of such heavy particles to the overall biological effect could be not negligible. In this context, we performed a systematic analysis aimed at an explicit assessment of the RBE (relative biological effectiveness, i.e., the ratio of photon to proton physical dose needed to achieve the same biological effect) contribution of target fragments in the biological dose calculations of proton fields. The TOPAS Monte Carlo code has been used to characterize the radiation field, i.e., for the scoring of primary protons and fragments in an exemplary water target. TRiP98, in combination with LEM IV RBE tables, was then employed to evaluate the RBE with a mixed field approach accounting for fragments' contributions. The results were compared with that obtained by considering only primary protons for the pristine beam and spread out Bragg peak (SOBP) irradiations, in order to estimate the relative weight of target fragments to the overall RBE. A sensitivity analysis of the secondary particles production cross-sections to the biological dose has been also carried out in this study. Finally, our modeling approach was applied to the analysis of a selection of cell survival and RBE data extracted from published in vitro studies. Our results indicate that, for high energy proton beams, the main contribution to the biological effect due to the secondary particles can be attributed to secondary protons, while the contribution of heavier fragments is mainly due to helium. The impact of target fragments on the biological dose is maximized in the entrance channels and for small α/ß values. When applied to the description of survival data, model predictions including all fragments allowed better agreement to experimental data at high energies, while a minor effect was observed in the peak region. An improved description was also obtained when including the fragments' contribution to describe RBE data. Overall, this analysis indicates that a minor contribution can be expected to the overall RBE resulting from target fragments. However, considering the fragmentation effects can improve the agreement with experimental data for high energy proton beams.

In-field and out-of-field microdosimetric characterisation of a 62 MeV proton beam at CATANA.

PURPOSE: A 5 and 10 µm thin silicon on insulator (SOI) 3D mushroom microdosimeter was used to characterize both the in-field and out-of-field of a 62 MeV proton beam. METHODS: The SOI mushroom microdosimeter consisted of an array of cylindrical sensitive volumes (SVs), developed by the Centre for Medical Radiation Physics, University of Wollongong, was irradiated with 62 MeV protons at the CATANA (Centro di AdroTerapia Applicazioni Nucleari Avanzate) facility in Catania, Italy, a facility dedicated to the radiation treatment of ocular melanomas. Dose mean lineal energy, ( y D ¯ ), values were obtained at various depths in PMMA along a pristine and spread out Bragg peak (SOBP). The measured microdosimetric spectra at each position were then used as inputs into the modified Microdosimetric Kinetic Model (MKM) to derive the RBE for absorbed dose in a middle of the SOBP 2Gy (RBED ). Microdosimetric spectra were obtained with both the 5 and 10 µm 3D SOI microdosimeters, with a focus on the distal part of the BP. The in-field and out-of-field measurement configurations along the Bragg curve were modeled in Geant4 for comparison with experimental results. Lateral out-of-field measurements were performed to study secondary particles' contribution to normal tissue's dose, up to 12 mm from the edge of the beam field, and quality factor and dose equivalent results were obtained. RESULTS: Comparison between experimental and simulation results showed good agreement between one another for both the pristine and SOBP beams in terms of y D ¯ and RBED. Though a small discrepancy between experiment and simulation was seen at the entrance of the Bragg curve, where experimental results were slightly lower than Geant4. The dose equivalent value measured 12 mm from the edge of the target volume was 1.27 ± 0.15 mSv/Gy with a Q ¯ value of 2.52 ± 0.30, both of which agree within uncertainty with Geant4 simulation. CONCLUSIONS: These results demonstrate that SOI microdosimeters are an effective tool to predict RBED in-field as well as dose equivalent monitoring out-of-field to provide insight to probability of second cancer generation.

Hypoxia Transcriptomic Modifications Induced by Proton Irradiation in U87 Glioblastoma Multiforme Cell Line.

