ETHICS (Pre-clinical Experimental and THeoretical studies to Improve treatment and protection by Charged particleS),

Medical applications of charged particles, such as hadrotherapy and radionuclide therapy, involve the exposure of normal cells composing the tissues and organs proximal to the tumour from either external or internal sources. Clinical implementation of biologically-optimised treatment plans and a safer use of cancer cell-targeting radionuclides are hampered by the uncertainties inherent to the radiobiology of healthy tissue response to densely ionising radiations, which may lead to increased risks of secondary cancers, hence needing to be urgently addressed. A better understanding of the effects on normal cells following the exposure to charged particles, on the other hand, may also benefit the general public because of the contribution to the collective dose from natural sources, such as indoor radon. The main objective of this research project is, therefore, to study the basic mechanisms underlying the biological effects brought about by charged particles that are of relevance for the integrity and normal functions of healthy tissues/organs. To achieve such a goal, both in vitro and in vivo experiments are planned at INFN as well as external facilities, involving a vast network of national and international collaborations and in conjunction with theoretical studies and medical physics-based approaches. The action of a number of ions will be investigated employing a wide array of assays and state-of-the art techniques. The ultimate aim is to help develop strategies that may limit high-LET radiation detrimental consequences for human health while improving their therapeutic benefits

An FTIR Microspectroscopy Ratiometric Approach for Monitoring X-ray Irradiation Effects on SH-SY5Y Human Neuroblastoma Cells

The ability of Fourier transform infrared (FTIR) spectroscopy in analyzing cells at a molecular level was exploited for investigating the biochemical changes induced in protein, nucleic acid, lipid, and carbohydrate content of cells after irradiation by graded X-ray doses. Infrared spectra from in vitro SH-SY5Y neuroblastoma cells following exposure to X-rays (0, 2, 4, 6, 8, 10 Gy) were analyzed using a ratiometric approach by evaluating the ratios between the absorbance of significant peaks. The spectroscopic investigation was performed on cells fixed immediately (t0 cells) and 24 h (t24 cells) after irradiation to study both the initial radiation-induced damage and the effect of the ensuing cellular repair processes. The analysis of infrared spectra allowed us to detect changes in proteins, lipids, and nucleic acids attributable to X-ray exposure. The ratiometric analysis was able to quantify changes for the protein, lipid, and DNA components and to suggest the occurrence of apoptosis processes. The ratiometric study of Amide I band indicated also that the secondary structure of proteins was significantly modified. The comparison between the results from t0 and t24 cells indicated the occurrence of cellular recovery processes. The adopted approach can provide a very direct way to monitor changes for specific cellular components and can represent a valuable tool for developing innovative strategies to monitor cancer radiotherapy outcome

NEPTUNE (Nuclear process-driven Enhancement of Proton Therapy UNravEled)

Protontherapy is an important radiation modality that has been used to treat cancer for over 60 years. In the last 10 years, clinical proton therapy has been rapidly growing with more than 80 facilities worldwide [1]. The interest in proton therapy stems from the physical properties of protons allowing for a much improved dose shaping around the target and greater healthy tissue sparing. One shortcoming of protontherapy is its inability to treat radioresistant cancers, being protons radiobiologically almost as effective as photons. Heavier particles, such as 12C ions, can overcome radioresistance but they present radiobiological and economic issues that hamper their widespread adoption. Therefore, many strategies have been designed to increase the biological effectiveness of proton beams. Examples are chemical radiosensitizing agents or, more recently, metallic nanoparticles. The goal of this project is to investigate the use of nuclear reactions triggered by protons generating short-range high- LET alpha particles inside the tumours, thereby allowing a highly localized DNA-damaging action. Specifically, we intend to consolidate and explain the promising results recently published in [2], where a significant enhancement of biological effectiveness was achieved by the p-11B reaction. Clinically relevant binary approaches were first proposed with Boron Neutron Capture Therapy (BNCT), which exploits thermal neutron capture in 10B, suitably accumulated into tumour before irradiation. The radiosensitising effects due to the presence of 10B will be compared to those elicited by p-11B, using the same carrier and relating the observed effects with intracellular 11B and 10B distribution as well as modelled particle action and measured dose deposition at the micro/nanometer scale. Moreover, the p-19F reaction, which also generates secondary particles potentially leading to local enhancement of proton effectiveness, will be investigated. The in-vivo imaging of 11B and 19F carriers will be studied, in particular by optimizing 19F-based magnetic resonance

Micro Sensing of pH Levels in Biological Samples by Graphene-Based Raman Spectroscopy

Graphene provides a unique way for sensing local pH level of substances on micrometric scale, with important implications for the monitoring of cellular metabolic activities where protonic excretion could occur. Doping modifications of graphene, induced by the contact of the graphene with different pH solutions were investigated by micro-Raman spectroscopy in order to develop a pH biosensor. To test the developed biosensor with real biological systems, the pH values of cell culture media in different conditions were evaluated

A New Low-Energy Proton Irradiation Facility to Unveil the Mechanistic Basis of the Proton-Boron Capture Therapy Approach

Protontherapy (PT) is a fast-growing cancer therapy modality thanks to much-improved normal tissue sparing granted by the charged particles' inverted dose-depth profile. Protons, however, exhibit a low biological effectiveness at clinically relevant energies. To enhance PT efficacy and counteract cancer radioresistance, Proton–Boron Capture Therapy (PBCT) was recently proposed. PBCT exploits the highly DNA-damaging α-particles generated by the p + 11B→3α (pB) nuclear reaction, whose cross-section peaks for proton energies of 675 keV. Although a significant enhancement of proton biological effectiveness by PBCT has been demonstrated for high-energy proton beams, validation of the PBCT rationale using monochromatic proton beams having energy close to the reaction cross-section maximum is still lacking. To this end, we implemented a novel setup for radiobiology experiments at a 3-MV tandem accelerator; using a scattering chamber equipped with an Au foil scatterer for beam diffusion on the biological sample, uniformity in energy and fluence with uncertainties of 2% and 5%, respectively, was achieved. Human cancer cells were irradiated at this beamline for the first time with 685-keV protons. The measured enhancement in cancer cell killing due to the 11B carrier BSH was the highest among those thus far observed, thereby corroborating the mechanistic bases of PBCT

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

An FTIR Spectroscopy Investigation on Different Methods of Lipid Extraction from HepG2 Cells

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Transcriptional modulations induced by proton irradiation in mice skin in function of adsorbed dose and distance

Hadron therapy by proton beams represents an advanced anti-cancer strategy due to its highly localized dose deposition allowing a greater sparing of normal tissue and/or organs at risk compared to photon/electron radiotherapy. However, it is not clear to what extent non-targeted effects such as transcriptional modulations produced along the beamline may diffuse and impact the surrounding tissue. In this work, we analyze the transcriptome of proton-irradiated mouse skin and choose two biomarker genes to trace their modulation at different distances from the beam's target and at different doses and times from irradiation to understand to what extent and how far it may propagate, using RNA-Seq and quantitative RT-PCR. In parallel, assessment of lipids alteration is performed by FTIR spectroscopy as a measure of tissue damage. Despite the observed high individual variability of expression, we can show evidence of transcriptional modulation of two biomarker genes at considerable distance from the beam's target where a simulation system predicts a significantly lower adsorbed dose. The results are compatible with a model involving diffusion of transcripts or regulatory molecules from high dose irradiated cells to distant tissue's portions adsorbing a much lower fraction of radiation