Proton therapy induces a local microglial neuroimmune response.

BACKGROUND AND PURPOSE: Although proton therapy is increasingly being used in the treatment of paediatric and adult brain tumours, there are still uncertainties surrounding the biological effect of protons on the normal brain. Microglia, the brain-resident macrophages, have been shown to play a role in the development of radiation-induced neurotoxicity. However, their molecular and hence functional response to proton irradiation remains unknown. This study investigates the effect of protons on microglia by comparing the effect of photons and protons as well as the influence of age and different irradiated volumes. MATERIALS AND METHODS: Rats were irradiated with 14 Gy to the whole brain with photons (X-rays), plateau protons, spread-out Bragg peak (SOBP) protons or to 50 % anterior, or 50 % posterior brain sub-volumes with plateau protons. RNA sequencing, validation of microglial priming gene expression using qPCR and high-content imaging analysis of microglial morphology were performed in the cortex at 12 weeks post irradiation. RESULTS: Photons and plateau protons induced a shared transcriptomic response associated with neuroinflammation. This response was associated with a similar microglial priming gene expression signature and distribution of microglial morphologies. Expression of the priming gene signature was less pronounced in juvenile rats compared to adults and slightly increased in rats irradiated with SOBP protons. High-precision partial brain irradiation with protons induced a local microglial priming response and morphological changes. CONCLUSION: Overall, our data indicate that the brain responds in a similar manner to photons and plateau protons with a shared local upregulation of microglial priming-associated genes, potentially enhancing the immune response to subsequent inflammatory challenges.

Impact of proton therapy on the DNA damage induction and repair in hematopoietic stem and progenitor cells.

Proton therapy is of great interest to pediatric cancer patients because of its optimal depth dose distribution. In view of healthy tissue damage and the increased risk of secondary cancers, we investigated DNA damage induction and repair of radiosensitive hematopoietic stem and progenitor cells (HSPCs) exposed to therapeutic proton and photon irradiation due to their role in radiation-induced leukemia. Human CD34+ HSPCs were exposed to 6 MV X-rays, mid- and distal spread-out Bragg peak (SOBP) protons at doses ranging from 0.5 to 2 Gy. Persistent chromosomal damage was assessed with the micronucleus assay, while DNA damage induction and repair were analyzed with the γ-H2AX foci assay. No differences were found in induction and disappearance of γ-H2AX foci between 6 MV X-rays, mid- and distal SOBP protons at 1 Gy. A significantly higher number of micronuclei was found for distal SOBP protons compared to 6 MV X-rays and mid- SOBP protons at 0.5 and 1 Gy, while no significant differences in micronuclei were found at 2 Gy. In HSPCs, mid-SOBP protons are as damaging as conventional X-rays. Distal SOBP protons showed a higher number of micronuclei in HSPCs depending on the radiation dose, indicating possible changes of the in vivo biological response.

A Monte-Carlo-based study of a single-2D-detector proton-radiography system.

PURPOSE: To assess the feasibility of a proton radiography (pRG) system based on a single thin pixelated detector for water-equivalent path length (WEPL) and relative stopping power (RSP) measurements. METHODS: A model of a pRG system consisting of a single pixelated detector measuring energy deposition and proton fluence was investigated in a Geant4-based Monte Carlo study. At the position directly after an object traversed by a broad proton beam, spatial 2D distributions are calculated of the energy deposition in, and the number of protons entering the detector. Their ratio relates to the 2D distribution of the average stopping power of protons in the detector. The system response is calibrated against the residual range in water of the protons to provide the 2D distribution of the WEPL of the object. The WEPL distribution is converted into the distribution of the RSP of the object. Simulations have been done, where the system has been tested on 13 samples of homogeneous materials of which the RSPs have been calculated and compared with RSPs determined from simulations of residual-range-in-water, which we refer to as reference RSPs. RESULTS: For both human-tissue- and non-human-tissue-equivalent materials, the RSPs derived with the detector agree with the reference values within 1%. CONCLUSION: The study shows that a pRG system based on one thin pixelated detection screen has the potential to provide RSP predictions with an accuracy of 1%.

