Radiation modulates bronchial epithelial progenitor activity as assessed in organoid formation assays

Radiation induced lung disease (RILI) is a serious side-effect of radiotherapy for lung cancer. We hypothesize that radiation-induced effects on the normal lung epithelium play a key role in development of RILI. Therefore, the aim of the present study was to study the effect of ionizing radiation (IR) on primary bronchial epithelial cells (PBEC). PBEC were cultured submerged undifferentiated (S-PBEC), differentiated at the Air-Liquid-Interface (ALI-PBEC), and as 3-dimensional organoids (3D-PBEC). Cells were exposed to IR (0.5, 2, 8Gy), dissociated and reseeded in an organoid model in similar densities 1h after IR to observe organoid formation. IR had a marked effect on organoid formation: organoid formation was markedly impaired following IR irrespective of culture model in which cells were exposed to IR (see figure for design and results). In contrast, IR did not increase markers of epithelial-mesenchymal transition (EMT; ZEB-1, SLUG, SNAIL, vimentin) or inflammation (IL-1β, IL-8, VEGF). Furthermore, there were no changes in morphology and TEER following IR, indicating that it did not cause cytotoxicity. IR did cause double stranded DNA breaks, increasing with radiation dose as measured by immunofluorescent staining of γH2Ax-foci. In conclusion, progenitor cell function is affected by radiation as shown by impaired organoid formation, especially in the ALI-PBEC. This cannot be explained by cytotoxicity, inflammation or EMT. This effect on progenitor cell function could be a lead to further delineate RILI.