Thermal Neutron Relative Biological Effectiveness Factors for Boron Neutron Capture Therapy from In Vitro Irradiations

cells Article Thermal Neutron Relative Biological Effectiveness Factors for Boron Neutron Capture Therapy from In Vitro Irradiations María Pedrosa-Rivera 1, Javier Praena 1, Ignacio Porras 1,* , Manuel P. Sabariego 1, Ulli Köster 2, Michael Haertlein 2,3, V. Trevor Forsyth 2,3,4, JoséC. Ramírez 5, Clara Jover 5, Daniel Jimena 5, Juan L. Osorio 5, Patricia Álvarez 6, Carmen Ruiz-Ruiz 6,* and María J. Ruiz-Magaña 6 1Departamento de Física Atómica, Molecular y Nuclear, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain; [email protected] (M.P.-R.); [email protected] (J.P.); [email protected] (M.P.S.) 2Institut Laue-Langevin, 71 Avenue des Martyrs, CEDEX 9, 38042 Grenoble, France; [email protected] (U.K.); [email protected] (M.H.); [email protected] (V.T.F.) 3Partnership for Structural Biology (PSB), CEDEX 9, 38042 Grenoble, France 4Faculty of Natural Sciences, Keele University, Staffordshire ST5 5BG, UK 5Servicio de Radiofísica y Protección Radiológica, Hospital Universitario Virgen de las Nieves, Avda. Fuerzas Armadas 2, 18014 Granada, Spain; [email protected] (J.C.R.); [email protected] (C.J.); [email protected].es (D.J.); [email protected] (J.L.O.) 6Departamento de Bioquímica y Biología Molecular III e Inmunología, Facultad de Medicina, Universidad de Granada, 18016 Granada, Spain; [email protected].es (P.Á.); [email protected] (M.J.R.-M.) *Correspondence: [email protected] (I.P.); [email protected] (C.R.-R.) Received: 30 July 2020; Accepted: 21 September 2020; Published: 23 September 2020   Abstract: The experimental determination of the relative biological effectiveness of thermal neutron factors is fundamental in Boron Neutron Capture Therapy. The present values have been obtained while using mixed beams that consist of both neutrons and photons of various energies. A common weighting factor has been used for both thermal and fast neutron doses, although such an approach has been questioned. At the nuclear reactor of the Institut Laue-Langevin a pure low-energy neutron beam has been used to determine thermal neutron relative biological effectiveness factors. Different cancer cell lines, which correspond to glioblastoma, melanoma, and head and neck squamous cell carcinoma, and non-tumor cell lines (lung fibroblast and embryonic kidney), have been irradiated while using an experimental arrangement designed to minimize neutron-induced secondary gamma radiation. Additionally, the cells were irradiated with photons at a medical linear accelerator, providing reference data for comparison with that from neutron irradiation. The survival and proliferation were studied after irradiation, yielding the Relative Biological Effectiveness that corresponds to the damage of thermal neutrons for the different tissue types. Keywords: boron neutron capture therapy; relative biological effectiveness; thermal neutrons 1. Introduction Boron Neutron Capture Therapy (BNCT) is currently undergoing a renaissance that may bring this therapy closer to hospital practice [ 1 ]. This is occurring as result of data from new accelerator sources, in combination with the promising results from previous clinical trials at research reactors [ 2 ]. A couple of this new accelerator-based sources in Japan (Kyoto Research Reactor Institute, Osaka, and Southern Tohoku General Hospital, Fukushima, Japan) have already started clinical trials [ 3 ]. In addition to this, the translation of recent research results from different disciplines to the clinical treatment may improve the therapeutic capability of this approach in the near future. Cells 2020,9, 2144; doi:10.3390/cells9102144 www.mdpi.com/journal/cells Cells 2020,9, 2144 2 of 14 Treatment planning is one line of improvement. In a clinical study that was performed at Helsinki University Central Hospital [ 4 ], the results from two cohorts of patients receiving a differently planning tumor volume (PTV) dosing clearly showed that a small increase in the dose may lead to a much improved therapeutic outcome. The dose in BNCT, and in particular the “photon-equivalent”, “photon-isoeffective”, or “biologically weighted” [ 5 ] dose, is a key problem because the dose that the organs at risk may tolerate is a