The Effects of Combined Exposure to Simulated Microgravity, Ionizing Radiation, and Cortisol on the In Vitro Wound Healing Process.

Human spaceflight is associated with several health-related issues as a result of long-term exposure to microgravity, ionizing radiation, and higher levels of psychological stress. Frequent reported skin problems in space include rashes, itches, and a delayed wound healing. Access to space is restricted by financial and logistical issues; as a consequence, experimental sample sizes are often small, which limits the generalization of the results. Earth-based simulation models can be used to investigate cellular responses as a result of exposure to certain spaceflight stressors. Here, we describe the development of an in vitro model of the simulated spaceflight environment, which we used to investigate the combined effect of simulated microgravity using the random positioning machine (RPM), ionizing radiation, and stress hormones on the wound-healing capacity of human dermal fibroblasts. Fibroblasts were exposed to cortisol, after which they were irradiated with different radiation qualities (including X-rays, protons, carbon ions, and iron ions) followed by exposure to simulated microgravity using a random positioning machine (RPM). Data related to the inflammatory, proliferation, and remodeling phase of wound healing has been collected. Results show that spaceflight stressors can interfere with the wound healing process at any phase. Moreover, several interactions between the different spaceflight stressors were found. This highlights the complexity that needs to be taken into account when studying the effect of spaceflight stressors on certain biological processes and for the aim of countermeasures development.

A simple microscopy setup for visualizing cellular responses to DNA damage at particle accelerator facilities.

Cellular responses to DNA double-strand breaks (DSBs) not only promote genomic integrity in healthy tissues, but also largely determine the efficacy of many DNA-damaging cancer treatments, including X-ray and particle therapies. A growing body of evidence suggests that activation of the mechanisms that detect, signal and repair DSBs may depend on the complexity of the initiating DNA lesions. Studies focusing on this, as well as on many other radiobiological questions, require reliable methods to induce DSBs of varying complexity, and to visualize the ensuing cellular responses. Accelerated particles of different energies and masses are exceptionally well suited for this task, due to the nature of their physical interactions with the intracellular environment, but visualizing cellular responses to particle-induced damage - especially in their early stages - at particle accelerator facilities, remains challenging. Here we describe a straightforward approach for real-time imaging of early response to particle-induced DNA damage. We rely on a transportable setup with an inverted fluorescence confocal microscope, tilted at a small angle relative to the particle beam, such that cells can be irradiated and imaged without any microscope or beamline modifications. Using this setup, we image and analyze the accumulation of fluorescently-tagged MDC1, RNF168 and 53BP1-key factors involved in DSB signalling-at DNA lesions induced by 254 MeV α-particles. Our results provide a demonstration of technical feasibility and reveal asynchronous initiation of accumulation of these proteins at different individual DSBs.

Feasibility of quasi-prompt PET-based range verification in proton therapy.

Compared to photon therapy, proton therapy allows a better conformation of the dose to the tumor volume with reduced radiation dose to co-irradiated tissues. In vivo verification techniques including positron emission tomography (PET) have been proposed as quality assurance tools to mitigate proton range uncertainties. Detection of differences between planned and actual dose delivery on a short timescale provides a fast trigger for corrective actions. Conventional PET-based imaging of 15O (T1/2 = 2 min) and 11C (T1/2 = 20 min) distributions precludes such immediate feedback. We here present a demonstration of near real-time range verification by means of PET imaging of 12N (T1/2 = 11 ms). PMMA and graphite targets were irradiated with a 150 MeV proton pencil beam consisting of a series of pulses of 10 ms beam-on and 90 ms beam-off. Two modules of a modified Siemens Biograph mCT PET scanner (21 × 21 cm2 each), installed 25 cm apart, were used to image the beam-induced PET activity during the beam-off periods. The modifications enable the detectors to be switched off during the beam-on periods. 12N images were reconstructed using planar tomography. Using a 1D projection of the 2D reconstructed 12N image, the activity range was obtained from a fit of the activity profile with a sigmoid function. Range shifts due to modified target configurations were assessed for multiples of the clinically relevant 108 protons per pulse (approximately equal to the highest intensity spots in the pencil beam scanning delivery of a dose of 1 Gy over a cubic 1 l volume). The standard deviation of the activity range, determined from 30 datasets obtained from three irradiations on PMMA and graphite targets, was found to be 2.5 and 2.6 mm (1σ) with 108 protons per pulse and 0.9 and 0.8 mm (1σ) with 109 protons per pulse. Analytical extrapolation of the results from this study shows that using a scanner with a solid angle coverage of 57%, with optimized detector switching and spot delivery times much smaller than the 12N half-life, an activity range measurement precision of 2.0 mm (1σ) and 1.3 mm (1σ) within 50 ms into an irradiation with 4 × 107 and 108 protons per pencil beam spot can be potentially realized. Aggregated imaging of neighboring spots or, if possible, increasing the number of protons for a few probe beam spots will enable the realization of higher precision range measurement.

