Evaluation of Proton-Induced Biomolecular Changes in MCF-10A Breast Cells by Means of FT-IR Microspectroscopy

Radiotherapy (RT) with accelerated beams of charged particles (protons and carbon ions), also known as hadrontherapy, is a treatment modality that is increasingly being adopted thanks to the several benefits that it grants compared to conventional radiotherapy (CRT) treatments performed by means of high-energy photons/electrons. Hence, information about the biomolecular effects in exposed cells caused by such particles is needed to better realize the underlying radiobiological mechanisms and to improve this therapeutic strategy. To this end, Fourier transform infrared microspectroscopy (-FT-IR) can be usefully employed, in addition to long-established radiobiological techniques, since it is currently considered a helpful tool for examining radiation-induced cellular changes. In the present study, MCF-10A breast cells were chosen to evaluate the effects of proton exposure using -FT-IR. They were exposed to different proton doses and fixed at various times after exposure to evaluate direct effects due to proton exposure and the kinetics of DNA damage repair. Irradiated and control cells were examined in transflection mode using low-e substrates that have been recently demonstrated to offer a fast and direct way to examine proton-exposed cells. The acquired spectra were analyzed using a deconvolution procedure and a ratiometric approach, both of which showed the different contributions of DNA, protein, lipid, and carbohydrate cell components. These changes were particularly significant for cells fixed 48 and 72 h after exposure. Lipid changes were related to variations in membrane fluidity, and evidence of DNA damage was highlighted. The analysis of the Amide III band also indicated changes that could be related to different enzyme contributions in DNA repair

FT-IR Transflection Micro-Spectroscopy Study on Normal Human Breast Cells after Exposure to a Proton Beam

Fourier transform infrared micro-spectroscopy (mu-FT-IR) is nowadays considered a valuable tool for investigating the changes occurring in human cells after exposure to ionizing radiation. Recently, considerable attention has been devoted to the use of this optical technique in the study of cells exposed to proton beams, that are being increasingly adopted in cancer therapy. Different experimental configurations are used for proton irradiation and subsequent spectra acquisition. To facilitate the use of mu-FT-IR, it may be useful to investigate new experimental approaches capable of speeding up and simplifying the irradiation and measurements phases. Here, we propose the use of low-e-substrates slides for cell culture, allowing the irradiation and spectra acquisition in transflection mode in a fast and direct way. In recent years, there has been a wide debate about the validity of these supports, but many researchers agree that the artifacts due to the presence of the electromagnetic standing wave effects are negligible in many practical cases. We investigated human normal breast cells (MCF-10 cell line) fixed immediately after the irradiation with graded proton radiation doses (0, 0.5, 2, and 4 Gy). The spectra obtained in transflection geometry showed characteristics very similar to those present in the spectra acquired in transmission geometry and confirm the validity of the chosen approach. The analysis of spectra indicates the occurrence of significant changes in DNA and lipids components of cells. Modifications in protein secondary structure are also evidenced

Proton-irradiated breast cells: molecular points of view

Breast cancer (BC) is the most common cancer in women, highly heterogeneous at both the clinical and molecular level. Radiation therapy (RT) represents an efficient modality to treat localized tumor in BC care, although the choice of a unique treatment plan for all BC patients, including RT, may not be the best option. Technological advances in RT are evolving with the use of charged particle beams (i.e. protons) which, due to a more localized delivery of the radiation dose, reduce the dose administered to the heart compared with conventional RT. However, few data regarding proton-induced molecular changes are currently available. The aim of this study was to investigate and describe the production of immunological molecules and gene expression profiles induced by proton irradiation. We performed Luminex assay and cDNA microarray analyses to study the biological processes activated following irradiation with proton beams, both in the non-tumorigenic MCF10A cell line and in two tumorigenic BC cell lines, MCF7 and MDA-MB-231. The immunological signatures were dose dependent in MCF10A and MCF7 cell lines, whereas MDA-MB-231 cells show a strong pro-inflammatory profile regardless of the dose delivered. Clonogenic assay revealed different surviving fractions according to the breast cell lines analyzed. We found the involvement of genes related to cell response to proton irradiation and reported specific cell line- and dose-dependent gene signatures, able to drive cell fate after radiation exposure. Our data could represent a useful tool to better understand the molecular mechanisms elicited by proton irradiation and to predict treatment outcome

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

Design and Status of the ELIMED Beam Line for Laser-Driven Ion Beams

Charged particle acceleration using ultra-intense and ultra-short laser pulses has gathered a strong interest in the scientific community and it is now one of the most attractive topics in the relativistic laser-plasma interaction research. Indeed, it could represent the future of particle acceleration and open new scenarios in multidisciplinary fields, in particular, medical applications. One of the biggest challenges consists of using, in a future perspective, high intensity laser-target interaction to generate high-energy ions for therapeutic purposes, eventually replacing the old paradigm of acceleration, characterized by huge and complex machines. The peculiarities of laser-driven beams led to develop new strategies and advanced techniques for transport, diagnostics and dosimetry of the accelerated particles, due to the wide energy spread, the angular divergence and the extremely intense pulses. In this framework, the realization of the ELIMED (ELI-Beamlines MEDical applications) beamline, developed by INFN-LNS (Catania, Italy) and installed in 2017 as a part of the ELIMAIA beamline at the ELI-Beamlines (Extreme Light Infrastructure Beamlines) facility in Prague, has the aim to investigate the feasibility of using laser-driven ion beams in multidisciplinary applications. ELIMED will represent the first user's open transport beam line where a controlled laser-driven ion beam will be used for multidisciplinary and medical studies. In this paper, an overview of the beamline, with a detailed description of the main transport elements, will be presented. Moreover, a description of the detectors dedicated to diagnostics and dosimetry will be reported, with some preliminary results obtained both with accelerator-driven and laser-driven beams