limiting factor in treatment planning and, therefore, it limits the dose delivered to PTV. Therefore, increasing the accuracy of the estimation of this dose can lead to an improvement of the clinical treatment. The biologically weighted dose is currently estimated while using fixed relative biological effectiveness (RBE) factors for the different components of the dose: thermal neutrons, fast (including epithermal) neutrons, boron, and gamma [ 6 ], although, recently, more accurate formalisms, such as the photon iso-effective dose [ 7 ] and intermediate models, have been proposed [ 8 ]. In all of these models, either the RBE factors or the alpha and beta radiobiological coefficients are required for both the BNCT dose components and for reference photon irradiation. Therefore, it is desirable to independently determine the experimental response to neutrons in the three energetic groups that were considered for BNCT: thermal (E n <0.5 eV), epithermal (0.5 eV ≤ E n≤ 10 keV), and fast (En>10 keV). A source of information of the response of cells to neutrons are in-vitro irradiation experiments, where different cell cultures are irradiated in a neutron beam at different doses and the biological end-point considered (the “survival”) is the fraction of cells that has the ability to form clones a few days after the irradiation (clonogenic assay). From the comparison of these survival curves from neutron irradiation with those that were obtained from a reference photon irradiation, the relative biological effectiveness of neutrons as compared to photons can be obtained. Some of these experiments can be found in Refs. [ 9 – 13 ]. However, in the previous measurements, the neutron irradiation was performed with a mixed beam that contained fast and thermal neutrons as well as gamma rays. In that case, and for estimating the pure effect of neutrons, the subtraction of the gamma component may lead to significant uncertainties if its contribution is large. Additionally, due to the mixing of fast and thermal neutrons in the previous experiments, a common RBE factor for both has been adopted, whilst there has been some evidence against this assumption [ 14 ]. A spectrum-dependence of the neutron effect would mean that the RBE of the fast neutron dose could depend on the particular facility being used, and it might be very different at reactor or accelerator-based sources. On the other hand, the RBE of thermal neutrons does not depend on the energy spectrum, because the biological effect is mostly due to the high-energy products of the 14 N(n,p) 14 C reaction (625.87 keV). Thus, in practice, a low energy neutron spectrum does not affect the energy of these products. Therefore, the RBE of thermal neutrons is a key factor in BNCT, because it does not depend on the facility. The aim of this work was to measure the biological response of different cell lines (cancer and normal tissues) under a pure low energy neutron beam, with almost no contamination of fast neutrons and gamma radiation, and then compare it with the response of the same cell lines under a reference photon irradiation from a hospital LINAC. Dedicated setups were designed for both kinds of irradiations. Two different biological end points have been studied for the determination of the neutron response, but only the common one (clonogenic ability) has been chosen for the thermal neutron RBE calculations. 2. Materials and Methods 2.1. Neutron Irradiations Neutron irradiations were carried out at the PF1B cold neutron beam line at the Institut Laue-Langevin (ILL) [ 15 ]. The arrangement that was developed for neutron irradiation of culture cells has been described elsewhere [ 16 ]. This cold beam (lower energy than thermal) is equivalent to a thermal neutron beam, because the energy that is delivered in the neutron interactions does not depend on its kinetic energy and, as a result of the 1/v behavior (where v is the neutron velocity) of neutron Cells 2020,9, 2144 3 of 14 capture in the cold/thermal energy range, the thermal equivalent flux can be used to characterize the beam [ 15 ]. Hence, in the following we discuss “thermal dose” even if the neutron spectrum used was actually “cold”. On PF1B, the epithermal neutron and gamma contributions are negligible as a result of the bent guide. The thermal