Low-LET Proton Irradiation of A549 Non-small Cell Lung Adenocarcinoma Cells: Dose Response and RBE Determination
TL;DR: Comparisons with X ray results indicate that proton irradiation at 10 keV/μm enhanced the tumor radiosensitivity with a significant dose-dependent decrease in the survival fraction.
Abstract: Since 1957, broad proton beam radiotherapy with a spread out Bragg peak has been used for cancer treatment. More recently, studies on the use of proton therapy in the treatment of non-small cell lung cancer (NSCLC) were performed and although the benefit of using protons for the treatment of NSCLC is recognized, more work is needed to gather additional data for the understanding of cell response. Human A549 cell survival was evaluated by colony forming assay 11 days after 10 keV/μm proton beam irradiation at 0.1 and 1 Gy/min. The residual energy of the proton beam at the location of the irradiated cells was 3.9 MeV. In parallel, early effects on the cell viability and DNA damage were assessed and DNA synthesis was measured. The survival curve obtained was fitted with both the linear and the induced-repair models, as a hyper-radiosensitivity was evidenced at very low doses. Above 0.5 Gy, a linear shape was observed with the α parameter equal to 0.824 ± 0.029 Gy−1. In addition, early cell death and cell pro...
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TL;DR: This review can serve as a source for defining input parameters for applying or refining biophysical models and to identify endpoints where additional radiobiological data are needed in order to reduce the uncertainties in proton RBE values to clinically acceptable levels.
Abstract: Proton therapy treatments are based on a proton RBE (relative biological effectiveness) relative to high-energy photons of 1.1. The use of this generic, spatially invariant RBE within tumors and normal tissues disregards the evidence that proton RBE varies with linear energy transfer (LET), physiological and biological factors, and clinical endpoint.Based on the available experimental data from published literature, this review analyzes relationships of RBE with dose, biological endpoint and physical properties of proton beams. The review distinguishes between endpoints relevant for tumor control probability and those potentially relevant for normal tissue complication. Numerous endpoints and experiments on sub-cellular damage and repair effects are discussed.Despite the large amount of data, considerable uncertainties in proton RBE values remain. As an average RBE for cell survival in the center of a typical spread-out Bragg peak (SOBP), the data support a value of ~1.15 at 2 Gy/fraction. The proton RBE increases with increasing LETd and thus with depth in an SOBP from ~1.1 in the entrance region, to ~1.15 in the center, ~1.35 at the distal edge and ~1.7 in the distal fall-off (when averaged over all cell lines, which may not be clinically representative). For small modulation widths the values could be increased. Furthermore, there is a trend of an increase in RBE as (α/β)x decreases. In most cases the RBE also increases with decreasing dose, specifically for systems with low (α/β)x. Data on RBE for endpoints other than clonogenic cell survival are too diverse to allow general statements other than that the RBE is, on average, in line with a value of ~1.1.This review can serve as a source for defining input parameters for applying or refining biophysical models and to identify endpoints where additional radiobiological data are needed in order to reduce the uncertainties to clinically acceptable levels.
664 citations
TL;DR: Particle radiobiology is now entering into a new phase, where beyond RBE, the tissue response is considered, and these results may open new applications for both cancer therapy and protection in deep space.
Abstract: Densely ionizing radiation has always been a main topic in radiobiology. In fact, α-particles and neutrons are sources of radiation exposure for the general population and workers in nuclear power plants. More recently, high-energy protons and heavy ions attracted a large interest for two applications: hadrontherapy in oncology and space radiation protection in manned space missions. For many years, studies concentrated on measurements of the relative biological effectiveness (RBE) of the energetic particles for different end points, especially cell killing (for radiotherapy) and carcinogenesis (for late effects). Although more recently, it has been shown that densely ionizing radiation elicits signalling pathways quite distinct from those involved in the cell and tissue response to photons. The response of the microenvironment to charged particles is therefore under scrutiny, and both the damage in the target and non-target tissues are relevant. The role of individual susceptibility in therapy and risk is obviously a major topic in radiation research in general, and for ion radiobiology as well. Particle radiobiology is therefore now entering into a new phase, where beyond RBE, the tissue response is considered. These results may open new applications for both cancer therapy and protection in deep space.
112 citations
Cites methods from "Low-LET Proton Irradiation of A549 ..."
...The single-fraction C-ion data point with the highest BED is described in Tsujii and Kamada,46 while the highest TCP has been achieved at Tsukuba University, Ibaraki, Japan, in a trial on 58 (T1/T2, 30/28) patients treated with 66Gy(RBE)/10 fractions for peripherally located and 72.6Gy(RBE)/22 fractions for centrally located tumours.42 BED for CPT was calculated using the formula above with d in Gy(RBE) as reported in the publications and the same a/b ratio as for X-rays....