In Glioblastoma Multiforme (GBM), hypoxia is associated with radioresistance and poor prognosis. Since standard GBM treatments are not always effective, new strategies are needed to overcome resistance to therapeutic treatments, including radiotherapy (RT). Our study aims to shed light on the biomarker network involved in a hypoxic (0.2% oxygen) GBM cell line that is radioresistant after proton therapy (PT). For cultivating cells in acute hypoxia, GSI's hypoxic chambers were used. Cells were irradiated in the middle of a spread-out Bragg peak with increasing PT doses to verify the greater radioresistance in hypoxic conditions. Whole-genome cDNA microarray gene expression analyses were performed for samples treated with 2 and 10 Gy to highlight biological processes activated in GBM following PT in the hypoxic condition. We describe cell survival response and significant deregulated pathways responsible for the cell death/survival balance and gene signatures linked to the PT/hypoxia configurations assayed. Highlighting the molecular pathways involved in GBM resistance following hypoxia and ionizing radiation (IR), this work could suggest new molecular targets, allowing the development of targeted drugs to be suggested in association with PT.

The Proton-Boron Reaction Increases the Radiobiological Effectiveness of Clinical Low- and High-Energy Proton Beams: Novel Experimental Evidence and Perspectives.

Protontherapy is a rapidly expanding radiotherapy modality where accelerated proton beams are used to precisely deliver the dose to the tumor target but is generally considered ineffective against radioresistant tumors. Proton-Boron Capture Therapy (PBCT) is a novel approach aimed at enhancing proton biological effectiveness. PBCT exploits a nuclear fusion reaction between low-energy protons and 11B atoms, i.e. p+11B→ 3α (p-B), which is supposed to produce highly-DNA damaging α-particles exclusively across the tumor-conformed Spread-Out Bragg Peak (SOBP), without harming healthy tissues in the beam entrance channel. To confirm previous work on PBCT, here we report new in-vitro data obtained at the 62-MeV ocular melanoma-dedicated proton beamline of the INFN-Laboratori Nazionali del Sud (LNS), Catania, Italy. For the first time, we also tested PBCT at the 250-MeV proton beamline used for deep-seated cancers at the Centro Nazionale di Adroterapia Oncologica (CNAO), Pavia, Italy. We used Sodium Mercaptododecaborate (BSH) as 11B carrier, DU145 prostate cancer cells to assess cell killing and non-cancer epithelial breast MCF-10A cells for quantifying chromosome aberrations (CAs) by FISH painting and DNA repair pathway protein expression by western blotting. Cells were exposed at various depths along the two clinical SOBPs. Compared to exposure in the absence of boron, proton irradiation in the presence of BSH significantly reduced DU145 clonogenic survival and increased both frequency and complexity of CAs in MCF-10A cells at the mid- and distal SOBP positions, but not at the beam entrance. BSH-mediated enhancement of DNA damage response was also found at mid-SOBP. These results corroborate PBCT as a strategy to render protontherapy amenable towards radiotherapy-resilient tumor. If coupled with emerging proton FLASH radiotherapy modalities, PBCT could thus widen the protontherapy therapeutic index.

A radiobiological study of carbon ions of different linear energy transfer in resistant human malignant cell lines.

PURPOSE: Analysis of elimination of four human radioresistant malignant cell lines to mono-energetic and non mono-energetic incoming carbon ion beams, characterized by different linear energy transfer (LET) qualities is performed. Comparisons with protons from the middle of the therapeutic spread out Bragg peak (SOBP) and reference γ-rays are also included. MATERIALS AND METHODS: HTB140 cells were irradiated at five positions, with different LET, along the 62 MeV carbon pristine Bragg peak. To provide reliable reproducibility of irradiations at INFN-LNS, as the carbon Bragg peak is very narrow, precise positioning of samples for desired LET value is complicated. The peak was slightly widened using two ripple filters. After defining irradiation position and LET at the peak itself where cell killing is almost the highest, irradiation position with the same LET value was found within somewhat broadened peak. HTB140, MCF-7, HTB177 and CRL5876 cells were irradiated at the two described positions. Additionally, irradiations in the middle of 62 MeV proton SOBP and reference γ-rays were performed. Doses ranged from 0.5 to 16 Gy. Cell survival and corresponding radiobiological parameters were assessed seven days after irradiations. RESULTS: When moving irradiation position along the carbon Bragg curve, LET rises from 85 to 747 keV/µm, while surviving fraction at 2 Gy (SF2) for HTB140 cells, falls from 0.72 to 0.57 further rising to 0.73 on the distal fall-off part of the curve. Improved cell radiosensitivity is seen for the doses below 4 Gy. Relative biological effectiveness (RBE) increases from 4.56 to 7.69 and drops to 4.23. Almost the highest cell killing LET, being ∼200 keV/µm, is used to irradiate HTB140, MCF-7, HTB177 and CRL5876 cells within the pristine and slightly broadened Bragg peak. After irradiations with protons of the mid SOBP, carbon ions of the pristine and slightly widened Bragg peak RBE ranges for HTB140 cells from 2.08, 4.81 to 7.06, for MCF-7 from 1.70, 3.28 to 4.17, for HTB177 from 1.98, 4.18 to 5.08 and for CRL5876 from 1.33, 2.57 to 3.51. CONCLUSIONS: Significant elimination of HTB140 cells is observed along the carbon Bragg curve. The highest one is achieved by LET that is at the level of already reported. For the same LET, mono-energetic carbon ions provide higher cell elimination than the non mono-energetic. For all cell lines, both carbon ion beams, more the monoenergetic one, express stronger killing rate than protons and especially γ-rays.