equivalent neutron flux at the sample position, as measured by gold foil irradiation, was 1.75 × 10 9 n thermal /(cm 2 s). Figure 1shows how this low energy spectrum, without any fast neutron contribution, is ideal for studies of the thermal factor, in contrast to the epithermal neutron beams used in BNCT. Cells 2020, 9, x FOR PEER REVIEW 3 of 14 depend on its kinetic energy and, as a result of the 1/v behavior (where v is the neutron velocity) of neutron capture in the cold/thermal energy range, the thermal equivalent flux can be used to characterize the beam [15]. Hence, in the following we discuss “thermal dose” even if the neutron spectrum used was actually “cold”. On PF1B, the epithermal neutron and gamma contributions are negligible as a result of the bent guide. The thermal equivalent neutron flux at the sample position, as measured by gold foil irradiation, was 1.75 × 109 nthermal/(cm2s). Figure 1 shows how this low energy spectrum, without any fast neutron contribution, is ideal for studies of the thermal factor, in contrast to the epithermal neutron beams used in BNCT. Figure 1. Simulated neutron spectrum at the end of the collimation system of the PF1B line at the Institut Laue-Langevin (ILL) (squares) when compared with neutron Boron Neutron Capture Therapy (BNCT) sources [17,18], such as the epithermal beam of Brookhaven Medical Research Reactor (BMRR), epithermal and thermal beams of Kyoto University Research Reactor Institute (KURR) and the epithermal beam in Finland Reactor 1 (FIR1). Data are expressed in neutron flux per unit of lethargy. The experimental arrangement that is shown in Figure 2 with two consecutive quartz cuvettes was designed for their simultaneous irradiation with the second one receiving lower dose than the first one. In each cuvette, the cells were attached to the surface perpendicular to the neutron beam. The gamma dose is due to the neutron capture on the elements of the quartz and the cell culture medium and it stays lower than the neutron dose, which makes this instrument a very appropriate place to study the effect of low-energy neutrons. The cells were irradiated homogenously during times ranging from 15 to 75 min. and at room temperature (23–25 °C). Figure 1. Simulated neutron spectrum at the end of the collimation system of the PF1B line at the Institut Laue-Langevin (ILL) (squares) when compared with neutron Boron Neutron Capture Therapy (BNCT) sources [ 17 , 18 ], such as the epithermal beam of Brookhaven Medical Research Reactor (BMRR), epithermal and thermal beams of Kyoto University Research Reactor Institute (KURR) and the epithermal beam in Finland Reactor 1 (FIR1). Data are expressed in neutron flux per unit of lethargy. The experimental arrangement that is shown in Figure 2with two consecutive quartz cuvettes was designed for their simultaneous irradiation with the second one receiving lower dose than the first one. In each cuvette, the cells were attached to the surface perpendicular to the neutron beam. The gamma dose is due to the neutron capture on the elements of the quartz and the cell culture medium and it stays lower than the neutron dose, which makes this instrument a very appropriate place to study the effect of low-energy neutrons. The cells were irradiated homogenously during times ranging from 15 to 75 min. and at room temperature (23–25 ◦C). Cells 2020,9, 2144 4 of 14 Cells 2020, 9, x FOR PEER REVIEW 4 of 14 Figure 2. Picture and schematic cut view of the experimental arrangement installed on the PF1B instrument at the institute Laue-Langevin (ILL). Two cuvettes containing cells were irradiated at the same time. All of the cells are irradiated homogeneously and the second cuvette receives a smaller dose than the first one. LiF is used as the first layer of collimators and as a beam stop, capturing neutrons without the generation of secondary gamma radiation. 