Validation of linear energy transfer computed in a Monte Carlo dose engine of a commercial treatment planning system.

The relative biological effectiveness (RBE) of protons is highly variable and difficult to quantify. However, RBE is related to the local ionization density, which can be related to the physical measurable dose weighted linear energy transfer (LETD). The aim of this study was to validate the LETD calculations for proton therapy beams implemented in a commercially available treatment planning system (TPS) using microdosimetry measurements and independent LETD calculations (Open-MCsquare (MCS)). The TPS (RayStation v6R) was used to generate treatment plans on the CIRS-731-HN anthropomorphic phantom for three anatomical sites (brain, nasopharynx, neck) for a spherical target (Ø = 5 cm) with uniform target dose to calculate the LETD distribution. Measurements were performed at the University Medical Center Groningen proton therapy center (Proteus Plus, IBA) using a µ +-probe utilizing silicon on insulator microdosimeters capable of detecting lineal energies as low as 0.15 keV µm-1 in tissue. Dose averaged mean lineal energy [Formula: see text] depth-profiles were measured for 70 and 130 MeV spots in water and for the three treatment plans in water and an anthropomorphic phantom. The [Formula: see text] measurements were compared to the LETD calculated in the TPS and MCS independent dose calculation engine. D · [Formula: see text] was compared to D · LETD in terms of a gamma-index with a distance-to-agreement criteria of 2 mm and increasing dose difference criteria to determine the criteria for which a 90% pass rate was accomplished. Measurements of D · [Formula: see text] were in good agreement with the D · LETD calculated in the TPS and MCS. The 90% passing rate threshold was reached at different D · LETD difference criteria for single spots (TPS: 1% MCS: 1%), treatment plans in water (TPS: 3% MCS: 6%) and treatment plans in an anthropomorphic phantom (TPS: 6% MCS: 1%). We conclude that D · LETD calculations accuracy in the RayStation TPS and open MCSquare are within 6%, and sufficient for clinical D · LETD evaluation and optimization. These findings remove an important obstacle in the road towards clinical implementation of D · LETD evaluation and optimization of proton therapy treatment plans. Novelty and significance The dose weighed linear energy transfer (LETD) distribution can be calculated for proton therapy treatment plans by Monte Carlo dose engines. The relative biological effectiveness (RBE) of protons is known to vary with the LETD distribution. Therefore, there exists a need for accurate calculation of clinical LETD distributions. Previous LETD validations have focused on general purpose Monte Carlo dose engines which are typically not used clinically. We present the first validation of mean lineal energy [Formula: see text] measurements of the LETD against calculations by the Monte Carlo dose engines of the Raystation treatment planning system and open MCSquare.

In vitro biological response of cancer and normal tissue cells to proton irradiation not affected by an added magnetic field.

To optimize beam delivery and conformality of proton therapy, MRI integration has been proposed. Therefore, we investigated if proton irradiation in a magnetic field would change biological responses. Our data in cancer cell lines and stem cell-derived organoid models suggest that a magnetic field does not modify the biological response.

Lack of DNA Damage Response at Low Radiation Doses in Adult Stem Cells Contributes to Organ Dysfunction.

