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,
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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 with 95% confidence intervals (CI), respectively.
Table I. Radiobiological parameters of intrinsic in vitro radiosensitivity of EMT6 cells.
Normoxic (n=15) Hypoxic (n=34) Hypoxic+ETZ (n=21)
Parameter Mean Median SD 95% CI Mean Median SD 95% CI Mean Median SD 95% CI
α (Gy–1) 0.257 0.223 0.188 0.162-0.351 0.0777* 0.0803 0.113 0.0396-0.116 0.182 0.179 0.116 0.143-0.222 β (Gy–2) 0.0159 0.0123 0.0208 0.0054-0.0265 0.0076 0.0062 0.0113 0.0038-0.01140.0062 0.0074 0.0077 0.0036-0.0088 α/β (Gy) −12.4 7.8 73.4 −49.5-24.8 18.1 0.1 114.6 −20.4-56.7 19.4 20.7 21.9 11.8-26.9 D50 (Gy) 3.21 2.46 2.53 1.93-4.49 6.44* 6.10 2.71 5.53-7.35 3.74 3.59 1.35 3.28-4.21 γ50 1.02 0.79 0.53 0.75-1.28 1.14 0.92 0.53 0.96-1.31 0.75 0.78 0.34 0.64-0.87
SD: Standard deviation; CI: confidence interval; ETZ: etanidazole. *p<0.01 compared to normoxic cell culture.
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Figure 3. Variations of calculated tumor control probability curves for EMT6 cells in vitro for normoxic (A, D), hypoxic (B, E), and hypoxic-plus- etanidazole (ETZ) (C, F) culture related to α and β coefficients and cell numbers. Fitted curves (solid line) in A-C represent the approximated 8 curves of α and β coefficients from exponential regression analysis with 95% confidence intervals (CI) (gray shaded area) in the case of N0=10 , respectively.
4624 Oita et al: Radiosensitivity Uncertainty Evaluation for Biophysical Modeling under normoxic conditions (Table I). It is apparent that the survival curves with low α coefficient. The effects of the TCP under such conditions was much lower than that at the variations of α and β coefficients, if either one were fixed to same dose under normoxic conditions. The number of cells the median value, are shown in Figure 2. Under hypoxic (proxy for tumor size) is another clinical factor of conditions, the effects of radiosensitizing by etanidazole are radioresistance, albeit less effective than the variations of α shown to increase the α coefficient, with a decrease of 8 and β coefficients for detecting early cancer (N0≤10 ) as variation in the α and β coefficients. However, under such shown in Figure 3D-F. conditions, the β coefficient’s contribution to radiosensitivity was relatively small. Consequently, the results suggest that Discussion etanidazole could lead to reduction of tumor heterogeneity at high doses, including the change of oxygen tension and The LQ formalism with α and β coefficients provides electron affinity. quantification of radiobiological response, which describes Figure 3 shows the variations of calculated TCP curves the radiation inactivation of cell mixtures of different accounting for the discrepancies of α and β coefficients and intrinsic radiosensitivity. However, in clinical setting, some cell numbers (tumor volume). Several authors have pointed- factors such as inappropriate derivation of α/β values from out that a precise prediction could potentially be possible by single in vitro assays, clarification of radiation-induced late assuming distribution of intrinsic radiosensitivity under effects, and variations in individual α/β values, might cause various cellular circumstances (14, 25). Another important unclear interpretation of the results obtained from early- and factor for determining the TCP is the number of cells that late-responding tissues (19). should be killed to result in a tumor cure; studies have shown Although the α coefficient is relatively constant a consistent cell density of 0.5-1.0×106/g (26, 27). throughout the interphase of the cell cycle (20), the intra- Consequently, the number of cells would be in the order of tumor heterogeneity could cause variations in intrinsic 107-109 in a typical tumor volume of 0.01-100 ml in clinical radiosensitivity at a single-fraction dose. Our data show that settings. In addition, non-proliferating tumor cells are the variation of the α coefficient was nearly the same as that considered to be more resistant to radiation than proliferating in previous studies (21). Consequently, it is suggested the cells (28). Proliferation is a highly important factor one the variation in the α coefficient is of the same order as influencing the TCP, especially for highly fractionated the radiosensitivity exhibited by asynchronous populations. treatment schedules (29). It could also be reasonable to It is thought that the β coefficient is relatively invariant and consider that the variations of intrinsic radiosensitivities of its contribution to cell death is much smaller at conventional cells in vitro, tumor cells extracted by biopsy, or the use of treatment doses (22). However, since hypo-fractionation tumor-bearing chick embryo (30), will predict the radiation techniques are clinically implemented, it is suggested the response of similar cells in vivo. The purpose of this study effects of the β coefficient will not be negligible. is not to claim that we fully-understand how to model the Wide ranges of the α/β ratios, which can lead to a cell- precise radiation response, but rather to show that important lethal dose, were shown in our study, including a negative biophysical parameters are useful for predicting tumor value. The results were caused by a negative β coefficient response. In conclusion, our results suggest that the through differing sensitivities of the cells in the distributions of the parameter values are required for heterogeneous populations at the high-dose region. biophysical modeling of applications for radiotherapy. Moreover, in the curve-fitting process, the data are often Further advancement would benefit from additional more complex than those described using the linear quadratic experiments employing different tumor models, and thus, in equation. In the case of single, very high-dose fraction vivo studies are necessary. experiments, the survival curve might be dependent on the experimental conditions. Acknowledgements Figure 1 shows the radiosensitivities in different cell cultures. The hypoxic cells appear to be twice as This work was supported by Grant-in-Aid for Young Scientists (B) radioresistant as normoxic cells by survival fraction. No. 25861105 from MEXT Japan. Intratumoral hypoxic cell proportions are a major complicating factor in cancer therapy, and are an important References target for anticancer drug design (23). 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