2.2. Photon Irradiations at Medical Linear Accelerator Photon irradiations were carried out at the medical linear accelerator (LINAC) of Virgen de las Nieves Hospital (HVN) in Granada. The Elekta Versa HD™ accelerator delivered a flattened 6 MV high energy photon beam [19]. The irradiation zone was adapted in order to carry out in vitro irradiations. The electronic equilibrium was secured by immersing the flasks in distilled water and placing under these 14 cm of solid water (see Figure 3). Two flasks were simultaneously irradiated at room temperature (23–25 °C), both within the field of the beam, and then receiving the same dose. Irradiations of 0.5–6 Gy were performed at 1 Gy/min. dose rate. Figure 2. Picture and schematic cut view of the experimental arrangement installed on the PF1B instrument at the institute Laue-Langevin (ILL). Two cuvettes containing cells were irradiated at the same time. All of the cells are irradiated homogeneously and the second cuvette receives a smaller dose than the first one. LiF is used as the first layer of collimators and as a beam stop, capturing neutrons without the generation of secondary gamma radiation. 2.2. Photon Irradiations at Medical Linear Accelerator Photon irradiations were carried out at the medical linear accelerator (LINAC) of Virgen de las Nieves Hospital (HVN) in Granada. The Elekta Versa HD ™ accelerator delivered a flattened 6 MV high energy photon beam [ 19 ]. The irradiation zone was adapted in order to carry out in vitro irradiations. The electronic equilibrium was secured by immersing the flasks in distilled water and placing under these 14 cm of solid water (see Figure 3). Two flasks were simultaneously irradiated at room temperature (23–25 ◦ C), both within the field of the beam, and then receiving the same dose. Irradiations of 0.5–6 Gy were performed at 1 Gy/min. dose rate. Cells 2020,9, 2144 5 of 14 Cells 2020, 9, x FOR PEER REVIEW 5 of 14 Figure 3. Experimental arrangements for cell irradiations with photons used at the medical linear accelerator in Granada. Two flasks with a layer of cells are irradiated at the same time with the same dose. 2.3. Dose Estimation Measurements of neutron flux were performed using gold foil activation data. Neutron doses at the cells were estimated with the MCNPX simulation code for the transport of the neutrons and the generated photons [20]. The geometry of the experimental arrangement was accurately simulated, calculating the dose by means of the kerma factor that depends on the nitrogen content of each cell line. Corrections due to charged particle disequilibrium effects, which are important, as shown by Bortolussi et al. [21], were included in the dose estimations. More details can be found in Ref. [16]. Photon doses at HVN medical LINAC were known from a computed tomography scan image and the clinically used Pinnacle treatment planning system (Philips, Amsterdam, The Netherlands) [22], which uses a collapsed cone convolution superposition algorithm. 2.4. Nitrogen Content Analysis At thermal energies, the neutron capture on nitrogen dominates the local dose deposition among all possible reactions in the tissue. Therefore, the nitrogen content of the cells is essential for the dose estimation. CHNS elemental analysis was performed with a THERMO SCIENTIFIC Flash 2000 (Thermo Fisher, Waltham, MA, USA) analyzer. CHNS elemental analyzers provide a means for the rapid determination of carbon, hydrogen, nitrogen, and sulphur in organic matrices and other types of materials. It is based on the dynamic combustion of a sample. The resultant gases are separated and detected by a thermal conductivity detector (TCD). This technique can determine the quantity of carbon, nitrogen, hydrogen, and sulphur with an error less than 3%. The nitrogen content of cells was measured by this method at the Centro de Instrumentación Científica (CIC) from the University of Granada. To this end, a dry pellet of 10 × 106 cells was prepared and 2 μg of the pellet were used for the CHNS elemental analysis. 2.5. Cells and Cell Culture The six irradiated cell lines were of human origin, four from cancer tissue and two from healthy tissue. The A375 cells are from malignant melanoma; Cal33 and SQ20 from head and neck squamous cell carcinoma; U87 from glioblastoma; HEK293 are from embryonic kidney; and, MRC5 are fibroblast from fetal lung tissue. The cell lines were kindly provided from Institute of Advanced Biosciences, Grenoble, except for HEK293, which was commercially acquired (Thermo Fisher). The selected cancer cell lines correspond to the type of tumors that have been typically treated with BNCT. All of them are adherent cells, which is also a requirement in our set-up. Figure 