Evaluation of proton beam radiation-induced skin injury in a murine model using a clinical SOBP.

The purpose of this paper is to characterize the skin deterministic damage due to the effect of proton beam irradiation in mice occurred during a long-term observational experiment. This study was initially defined to evaluate the insurgence of myelopathy irradiating spinal cords with the distal part of a Spread-out Bragg peak (SOBP). To the best of our knowledge, no study has been conducted highlighting high grades of skin injury at the dose used in this paper. Nevertheless these effects occurred. In this regard, the experimental evidence of significant insurgence of skin injury induced by protons using a SOBP configuration will be shown. Skin damages were classified into six scores (from 0 to 5) according to the severity of the injuries and correlated to ED50 (i.e. the radiation dose at which 50% of animals show a specific score) at 40 days post-irradiation (d.p.i.). The effects of radiation on the overall animal wellbeing have been also monitored and the severity of radiation-induced skin injuries was observed and quantified up to 40 d.p.i.

High-current stream of energetic α particles from laser-driven proton-boron fusion.

The nuclear reaction known as proton-boron fusion has been triggered by a subnanosecond laser system focused onto a thick boron nitride target at modest laser intensity (∼10^{16}W/cm^{2}), resulting in a record yield of generated α particles. The estimated value of α particles emitted per laser pulse is around 10^{11}, thus orders of magnitude higher than any other experimental result previously reported. The accelerated α-particle stream shows unique features in terms of kinetic energy (up to 10 MeV), pulse duration (∼10 ns), and peak current (∼2 A) at 1 m from the source, promising potential applications of such neutronless nuclear fusion reactions. We have used a beam-driven fusion scheme to explain the total number of α particles generated in the nuclear reaction. In this model, protons accelerated inside the plasma, moving forward into the bulk of the target, can interact with ^{11}B atoms, thus efficiently triggering fusion reactions. An overview of literature results obtained with different laser parameters, experimental setups, and target compositions is reported and discussed.

Sensitization of chondrosarcoma cells with PARP inhibitor and high-LET radiation.

Chondrosarcoma is a malignant tumor that arises from cartilaginous tissue and is radioresistant and chemoresistant to conventional treatments. The preferred treatment consists of surgical resection, which might cause severe disabilities for the patient; in addition, this procedure might be impossible for inoperable locations, such as the skull base. Carbon ion irradiation (hadron therapy) has been proposed as an alternative treatment, primarily due to its greater biological effectiveness and improved ballistic properties compared with conventional radiotherapy with X-rays. The goal of this study was to characterize the genetic mutations of a grade III chondrosarcoma cell line (CH2879) and examine the cellular responses to conventional radiotherapy (X-rays) and hadron therapy (proton and carbon ions) in the presence of the PARP inhibitor Olaparib. To better understand PARP inhibition, we first analyzed the formation of poly-ADP ribose chains by western blot; we observed an increase in its signal after irradiation, which disappeared on addition of the PARP inhibitor. PARPi enhanced ratio of approximately 1.3, 1.8, and 1.5 following irradiation of cells with X-rays, protons, and C-ions, respectively, as detected by clonogenic assay. The decrease in cell survival was confirmed by proliferation assay. The radiosensitivity of CH2879 cells was associated with mutations in homologous recombination repair genes, such as RAD50, SMARCA2 and NBN. This study demonstrates the capacity of the PARP inhibitor Olaparib to radiosensitize mutated chondrosarcoma cells to conventional photon irradiation, proton and carbon ion irradiation.