PURPOSE: Radiotherapy for head and neck cancer may result in serious side effects, such as hyposalivation, impairing the patient's quality of life. Modern radiotherapy techniques attempt to reduce the dose to salivary glands, which, however, results in low-dose irradiation of the tissue stem cells. Here we assess the low-dose sensitivity of tissue stem cells and the consequences for tissue function. EXPERIMENTAL DESIGN: Postirradiation rat salivary gland secretory function was determined after pilocarpine induction. Murine and patient-derived salivary gland and thyroid gland organoids were irradiated and clonogenic survival was assessed. The DNA damage response (DDR) was analyzed in organoids and modulated using different radiation modalities, chemical inhibition, and genetic modification. RESULTS: Relative low-dose irradiation to the high-density stem cell region of rat salivary gland disproportionally impaired function. Hyper-radiosensitivity at doses <1 Gy, followed by relative radioresistance at doses ≥1 Gy, was observed in salivary gland and thyroid gland organoid cultures. DDR modulation resulted in diminished, or even abrogated, relative radioresistance. Furthermore, inhibition of the DDR protein ATM impaired DNA repair after 1 Gy, but not 0.25 Gy. Irradiation of patient-derived salivary gland organoid cells showed similar responses, whereas a single 1 Gy dose to salivary gland-derived stem cells resulted in greater survival than clinically relevant fractionated doses of 4 × 0.25 Gy. CONCLUSIONS: We show that murine and human glandular tissue stem cells exhibit a dose threshold in DDR activation, resulting in low-dose hyper-radiosensitivity, with clinical implications in radiotherapy treatment planning. Furthermore, our results from patient-derived organoids highlight the potential of organoids to study normal tissue responses to radiation.

Comparative study of the effects of different radiation qualities on normal human breast cells.

BACKGROUND: As there is a growing number of long-term cancer survivors, the incidence of carcinogenesis as a late effect of radiotherapy is getting more and more into the focus. The risk for the development of secondary malignant neoplasms might be significantly increased due to exposure of healthy tissue outside of the target field to secondary neutrons, in particular in proton therapy. Thus far, the radiobiological effects of these neutrons and a comparison with photons on normal breast cells have not been sufficiently characterised. METHODS: MCF10A cells were irradiated with doses of up to 2 Gy with neutrons of different energy spectra and X-rays for comparison. The biological effects of neutrons with a broad energy distribution ( = 5.8 MeV), monoenergetic neutrons (1.2 MeV, 0.56 MeV) and of the mixed field of gamma's and secondary neutrons ( = 70.5 MeV) produced by 190 MeV protons impinging on a water phantom, were analysed. The clonogenic survival and the DNA repair capacity were determined and values of relative biological effectiveness were compared. Furthermore, the influence of radiation on the sphere formation was observed to examine the radiation response of the potential fraction of stem like cells within the MCF10A cell population. RESULTS: X-rays and neutrons caused dose-dependent decreases of survival fractions after irradiations with up to 2 Gy. Monoenergetic neutrons with an energy of 0.56 MeV had a higher effectiveness on the survival fraction with respect to neutrons with higher energies and to the mixed gamma - secondary neutron field induced by proton interactions in water. Similar effects were observed for the DNA repair capacity after exposure to ionising radiation (IR). Both experimental endpoints provided comparable values of the relative biological effectiveness. Significant changes in the sphere formation were notable following the various radiation qualities. CONCLUSION: The present study compared the radiation response of MCF10A cells after IR with neutrons and photons. For the first time it was shown that monoenergetic neutrons with energies around 1 MeV have stronger radiobiological effects on normal human breast cells with respect to X rays, to neutrons with a broad energy distribution ( = 5.8 MeV), and to the mixed gamma - secondary neutron field given by interactions of 190 MeV protons in water. The results of the present study are highly relevant for further investigations of radiation-induced carcinogenesis and are very important in perspective for a better risk assessment after secondary neutron exposure in the field of conventional and proton radiotherapy.

Improving proton therapy by metal-containing nanoparticles: nanoscale insights.