3. Experimental arrangements for cell irradiations with photons used at the medical linear accelerator in Granada. Two flasks with a layer of cells are irradiated at the same time with the same dose. 2.3. Dose Estimation Measurements of neutron flux were performed using gold foil activation data. Neutron doses at the cells were estimated with the MCNPX simulation code for the transport of the neutrons and the generated photons [ 20 ]. The geometry of the experimental arrangement was accurately simulated, calculating the dose by means of the kerma factor that depends on the nitrogen content of each cell line. Corrections due to charged particle disequilibrium effects, which are important, as shown by Bortolussi et al. [21], were included in the dose estimations. More details can be found in Ref. [16]. Photon doses at HVN medical LINAC were known from a computed tomography scan image and the clinically used Pinnacle treatment planning system (Philips, Amsterdam, The Netherlands) [ 22 ], which uses a collapsed cone convolution superposition algorithm. 2.4. Nitrogen Content Analysis At thermal energies, the neutron capture on nitrogen dominates the local dose deposition among all possible reactions in the tissue. Therefore, the nitrogen content of the cells is essential for the dose estimation. CHNS elemental analysis was performed with a THERMO SCIENTIFIC Flash 2000 (Thermo Fisher, Waltham, MA, USA) analyzer. CHNS elemental analyzers provide a means for the rapid determination of carbon, hydrogen, nitrogen, and sulphur in organic matrices and other types of materials. It is based on the dynamic combustion of a sample. The resultant gases are separated and detected by a thermal conductivity detector (TCD). This technique can determine the quantity of carbon, nitrogen, hydrogen, and sulphur with an error less than 3%. The nitrogen content of cells was measured by this method at the Centro de Instrumentaci ó n Cient í fica (CIC) from the University of Granada. To this end, a dry pellet of 10 × 10 6 cells was prepared and 2 µg of the pellet were used for the CHNS elemental analysis. 2.5. Cells and Cell Culture The six irradiated cell lines were of human origin, four from cancer tissue and two from healthy tissue. The A375 cells are from malignant melanoma; Cal33 and SQ20 from head and neck squamous cell carcinoma; U87 from glioblastoma; HEK293 are from embryonic kidney; and, MRC5 are fibroblast from fetal lung tissue. The cell lines were kindly provided from Institute of Advanced Biosciences, Grenoble, except for HEK293, which was commercially acquired (Thermo Fisher). The selected cancer Cells 2020,9, 2144 6 of 14 cell lines correspond to the type of tumors that have been typically treated with BNCT. All of them are adherent cells, which is also a requirement in our set-up. The cells were cultured in DMEM medium (HyClone, Logan, UT, USA) that contained 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA), 1 µ M L-glutamine (Gibco), 100 IU/mL penicillin, and 100 IU/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) (complete medium) at 37 ◦ C in a humidified CO 2 95% air incubator. The cells were sub-cultured at a ratio of 1:3 and they were grown to 90% confluence. The medium was replaced every 2–3 days. Twenty-four hours before irradiations, cells were cultured in complete medium at 70% confluence in either quartz cuvettes (between 1.5 × 10 5 and 2 × 10 5 cells in 200 µ L culture medium) for neutron irradiation or T25 flasks (between 0.7 × 10 6 and 1.4 × 10 6 cells) for photon irradiation. The medium was replaced with fresh complete medium before irradiating the cells. 2.6. Clonogenic Assays The irradiated cells were detached with 1% trypsin-EDTA (Sigma-Aldrich) and then prepared for clonogenic assays. Cells were seeded in triplicate on six-well plates at appropriate numbers (between 2×102 and 8 × 10 3 cells/well), based on the irradiation dose and the growing characteristics of the cell line, to form colonies of more than 50 cells in around two weeks. Every four days, the medium was exchanged with fresh complete medium. Colonies were fixed with 90% ethanol, stained with crystal violet, and then counted using the open access automatic counter program by Nghia Ho [23]. 