ANTICANCER RESEARCH 34: 4621-4626 (2014) Radiosensitivity Uncertainty Evaluation for the In Vitro Biophysical Modeling of EMT6 Cells MASATAKA OITA1,2, YOSHIHIRO UTO2, MASAHIDE TOMINAGA3, MOTOHARU SASAKI4, YASUO HARA4, TARO KISHI4 and HITOSHI HORI2 1Department of Radiological Technology, Graduate School of Health Sciences, Okayama University, Okayama, Japan; 2Department of Life System, Institute of Technology and Science, and 3Department of Radiological Technology, Institute of Health Biosciences, Graduate School, The University of Tokushima, Tokushima, Japan; 4Department of Radiology, Tokushima University Hospital, Tokushima, Japan Abstract. Background/Aim: The aims of this study were to radiobiological parameters and molecular biological data evaluate the cell survival uncertainty distribution of radiation from tumor and critical organs could be used in clinical and to assess the accuracy of predictions of tumor response practice for better treatment (6, 7). by using three different in vitro experimental cell cultures with Applying biophysical models to treatment planning in radiosensitizers (including etanidazole). Materials and radiotherapy could increase tumor controllability and reduce Methods: Using EMT6 cells and X-rays, the cell survival toxicity to normal tissues (8, 9). It is well-recognized that fraction was obtained from 15, 34, and 21 different radiation sensitivity is affected by cell heterogeneity and experiments under normoxic, hypoxic, and hypoxic-plus- radiosensitizing agents (10-12). Nevertheless, there is radiosensitizer culture, respectively. Results: The α uncertainty surrounding radiosensitivity because of the coefficients were 0.257±0.188, 0.078±0.080, and 0.182±0.116 factors involved are not well-accounted for in biophysical Gy–1, respectively. The β coefficients were 0.0159±0.0208, modeling (13, 14). In particular, hypoxic cells in tumors 0.0076±0.0113, and 0.0062±0.0077 Gy–2, respectively. The α lead to increased radioresistance of clonogens, which affects coefficient and the dose that killed half of the clonogens clinical outcome (15, 16). Etanidazole and misonidazole are population (D50) were significantly different between well-known hypoxic cell radiosensitizers but have had normoxic cell and hypoxic cell cultures (p<0.01), respectively. limited therapeutic impact on radiotherapy because of side- The use of radiosensitizers under hypoxic conditions effects such as neurotoxicity (17). For biophysical improved radiosensitivity. Conclusion: Our results suggest evaluation of physical dose prescriptions, including dose that parameter value distributions are required for fractions and biological equivalent dose (BED), the linear biophysical modeling of applications for radiotherapy. quadratic (LQ) model based on intrinsic radiosensitivity is commonly used in practical radiotherapy (18). However, to In recent years, there have been rapid advances in make precise predictions on outcomes after radiotherapy at radiotherapy technologies, such as volumetric modulated arc the treatment planning stage, the above-mentioned therapy, stereotactic body radiotherapy, and image-guided uncertainties of radiation sensitivity would need to be radiotherapy (1-5). Because clinical failures after radiation carefully considered. therapy (due to hypoxia, intrinsic radioresistance, and In this study, we evaluated the distribution of uncertainty cellular proliferation) are known to induce genetic changes, of cell survival due to radiation and assessed tumor response predictions using three different in vitro experimental cell cultures. We then discuss the relationship between in vitro radiosensitizing activities and uncertainties in cell survival Correspondence to: Dr. Masataka Oita, Department of Radiological characteristics using radiobiological parameters. Technology, Graduate School of Health Sciences, Okayama University, 2-5-1 Shikata-tyo, Kita-ku, Okayama 700-8558, Japan. Tel: +81 862356871, Fax: +81 862223717, e-mail: [email protected] Materials and Methods u.ac.jp Development of the experimental system. Etanidazole was Key Words: Radiotherapy, biophysical modelling, radiosensitivity synthesized in our laboratory. EMT6/KU mouse mammary tumor uncertainty, in vitro. cells (supplied by Dr. Shinichiro Masunaga, Kyoto University, 0250-7005/2014 $2.00+.40 4621 ANTICANCER RESEARCH 34: 4621-4626 (2014) Kyoto, Japan) were maintained in Eagle’s minimal essential medium (E-MEM) (Sigma-Aldrich Japan, Tokyo, Japan) supplemented with 10% fetal bovine serum (JR Scientific, Inc., Woodland, CA, USA). For single-cell experiments, exponentially- growing cells were harvested from normoxic cell culture dishes by trypsinization, and suspended in test tubes containing 1 ml of E- MEM (2×106 cells/ml). The hypoxic cultures were