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...Slow protons aremore effective than X-rays in the inactivation of human tumour cell lines.47 A recent experiment in a human NSCLC cell line reported an RBE5 1.9 for 3.9MeV protons compared with X-rays.48 The Tsukuba proton beam line used for the NSCLC treatment42 has been used for in vitro experiments in different human cell lines.49 Apoptosis induction was greater than two-fold the level induced by 10MeV X-ray.49 Other experiments have shown that the signalling cascade following exposure to protons can substantially differ from the damage response to photons.50 A potential advantage of CPT in hypofractionation is the reduced oxygen enhancement ratio (OER) using high-LET radiation....
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...There could be a dose threshold, and it seems to be charge- and energy-dependent, and defining an RBE is even more difficult, considering that most of these end points are not observed at all with X-rays.131 These observations have therefore triggered a number of cellular and molecular studies on neural cells and tissue, to gain understanding of the mechanisms of heavy ion-induced damage in the brain....
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...9MeV protons compared with X-rays.(48) The Tsukuba proton beam line used for the NSCLC treatment(42) has been used for in vitro experiments in different human cell lines....
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TL;DR: It is demonstrated that the phenomenon-based MultiScale Approach to the assessment of radiation damage with ions gives a positive answer to the question whether it is possible to quantitatively predict macroscopic biological effects caused by ion radiation on the basis of physical and chemical effects related to the ion-medium interactions on a nanometre scale.
Abstract: Ion-beam therapy provides advances in cancer treatment, offering the possibility of excellent dose localization and thus maximising cell-killing within the tumour. The full potential of such therapy can only be realised if the fundamental mechanisms leading to lethal cell damage under ion irradiation are well understood. The key question is whether it is possible to quantitatively predict macroscopic biological effects caused by ion radiation on the basis of physical and chemical effects related to the ion-medium interactions on a nanometre scale. We demonstrate that the phenomenon-based MultiScale Approach to the assessment of radiation damage with ions gives a positive answer to this question. We apply this approach to numerous experiments where survival curves were obtained for different cell lines and conditions. Contrary to other, in essence empirical methods for evaluation of macroscopic effects of ionising radiation, the MultiScale Approach predicts the biodamage based on the physical effects related to ionisation of the medium, transport of secondary particles, chemical interactions, thermo-mechanical pathways of biodamage, and heuristic biological criteria for cell survival. We anticipate this method to give great impetus to the practical improvement of ion-beam cancer therapy and the development of more efficient treatment protocols.
69 citations
TL;DR: Analysis indicated that non‐linear models could give a better representation of the RBE‐LET relationship, as differences between the models were observed for the SOBP scenario, and both non‐ linear LET spectrum‐ and linear LETd based models should be further evaluated in clinically realistic scenarios.
Abstract: Purpose
The relative biological effectiveness (RBE) of protons varies with the radiation quality, quantified by the linear energy transfer (LET). Most phenomenological models employ a linear dependency of the dose-averaged LET (LETd) to calculate the biological dose. However, several experiments have indicated a possible non-linear trend. Our aim was to investigate if biological dose models including non-linear LET dependencies should be considered, by introducing a LET spectrum based dose model.
Method
The RBE-LET relationship was investigated by fitting of polynomials from 1st to 5th degree to a database of 85 data points from aerobic in vitro experiments. We included both unweighted and weighted regression, the latter taking into account experimental uncertainties. Statistical testing was performed to decide whether higher degree polynomials provided better fits to the data as compared to lower degrees. The newly developed models were compared to three published LETd based models for a simulated spread out Bragg peak (SOBP) scenario.
Results
The statistical analysis of the weighted regression analysis favoured a non-linear RBE-LET relationship, with the quartic polynomial found to best represent the experimental data (p=0.010). The results of the unweighted regression analysis were on the borderline of statistical significance for non-linear functions (p=0.053), and with the current database a linear dependency could not be rejected. For the SOBP scenario, the weighted non-linear model estimated a similar mean RBE value (1.14) compared to the three established models (1.13-1.17). The unweighted model calculated a considerably higher RBE value (1.22).
Conclusion
The analysis indicated that non-linear models could give a better representation of the RBE-LET relationship. However, this is not decisive, as inclusion of the experimental uncertainties in the regression analysis had a significant impact on the determination and ranking of the models. As differences between the models were observed for the SOBP scenario, both non-linear LET spectrum- and linear LETd based models should be further evaluated in clinically realistic scenarios.
This article is protected by copyright. All rights reserved.
34 citations
TL;DR: The effect of proton- and gamma- irradiation on cell cycle, death, epithelial-mesenchymal transition (EMT) and "stemness" in human non-small cell lung carcinoma cells (A549) is investigated.
Abstract: Proton beam therapy is a cutting edge modality over conventional gamma radiotherapy because of its physical dose deposition advantage. However, not much is known about its biological effects vis-a-vis gamma irradiation. Here we investigated the effect of proton- and gamma- irradiation on cell cycle, death, epithelial-mesenchymal transition (EMT) and "stemness" in human non-small cell lung carcinoma cells (A549). Proton beam (3MeV) was two times more cytotoxic than gamma radiation and induced higher and longer cell cycle arrest. At equivalent doses, numbers of genes responsive to proton irradiation were ten times higher than those responsive to gamma irradiation. At equitoxic doses, the proton-irradiated cells had reduced cell adhesion and migration ability as compared to the gamma-irradiated cells. It was also more effective in reducing population of Cancer Stem Cell (CSC) like cells as revealed by aldehyde dehydrogenase activity and surface phenotyping by CD44(+), a CSC marker. These results can have significant implications for proton therapy in the context of suppression of molecular and cellular processes that are fundamental to tumor expansion.