2.7. Proliferation Assays After irradiation, the cells were cultured in triplicate in 96-well plates at a density of 1 × 10 3 cells/well and incubated for four days. Proliferative ability was determined by a 5-bromo-2-deoxyuridine (BrdU) ELISA kit (Roche, Mannheim, Germany), following the manufacturer’s instruction. 3. Results 3.1. Dosimetry For ILL neutron irradiation studies, with nitrogen concentration in the cell lines ranging from 1.0 to 2.3%, the simulated neutron thermal doses rates varied from 0.005 to 0.026 Gy/min., depending on the tissue type. The elemental analyzer method is validated, as the nitrogen content of HEK293 cell line measured is in good agreement with the reference in ICRU report 46 for fetus kidney [ 24 ]. Table 1 indicates the nitrogen content for the remaining cell lines. Similar quantities of nitrogen, around 2%, are found in most of the cell lines, except for the MRC5 healthy cell line, where the amount of measured nitrogen is lower. It should be noted that the thermal dose is nearly entirely ( ≈ 96% contribution) due to neutron captures on nitrogen [ 25 ]. Hence, a variation in nitrogen concentration leads in first order to a proportional change of thermal dose. The assumed nitrogen content and resulting thermal dose are both shown in Table 1. Table 1. Nitrogen content and dose components of the cell lines irradiated using the PF1B neutron beam instrument at the Institut Laue-Langevin (ILL). Campaign of June 2018. Cell Line Nitrogen Content (%) Thermal Dose, Dn(Gy/min) Gamma Dose, Dγ(Gy/min) Cuvette 1 Cuvette 2 Cuvette 1 Cuvette 2 A375 2.2 0.026 0.012 0.013 0.010 Cal33 2.3 0.024 0.011 0.013 0.010 U87 2.0 0.021 0.010 0.013 0.010 SQ20 2.3 0.024 0.011 0.013 0.010 HEK293 1.7 0.018 0.008 0.013 0.010 MRC5 1.0 0.011 0.005 0.013 0.010 Cells 2020,9, 2144 7 of 14 Gamma doses, mainly from neutron interaction with the experimental system, are tissue-independent and, therefore, are the same for all cell lines. In most of the cases, the thermal dose component remained higher than the gamma dose component (see Table 1). The main objective of the irradiations, which was to have a dose mostly due to low-energy neutrons, was achieved. The biological effect of the secondary gamma radiation would be expected to be less than in higher dose rate LINAC irradiations due to the low gamma dose rate. Indeed, this could be expected in long irradiation, because of repair effects. Nevertheless, the RBE of the gamma dose is set equal to 1 due to the lack of data for the studied cell lines at the present dose rate. Additionally, conclusive data for the possible synergies between the high-LET and low-LET radiations were not found. Thus, a cross term for describing this effect in the survival curves was not considered. 3.2. Results for Neutron Irradiation Two end-points were studied for the neutron irradiations: survival, by means of clonogenic assay, and proliferation, by using BrdU incorporation assay, around two weeks and four days, respectively, after the irradiation. Figure 4displays plates for the visual appearance of these assays with A375 cells. Cells 2020, 9, x FOR PEER REVIEW 7 of 14 Gamma doses, mainly from neutron interaction with the experimental system, are tissue-independent and, therefore, are the same for all cell lines. In most of the cases, the thermal dose component remained higher than the gamma dose component (see Table 1). The main objective of the irradiations, which was to have a dose mostly due to low-energy neutrons, was achieved. The biological effect would be expected to be less than in higher dose rate LINAC irradiations due to the low gamma dose rate. Indeed, this could be expected in long irradiation, because of repair effects. Nevertheless, the RBE of the gamma dose is set equal to 1 due to the lack of data for the studied cell lines at the present dose rate. Additionally, conclusive data for the possible synergies between the high-LET and low-LET radiations were not found. Thus, a cross term for describing this effect in the survival curves was not considered. 