treated with 95% N2–5% CO2 gas for 30 min. In vitro radiosensitization was also measured in single EMT6 cells by adding radiosensitizer under hypoxic conditions. Radiation procedure. X-Ray irradiation was carried out using an X- ray unit (Hitachi X-ray unit, model MBR-1505R2) with 0.5 mm Al/1.0 mm Cu filter (150 kV, 4 Gy/min). In in vitro assays, cells on the dish were irradiated with 4-28 Gy. After irradiation, colony formation assays were performed. Biophysical modeling. The LQ formalism is used for tumor cure probability curves, which reflect the various tumor parameters. The probability of cells surviving a single dose is given by: Figure 1. Surviving fractions of EMT6 cells in vitro under normoxia, 2 hypoxia, and hypoxia-plus-etanidazole (ETZ) culture. Fitted curves N=N0(−αD−βD ) (Eq. 1) (solid line) represent the approximated curves of α and β coefficients from exponential regression analysis. where N equals the number of surviving cells, N0 equals the number of tumor cells at the start of experiment, α and β are the estimates of radiosensitivity, and D is the total dose given. It follows directly from the above assumptions that the effect of n equally sized Results fractions and d equally dosed fractions can be shown by: 2 β N=N0exp(−nαd−nβd )=N0exp{−αD(1+ /αd)} (Eq. 2) Radiation response of the single cell. Single-cell survival after single-dose fraction in different cell cultures is shown in The Poisson probability of there being zero surviving cells at the Figure 1. Fitted curves using median parameter values of α end of a fractionated treatment is then given as the tumor control and β coefficients from exponential regression analysis (solid probability (TCP) by: and dotted lines, respectively), and the effect of each β coefficient on the curves (α contribution: dark gray, β TCP=exp[N0exp{−αD(1+ /αd)}] (Eq. 3) contribution: light gray) are presented in Figure 2. It can be where the other parameters are as given in Eq. 1 and 2. seen that etanidazole enhanced the radiosensitivity of EMT6 cells in a dose-dependent manner under hypoxic conditions. To evaluate the effects of the radiation sensitizer and the The variations in predicted survival at higher single doses varying sensitivities due to the different cell cultures, data were may be increased; however, this increase could be suppressed obtained from 15, 34, and 21 different experiments under normoxic cell culture, hypoxic cell culture, and hypoxic cell by radiosensitizers. culture-plus-radiosensitizer, respectively. For each cell culture, the averaged survival fraction (SF) after a single fraction was Comparisons of radiobiological parameters. The calculated based on three repeated experiments and entered into radiobiological parameters from these experiments are the model. Then summarized dose survival data by each cultured summarized in Table I. The α and D50 values were experiment were fitted by the model, and biological parameters, significantly different between normoxic and hypoxic cell α, β, D , and γ were calculated. D and γ represented the 50 50 50 50 cultures (p<0.01). dose that killed half of the clonogens population and the steepness of a dose–response level, which was defined as SF=0.5, respectively. The effects of cell proliferation and repair were not Effects of cell survival and tumor control probability on taken into consideration in this study. variation in radiobiological parameters. Figure 2 shows that when the α coefficient values were fixed (light gray), the Statistical analysis. Exponential regression analyses were used to variations in cell survival curves at high doses were assess α and β coefficients, respectively. Statistical comparisons of increased; a similar trend is seen for the β coefficient values mean values were analyzed by two-sample independent t-test. All (dark gray) in all cell cultures. The effects of α and β the data were analyzed using OriginLab (OriginLab Corporation, Northampton, MA, USA) scientific graphing and statistical analysis coefficients on TCP (gray shaded area) are shown in Figure software. A p-value of less than 0.05 was considered statistically 3A-C. Under hypoxic conditions, the mean values of α and β significant. coefficients were smaller and the D50 much higher than 4622 Oita et al: Radiosensitivity Uncertainty Evaluation for Biophysical Modeling Figure 2. Surviving fractions of EMT6 cells in vitro for cells cultured under normoxic (A), hypoxic (B), and hypoxic-plus-etanidazole (ETZ) (C) conditions. Fitted curves (solid line) represent the approximated curves of α and β coefficients from exponential regression analysis (solid line and dotted line). The effect of each coefficient on the curves are represent by dark gray shading for the α contribution and by light gray shading for the β contribution