34 citations
References
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TL;DR: It is shown that hypoxia protected HepG2 cells against etoposide-induced apoptosis and the possible implication of HIF-1 in the anti-apoptotic role of Hypoxia was investigated using Hif-1alpha siRNA, which indicated that H IF-1 is not involved in thehypoxia-induced anti-APoptotic pathway.
54 citations
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11 Jul 2000
45 citations
"Low-LET Proton Irradiation of A549 ..." refers background in this paper
...Besides traditional treatment of this disease, such as surgery or chemotherapy and/or radiotherapy, new modalities are under investigation like radio-immunotherapy (RIT) (2, 3) or external radiotherapy with heavy charged particles like protons....
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...Besides traditional treatment of this disease, such as surgery or chemotherapy and/or radiotherapy, new modalities are under investigation such as radio-immunotherapy (RIT) (2, 3) and external radiotherapy with charged particles such as protons....
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TL;DR: Although from a physical point of view PT is a good option for the treatment of NSCLC, limited data are available on its application in the clinical practice and the application of PT to lung cancer does present technical challenges.
45 citations
"Low-LET Proton Irradiation of A549 ..." refers background in this paper
...More recently, studies on the use of proton therapy in the treatment of nonsmall-cell lung cancer (NSCLC) have been performed and reviewed in (8-10)....
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TL;DR: No significant differences in RBE between pulsed and continuous proton irradiation in HeLa cells were detected, well in line with the data on micronucleus induction in He La cells.
Abstract: In particle tumor therapy including beam scanning at accelerators, the dose per voxel is delivered within about 100 ms. In contrast, the new technology of laser plasma acceleration will produce ultimately shorter particle packages that deliver the dose within a nanosecond. Here, possible differences for relative biological effectiveness in creating DNA double-strand breaks in pulsed or continuous irradiation mode are studied. HeLa cells were irradiated with 1 or 5 Gy of 20-MeV protons at the Munich tandem accelerator, either at continuous mode (100 ms), or applying a single pulse of 1-ns duration. Cells were fixed 1 h after 1-Gy irradiation and 24 h after 5-Gy irradiation, respectively. A dose-effect curve based on five doses of X-rays was taken as reference. The total number of phosphorylated histone H2AX (gamma-H2AX) foci per cell was determined using a custom-made software macro for gamma-H2AX foci counting. For 1 h after 1-Gy 20-MeV proton exposures, values for the relative biological effectiveness (RBE) of 0.97 ± 0.19 for pulsed and 1.13 ± 0.21 for continuous irradiations were obtained in the first experiment 1.13 ± 0.09 and 1.16 ± 0.09 in the second experiment. After 5 Gy and 24 h, RBE values of 0.99 ± 0.29 and 0.91 ± 0.23 were calculated, respectively. Based on the gamma-H2AX foci numbers obtained, no significant differences in RBE between pulsed and continuous proton irradiation in HeLa cells were detected. These results are well in line with our data on micronucleus induction in HeLa cells.
40 citations
"Low-LET Proton Irradiation of A549 ..." refers background in this paper
...Proton irradiation has already been shown to induce DNA double-strand breaks (26), and we observed an increase in DNA damage with increasing doses....
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15 Dec 2011-Nuclear Instruments & Methods in Physics Research Section B-beam Interactions With Materials and Atoms
TL;DR: In this paper, a broad beam in vitro irradiation station for use in radiobiological experiments is presented, where cells are handled in GLP conditions and can be irradiated at various fluxes with ions ranging from hydrogen to carbon.
Abstract: The study of the interaction of charged particles with living matter is of prime importance to the fields of radiotherapy, radioprotection and space radiobiology. Particle accelerators and their associated equipment are proven to be helpful tools in performing basic science in all these fields. Indeed, they can accelerate virtually any ions to a given energy and flux and let them interact with living matter either in vivo or in vitro. In this context, the University of Namur has developed a broad beam in vitro irradiation station for use in radiobiological experiments. Cells are handled in GLP conditions and can be irradiated at various fluxes with ions ranging from hydrogen to carbon. The station is mounted on a 2 MV tandem accelerator, and the energy range can be set up in the linear energy transfer (LET) ranges that are useful for radiobiological experiments. This paper describes the current status of the hardware that has been developed, and presents results related to its performance in term of dose-rate, energy range and beam uniformity for protons, alpha particles and carbon ions. The results of clonogenic assays of A549 lung adenocarcinoma cells irradiated with protons and alpha particles are also presented and compared with literature. © 2011 Elsevier B.V. All rights reserved.
32 citations
"Low-LET Proton Irradiation of A549 ..." refers methods in this paper
...The plating efficiency (PE) was determined for each dose and the surviving fraction was calculated as the ratio of the PE for the irradiated cells to the control cells (12)....
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