3.2. Results for Neutron Irradiation Two end-points were studied for the neutron irradiations: survival, by means of clonogenic assay, and proliferation, by using BrdU incorporation assay, around two weeks and four days, respectively, after the irradiation. Figure 4 displays plates for the visual appearance of these assays with A375 cells. Both end-points showed different results when data were fitted following a linear quadratic equation, as shown in Figure 5. The biggest difference between the survival and proliferation curves was observed in the head and neck cancer cell lines. Figure 4. A375 plates for both clonogenic and proliferative assays. Left, plate for clonogenic assay with the control sample (CT) and the sample irradiated with a neutron dose of 4 Gy at day seven after irradiation. Right, BrdU cell proliferation assay of control and irradiated cells at day four after irradiation. The samples were analyzed in triplicate for each data point. Because a correlation between the two different end-points could not be established, probably because of the time after irradiation at which they are measured, the comparison with photon irradiation, and the calculations of RBE factors are limited to the most common end-point used, which is the long-term survival by clonogenic assay. Figure 4. A375 plates for both clonogenic and proliferative assays. Left, plate for clonogenic assay with the control sample (CT) and the sample irradiated with a neutron dose of 4 Gy at day seven after irradiation. Right, BrdU cell proliferation assay of control and irradiated cells at day four after irradiation. The samples were analyzed in triplicate for each data point. Both end-points showed different results when data were fitted following a linear quadratic equation, as shown in Figure 5. The biggest difference between the survival and proliferation curves was observed in the head and neck cancer cell lines. Because a correlation between the two different end-points could not be established, probably because of the time after irradiation at which they are measured, the comparison with photon irradiation, and the calculations of RBE factors are limited to the most common end-point used, which is the long-term survival by clonogenic assay. Cells 2020,9, 2144 8 of 14 Cells 2020, 9, x FOR PEER REVIEW 8 of 14 Figure 5. Proliferation and survival data for the different cell lines in response to neutron irradiation. Proliferation (expressed as absorbance relative to control (CT), as determined by BrdU assay), and survival (based on clonogenic assays) are represented for each cell line as a function of the total absorbed dose after neutron irradiation at ILL. Data were obtained from between two and four individual experiments with three replicate dishes plated per point per experiment. 3.3. Cell Survival Survival data after irradiation, S, as studied by clonogenic assays, were fitted using the linear quadratic formula [26]: −ln𝑆 = 𝛼𝐷 + 𝛽𝐷2 (1) where D is the total absorbed dose and α and β are the parameters that describe the behavior of the survival. These parameters are constants that depend on the tissue/cell line and end-point. For irradiations at the medical LINAC, the total dose will correspond to that for the photons only, Dγ, while the survival following irradiations at ILL is due to a dose combination of low-energy neutrons and photons, DILL. Thus, the total absorbed dose at ILL beam corresponds to the sum of neutron and gamma dose, DILL= Dn + Dγ. From the two fitted survival data, SILL and Sγ, the survival that corresponds to the neutron effect alone, Sn, can be calculated as: 𝑆𝑛=𝑒−(𝛼𝐼𝐿𝐿𝐷𝐼𝐿𝐿−𝛽𝐼𝐿𝐿𝐷𝐼𝐿𝐿 2) 𝑒−(𝛼𝛾𝐷𝛾−𝛽𝛾𝐷𝛾 2) (2) This survival describes the effect if the dose is due to low-energy neutrons alone, Dn. The data can be fitted to a quadratic function, but the parameter β is assumed to be zero given the characteristics of high-LET radiation and the better results found with a linear fitting. Figure 5. Proliferation and survival data for the different cell lines in response to neutron irradiation. Proliferation (expressed as absorbance relative to control (CT), as determined by BrdU assay), and survival (based on clonogenic assays) are represented for each cell line as a function of the total absorbed dose after neutron irradiation at ILL. Data were obtained from between two and four individual experiments with three replicate dishes plated per point per experiment. 