In Vitro Reaction of Cells Derived from Human Normal Lung Tis- sues to Carbon-Ion Beam Irradiation

Background: Radiation-induced lung injury (RILI) is a serious concern in carbon-ion radiation therapy (CIRT) for thoracic malignancies. To estimate the induction of RILI after CIRT, translation of evidence in X-ray radiation therapy to CIRT is of great importance. However, the relative biological effectiveness (RBE) of carbon-ion beams in normal lung tissues is not fully elucidated, making the translation difficult. This is in part due to the absence of in vitro assays that can determine the RBE values in normal cells lacking clonogenic ability. Materials and methods: Immortalized human small airway epithelial cells (iSAECs), human lung fibroblasts HFL-I, and A549 lung cancer cellswere irradiated with X-rays or carbon-ion beams, cultured for 10 days, fixed with methanol, and then subjected to crystal violet staining (CVS). The stained cells were solubilized and the absorbance of the solutions was measured using a spectrophotometer. The data plots were fitted to the linear-quadratic model to generate survival curves. Results: The absorbance values in the CVS assay were highly correlated withthe number of colonies in the clonogenic survival assay for A549 cells (R = 0.905, P < 0.01), resulting in strong association between the survival curves generated using the two assays. In iSAECs, the D50 values for X-ray and carbon-ion beam irradiation were 8.3 Gy and 2.6 Gy, respectively, and the RBE was 3.2. In HFL-I cells, the D50 values were 3.3 Gy and 1.5 Gy, respectively, and the RBE was 2.2. Conclusion: The CVS assay may provide an alternative to the clonogenic survival assay for the assessment of the RBE of carbon ion beam irradiationof normal cells lacking clonogenic ability, and will help understand the biological effect of CIRT on human normal lung tissues.