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.
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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: The mechanism of induced cellular radioresistance appears to be initiated after a certain amount of energy is deposited in the cell nucleus, which depends on both radiation quality and the number of particles traversing the cell.
Abstract: Purpose : To examine the low-dose sensitivity of V79 cells under exposure to γ-rays and 4 He ions of different energies. Materials and methods : Cell survival and cytogenetic analysis using the Giemsa technique were studied following irradiation to doses of 0-3 Gy at the INFN-LNL facilities. Results : Low-dose hyper-radiosensitivity (HRS) of V79 was demonstrated after irradiation with γ-rays and α -particles of various linear energy transfers (LET) (58.9, 79.3 and 101.7 keV μ m -1) . Cytogenetic analysis showed an LET dependence of aberrations at a dose of 1Gy; the frequency of chromatid fragments appeared to vary with the number of α -particles traversing the cell nucleus. The results of both studies fit together to give a better understanding of so-called 'induced radioresistance' phenomenon. Conclusions : The mechanism of induced cellular radioresistance appears to be initiated after a certain amount of energy is deposited in the cell nucleus. This amount depends on both radiation quality and the numbe...
32 citations
TL;DR: The present results with protons and heavier charged particles are promising, however, the current lack of evidence on the clinical (cost-)effectiveness of particle therapy emphasizes the need to investigate the efficiency of particle Therapy in an adequate manner.
Abstract: Background. The societal burden of lung cancer is high because of its high incidence and high lethality. From a theoretical point of view, radiotherapy with beams of protons and heavier charged particles, for example, carbon ions (C-ions), should lead to superior results, compared with photon beams. In this review, we searched for clinical evidence to justify implementa
26 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: An improved method of predicting cell survival for modulated x-ray radiation treatment is provided, and the limitations of the LQ model are highlighted, particularly in its ability to describe the biological response of cells irradiated under these conditions.
Abstract: In this study we present two prediction methods, mean dose and summed dose, for predicting the number of A549 cells that will survive after modulated x-ray irradiation. The prediction methods incorporate the dose profile from the modulated x-ray fluence map applied across the cell sample and the linear quadratic (LQ) model. We investigated the clonogenic survival of A549 cells when irradiated using two different modulated x-ray fluence maps. Differences between the measured and predicted surviving fraction were observed for modulated x-ray irradiation. When the x-ray fluence map produced a steep dose gradient across the sample, fewer cells survived in the unirradiated region than expected. When the x-ray fluence map produced a less steep dose gradient across the sample, more cells survived in the unirradiated region than expected. Regardless of the steepness of the dose gradient, more cells survived in the irradiated region than expected for the reference dose range of 1–10 Gy. The change in the cell survival for the unirradiated regions of the two different dose gradients may be an important factor to consider when predicting the number of cells that will survive at the edge of modulated x-ray fields. This investigation provides an improved method of predicting cell survival for modulated x-ray radiation treatment. It highlights the limitations of the LQ model, particularly in its ability to describe the biological response of cells irradiated under these conditions.
26 citations
"Low-LET Proton Irradiation of A549 ..." refers background or methods in this paper
...6, a comparison is made between survival curves obtained for protons at 10 keV/lm, 25 keV/lm, and 250 kV X rays and at 6 MV X rays (22)....
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...(22) is used for comparison with data from the literature....
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TL;DR: BED and SCP models are used to analyze the advantages of injecting radioactive nanoparticles instead of a single radionuclide per vector in radioimmunotherapy and highlight the important benefit in replacing the single beta-emitter 90Y attached to each antibody by a 90Y2O3 nanoparticle.
Abstract: PURPOSE: Radioactive atoms attached to monoclonal antibodies are used in radioimmunotherapy to treat cancer while limiting radiation to healthy tissues. One limitation of this method is that only one radioactive atom is linked to each antibody and the deposited dose is often insufficient to eradicate solid and radioresistant tumors. In a previous study, simulations with the Monte Carlo N-Particle eXtended code showed that physical doses up to 50 Gy can be delivered inside tumors by replacing the single radionuclide by a radioactive nanoparticle of 5 nm diameter containing hundreds of radioactive atoms. However, tumoral and normal tissues are not equally sensitive to radiation, and previous works did not take account the biological effects such as cellular repair processes or the presence of less radiosensitive cells such as hypoxic cells. METHODS: The idea is to adapt the linear-quadratic expression to the tumor model and to determine biological effective doses (BEDs) delivered through and around a tumor. This BED is then incorporated into a Poisson formula to determine the shell control probability (SCP) which predicts the cell cluster-killing efficiency at different distances "r" from the center of the tumor. BED and SCP models are used to analyze the advantages of injecting radioactive nanoparticles instead of a single radionuclide per vector in radioimmunotherapy. RESULTS: Calculations of BED and SCP for different distances r from the center of a solid tumor, using the non-small-cell lung cancer as an example, were investigated for 90Y2O3 nanoparticles. With a total activity of about 3.5 and 20 MBq for tumor radii of 0.5 and 1.0 cm, respectively, results show that a very high BED is deposited in the well oxygenated part of the spherical carcinoma. CONCLUSIONS: For either small or large solid tumors, BED and SCP calculations highlight the important benefit in replacing the single beta-emitter 90Y attached to each antibody by a 90Y2O3 nanoparticle.
23 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 such as radio-immunotherapy (RIT) (2, 3) and external radiotherapy with charged particles such as 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: Alpha particle irradiations are more effective than X-rays and gene expression changes observed in both cell types after alpha particle or X-ray exposure showed possible crosstalk between both cell type that may induce the development of radioresistance.
22 citations