3.3. Cell Survival Survival data after irradiation, S, as studied by clonogenic assays, were fitted using the linear quadratic formula [26]: −ln S=αD+βD2(1) where Dis the total absorbed dose and α and β are the parameters that describe the behavior of the survival. These parameters are constants that depend on the tissue/cell line and end-point. For irradiations at the medical LINAC, the total dose will correspond to that for the photons only, D γ , while the survival following irradiations at ILL is due to a dose combination of low-energy neutrons and photons, D ILL . Thus, the total absorbed dose at ILL beam corresponds to the sum of neutron and gamma dose, DILL =Dn+Dγ. From the two fitted survival data, S ILL and S γ , the survival that corresponds to the neutron effect alone, Sn, can be calculated as: Sn=e−(αILLDILL−βILLD2 ILL) e−(αγDγ−βγD2 γ)(2) This survival describes the effect if the dose is due to low-energy neutrons alone, D n . The data can be fitted to a quadratic function, but the parameter β is assumed to be zero given the characteristics of high-LET radiation and the better results found with a linear fitting. Cells 2020,9, 2144 9 of 14 Figure 6shows the survival after neutrons irradiations at ILL (S ILL ) and the survival after photon irradiations at HVN medical LINAC (S γ ). As a result of the neutron-induced gamma production in the experimental arrangement, he survival (S n ) that was associated with the neutron dose alone was obtained by the deduction of the gamma effect, as indicated in Equation (2). Table 2shows the alpha and beta parameters for each one. Cells 2020, 9, x FOR PEER REVIEW 9 of 14 Figure 6 shows the survival after neutrons irradiations at ILL (SILL) and the survival after photon irradiations at HVN medical LINAC (Sγ). As a result of the neutron-induced gamma production in the experimental arrangement, he survival (Sn) that was associated with the neutron dose alone was obtained by the deduction of the gamma effect, as indicated in Equation (2). Table 2 shows the alpha and beta parameters for each one. Figure 6. Clonogenic survival of six cell lines as a function of the absorbed dose (Gy) following irradiation with photons at the medical linear accelerator, Sγ, with neutrons at the ILL beam, SILL, and, derived, ILL neutrons alone, Sn. Sγ for U87 from [27] and Sγ for SQ20 from the one obtained for Cal33. The data were obtained from between two and seven individual experiments after photon irradiation and neutron irradiation. Cells were seeded in triplicate for each data point in each experiment. Table 2. Alpha and beta values (standard error) for medical linear accelerator irradiations and for the different dose components at ILL. Cell Line ILL, Total Medical LINAC, Photons ILL, Pure Neutrons αILL (Gy−1) βILL (Gy−2) αγ (Gy−1) βγ (Gy−1) αn (Gy−1) A375 1.22  0.16 0.04 0.06 0.25  0.03 0.075  0.012 1.86  0.06 Cal33 0.56  0.09 0.21  0.04 0.03  0.02 0.047  0.007 1.65  0.05 U87 0.98  0.43 0.07  0.21 0.23  0.02 0.012  0.005 1.74  0.19 SQ20 0.44  0.27 0.31  0.13 0.03  0.02 0.047  0.007 1.63  0.13 HEK293 1.18  0.10 0.14  0.06 0.16  0.01 0.091  0.002 2.37  0.09 MRC5 1.41  0.50 0.00  0.50 0.34  0.08 0.011  0.015 3.40  0.45 The U87 cell line was not photon irradiated and the results from Bayart et al. [27], obtained with a Varian NDI 226 X-ray tube of 200 kVp (kilovolt peak) at a dose rate of 1.2 Gy/min, were used. Figure 6. Clonogenic survival of six cell lines as a function of the absorbed dose (Gy) following irradiation with photons at the medical linear accelerator, S γ , with neutrons at the ILL beam, S ILL, and, derived, ILL neutrons alone, S n .S γ for U87 from [ 27 ] and S γ for SQ20 from the one obtained for Cal33. The data were obtained from between two and seven individual experiments after photon irradiation and neutron irradiation. Cells were seeded in triplicate for each data point in each experiment. Table 2. Alpha and beta values ( ± standard error) for medical linear accelerator irradiations and for the different dose components at ILL. Cell Line ILL, Total Medical LINAC, Photons ILL, Pure Neutrons αILL (Gy−1)βILL (Gy−2)αγ(Gy−1)βγ(Gy−1)αn(Gy−1) A375 1.22 ±0.16 0.04 ±0.06 0.25 ±0.03 0.075 ±0.012 1.86 ±0.06 Cal33 0.56 ±0.09 0.21 ±0.04 0.03 ±0.02 0.047 ±0.007 1.65 ±0.05 U87 0.98 ±0.43 0.07 ±0.21 0.23 ±0.02 0.012 ±0.005 1.74 ±0.19 SQ20 0.44 ±0.27 0.31 ±0.13 0.03 ±0.02 0.047 ±0.007 1.63 ±0.13 HEK293 1.18 ±0.10 0.14 ±0.06 0.16 ±0.01 0.091 ±0.002 2.37 ±0.09 MRC5 1.41 ±0.50 0.00 ±0.50 0.34 ±0.08 0.011 ±0.015 3.40 ±0.45