The use of nanoparticles to enhance the effect of radiation-based cancer treatments is a growing field of study and recently, even nanoparticle-induced improvement of proton therapy performance has been investigated. Aiming at a clinical implementation of this approach, it is essential to characterize the mechanisms underlying the synergistic effects of nanoparticles combined with proton irradiation. In this study, we investigated the effect of platinum- and gadolinium-based nanoparticles on the nanoscale damage induced by a proton beam of therapeutically relevant energy (150 MeV) using plasmid DNA molecular probe. Two conditions of irradiation (0.44 and 3.6 keV/µm) were considered to mimic the beam properties at the entrance and at the end of the proton track. We demonstrate that the two metal-containing nanoparticles amplify, in particular, the induction of nanosize damages (>2 nm) which are most lethal for cells. More importantly, this effect is even more pronounced at the end of the proton track. This work gives a new insight into the underlying mechanisms on the nanoscale and indicates that the addition of metal-based nanoparticles is a promising strategy not only to increase the cell killing action of fast protons, but also to improve tumor targeting.

The In Vitro Response of Tissue Stem Cells to Irradiation With Different Linear Energy Transfers.

PURPOSE: A reduction in the dose, irradiated volume, and sensitivity of, in particular, normal tissue stem cells is needed to advance radiation therapy. This could be obtained with the use of particles for radiation therapy. However, the radiation response of normal tissue stem cells is still an enigma. Therefore, in the present study, we developed a model to investigate the in vitro response of stem cells to particle irradiation. METHODS AND MATERIALS: We used the immortalized human salivary gland (HSG) cell line resembling salivary gland (SG) cells to translate the radiation response in 2-dimensional (2D) to 3-dimensional (3D) conditions. This response was subsequently translated to the response of SG stem cells (SGSCs). Dispersed single cells were irradiated with photons or carbon ions at different linear energy transfers (LETs; 48.76 ± 2.16, 149.9 ± 10.8, and 189 ± 15 keV/µm). Subsequently, 2D or 3D clonogenicity was determined by counting the colonies or secondary stem cell-derived spheres in Matrigel. γH2AX immunostaining was used to assess DNA double strand break repair. RESULTS: The 2D response of HSG cells showed a similar increase in dose response to increasing higher LET irradiation as other cell lines. The 3D response of HSG cells to increasing LET irradiation was reduced compared with the 2D response. Finally, the response of mouse SGSCs to photons was similar to the 3D response of HSG cells. The response to higher LET irradiation was reduced in the stem cells. CONCLUSIONS: Mouse SGSC radiosensitivity seems reduced at higher LET radiation compared with transformed HSG cells. The developed model to assess the radiation response of SGSCs offers novel possibilities to study the radiation response of normal tissue in vitro.

Proton Radiography With Timepix Based Time Projection Chambers.

The development of a proton radiography system to improve the imaging of patients in proton beam therapy is described. The system comprises gridpix based time projection chambers, which are based on the Timepix chip designed by the Medipix collaboration, for tracking the protons. This type of detector was chosen to have minimal impact on the actual determination of the proton tracks by the tracking detectors. To determine the residual energy of the protons, a BaF 2 crystal with a photomultiplier tube is used. We present data taken in a feasibility experiment with phantoms that represent tissue equivalent materials found in the human body. The obtained experimental results show a good agreement with the performed simulations.

Relative electron density determination using a physics based parameterization of photon interactions in medical DECT.

Radiotherapy and particle therapy treatment planning require accurate knowledge of the electron density and elemental composition of the tissues in the beam path to predict the local dose deposition. We describe a method for the analysis of dual energy computed tomography (DECT) images that provides the electron densities and effective atomic numbers of tissues. The CT measurement process is modelled by system weighting functions, which apply an energy dependent weighting to the parameterization of the total cross section for photon interactions with matter. This detailed parameterization is based on the theoretical analysis of Jackson and Hawkes and deviates, at most, 0.3% from the tabulated NIST values for the elements H to Zn. To account for beam hardening in the object as present in the CT image we implemented an iterative process employing a local weighting function, derived from the method proposed by Heismann and Balda. With this method effective atomic numbers between 1 and 30 can be determined. The method has been experimentally validated on a commercially available tissue characterization phantom with 16 inserts made of tissue substitutes and aluminium that has been scanned on a dual source CT system with tube potentials of 100 kV and 140 kV using a clinical scan protocol. Relative electron densities of all tissue substitutes have been determined with accuracy better than 1%. The presented DECT analysis method thus provides high accuracy electron densities and effective atomic numbers for radiotherapy and especially particle therapy treatment planning.