Radiobiological Outcomes, Microdosimetric Evaluations and Monte Carlo Predictions in Eye Proton Therapy

CATANA (Centro di AdroTerapia ed Applicazioni Nucleari Avanzate) was the first Italian protontherapy facility dedicated to the treatment of ocular neoplastic pathologies. It is in operation at the LNS Laboratories of the Italian Institute for Nuclear Physics (INFN-LNS) and to date, 500 patients have been successfully treated. Even though proton therapy has demonstrated success in clinical settings, there is still a need for more accurate models because they are crucial for the estimation of clinically relevant RBE values. Since RBE can vary depending on several physical and biological parameters, there is a clear need for more experimental data to generate predictions. Establishing a database of cell survival experiments is therefore useful to accurately predict the effects of irradiations on both cancerous and normal tissue. The main aim of this work was to compare RBE values obtained from in-vitro experimental data with predictions made by the LEM II (Local Effect Model), Monte Carlo approaches, and semi-empirical models based on LET experimental measurements. For this purpose, the 92.1 uveal melanoma and ARPE-19 cells derived from normal retinal pigmented epithelium were selected and irradiated in the middle of clinical SOBP of the CATANA proton therapy facility. The remarkable results show the potentiality of using microdosimetric spectrum, Monte Carlo simulations and LEM model to predict not only the RBE but also the survival curves

Generation of α-Particle Beams With a Multi-kJ, Peta-Watt Class Laser System

We present preliminary results on generation of energetic α-particles driven by lasers. The experiment was performed at the Institute of Laser Engineering in Osaka using the short-pulse, high-intensity, high-energy, PW-class laser. The laser pulse was focused onto a thin plastic foil (pitcher) to generate a proton beam by the well-known TNSA mechanism which, in turn, was impinging onto a boron-nitride (BN) target (catcher) to generated alpha-particles as a result of proton-boron nuclear fusion events. Our results demonstrate generation of α-particles with energies in the range 8–10 MeV and with a flux around 5 × 10^9 sr^−1

A Hydrogenated amorphous silicon detector for Space Weather Applications

The characteristics of a hydrogenated amorphous silicon (a-Si:H) detector are presented here for monitoring in space solar flares and the evolution of large energetic proton events up to hundreds of MeV. The a-Si:H presents an excellent radiation hardness and finds application in harsh radiation environments for medical purposes, for particle beam characterization and in space weather science and applications. The critical flux detection threshold for solar X rays, soft gamma rays, electrons and protons is discussed in detail.Comment: 32 pages, 13 figures, submitted to Experimental Astronom

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 10^9 Gy/s, and predicted oxygen depletion effects on cellular response can be tested. Access to appropriate experimental environments, 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) and patient-derived glioblastoma (E2) cancer stem 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 exposed to cyclotron-accelerated protons and X-rays in the normal fibroblast and radioresistant GBM stem cells. Results: Oxygen measurements showed that our chambers maintained a radiobiological hypoxic environment for at least 45 minutes and pathological hypoxia for up to 24 hrs 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 minutes post-irradiation. Under hypoxic irradiations, the Laser-driven protons induced significant residual DNA DSB damage in hypoxic AG01522 cells compared to the conventional dose rate protons suggesting an important impact of these extreme high dose-rate exposures. We obtained an oxygen enhancement ratio (OER) of 2.1 ± 0.108 and 2.501 ±0.125 respectively for the AG01522 and patient derived GBM stem cells for the X-rays using our hypoxia chambers for irradiation. Conclusion:We demonstrated the design and application of portable hypoxia chambers for studying cellular radiobiological endpoints after laser-driven protons at ultra-high dose, conventional protons and X-ray exposures. Good levels of reduced oxygen concentration could be maintained in the absence of external gassing to quantify hypoxic effects and the data obtained provided an indication of an enhanced residual DNA DSB damage under hypoxic conditions at ultra-high dose rate compared to the conventional protons or X-rays

Clinical and Research Activities at the CATANA Facility of INFN-LNS: From the Conventional Hadrontherapy to the Laser-Driven Approach

The CATANA proton therapy center was the first Italian clinical facility making use of energetic (62 MeV) proton beams for the radioactive treatment of solid tumors. Since the date of the first patient treatment in 2002, 294 patients have been successful treated whose majority was affected by choroidal and iris melanomas. In this paper, we report on the current clinical and physical status of the CATANA facility describing the last dosimetric studies and reporting on the last patient follow-up results. The last part of the paper is dedicated to the description of the INFN-LNS ongoing activities on the realization of a beamline for the transport of laser-accelerated ion beams for future applications. The ELIMED (ELI-Beamlines MEDical and multidisciplinary applications) project is introduced and the main scientific aspects will be described