High and low LET radiation differentially induce normal tissue damage signals.

PURPOSE: Radiotherapy using high linear energy transfer (LET) radiation is aimed at efficiently killing tumor cells while minimizing dose (biological effective) to normal tissues to prevent toxicity. It is well established that high LET radiation results in lower cell survival per absorbed dose than low LET radiation. However, whether various mechanisms involved in the development of normal tissue damage may be regulated differentially is not known. Therefore the aim of this study was to investigate whether two actions related to normal tissue toxicity, p53-induced apoptosis and expression of the profibrotic gene PAI-1 (plasminogen activator inhibitor 1), are differentially induced by high and low LET radiation. METHODS AND MATERIALS: Cells were irradiated with high LET carbon ions or low LET photons. Cell survival assays were performed, profibrotic PAI-1 expression was monitored by quantitative polymerase chain reaction, and apoptosis was assayed by annexin V staining. Activation of p53 by phosphorylation at serine 315 and serine 37 was monitored by Western blotting. Transfections of plasmids expressing p53 mutated at serines 315 and 37 were used to test the requirement of these residues for apoptosis and expression of PAI-1. RESULTS: As expected, cell survival was lower and induction of apoptosis was higher in high -LET irradiated cells. Interestingly, induction of the profibrotic PAI-1 gene was similar with high and low LET radiation. In agreement with this finding, phosphorylation of p53 at serine 315 involved in PAI-1 expression was similar with high and low LET radiation, whereas phosphorylation of p53 at serine 37, involved in apoptosis induction, was much higher after high LET irradiation. CONCLUSIONS: Our results indicate that diverse mechanisms involved in the development of normal tissue damage may be differentially affected by high and low LET radiation. This may have consequences for the development and manifestation of normal tissue damage.

Development of a facility for high-precision irradiation of cells with carbon ions.

PURPOSE: Compared to photons, using particle radiation in radiotherapy reduces the dose and irradiated volume of normal tissues, potentially reducing side effects. The biological effect of dose deposited by particles such as carbon ions, however, differs from that of dose deposited by photons. The inaccuracy in models to estimate the biological effects of particle radiation remains the most important source of uncertainties in particle therapy. Improving this requires high-precision studies on biological effects of particle radiation. Therefore, the authors aimed to develop a facility for reproducible and high-precision carbon-ion irradiation of cells in culture. The combined dose nonuniformity in the lateral and longitudinal direction should not exceed +/-1.5%. Dose to the cells from particles than other carbon ions should not exceed 5%. METHODS: A uniform lateral dose distribution was realized using a single scatter foil and quadrupole magnets. A modulator wheel was used to create a uniform longitudinal dose distribution. The choice of beam energy and the optimal design of these components was determined using GEANT4 and SRIM Monte Carlo simulations. Verification of the uniformity of the dose distribution was performed using a scintillating screen (lateral) and a water phantom (longitudinal). The reproducibility of dose delivery between experiments was assessed by repeated measurements of the spatial dose distribution. Moreover, the reproducibility of dose-response measurements was tested by measuring the survival of irradiated HEK293 cells in three independent experiments. RESULTS: The relative contribution of dose from nuclear reaction fragments to the sample was found to be <5% when using 90 MeV/u carbon ions. This energy still allows accurate dosimetry conforming to the IAEA Report TRS-398, facilitating comparison to dose-effect data obtained with other radiation qualities. A 1.3 mm long spread-out Bragg peak with a diameter of 30 mm was created, allowing the irradiation of cell samples with the specified accuracy. Measurements of the transverse and longitudinal dose distribution showed that the dose variation over the sample volume was +/-0.8% and +/-0.7% in the lateral and longitudinal directions, respectively. The track-averaged LET of 132 +/- 10 keV/microm and dose-averaged LET of 189 +/- 15 keV/microm at the position of the sample were obtained from a GEANT4 simulation, which was validated experimentally. Three separately measured cell-survival curves yielded nearly identical results. CONCLUSIONS: With the new facility, high-precision carbon-ion irradiations of biological samples can be performed with highly reproducible results.