IntroductionLung cancer is the leading cause of cancer death in western countries. While significant progress has been made in terms of treatment, radiotherapy is limited by dose-limiting side-effects. Reducing side-effects may improve tumour control by dose-escalation and treatment-time. The NOTCH signalling pathway plays an important role in differentiation of the airway epithelium and its deregulation is associated with lung cancer. However, the mechanism by which NOTCH inhibition integrates with airway repair is unknown. What is currently lacking are primary human lung tissue models that enable robust evaluation of the effects of treatment which can guide the selection of combination treatments for lung cancer.Material and methodsWe therefore investigated the effects of inhibiting NOTCH in human basal stem cells from normal primary bronchial epithelial cells isolated from lung cancer patients undergoing lobectomy. Basal stem cells isolated from 3 different patients were treated with the pan NOTCH inhibitor (DBZ) and irradiated with 2–4 Gy. Incucyte experiment, Edu/PI staining and clonogenic assay were used to evaluate proliferation and clonal capacity. To evaluate DNA damage response 53 bp1 staining was performed. To study Notch inhibition in basal cells differentiation the tracheal epithelium of 2–5 Gy irradiated mice was seeded in ALI system and confocal stainings were performed.Results and discussionsIncucyte experiment revealed a reduced proliferation of stem cells upon NOTCH inhibition which is further reduced upon irradiation. NOTCH inhibition induces accumulation of the cells in GO-G1 phase and a significant reduction in S phase which was exacerbated upon irradiation. The reduction in proliferation also led to a reduction in long term survival. We observed activation of the DNA damage response pathway by expression of pCHK2, pATM upon NOTCH inhibition which was enhanced by radiation. 53 BP1 foci 24 hours after RT was higher when NOTCH was inhibited demonstrating that NOTCH is required for the DNA damage response. In cultured primary stem cells from irradiated murine tracheas NOTCH inhibition induced a fate change by blocking mucous differentiation and inducing cillilary differentiation and enhancing progenitor p63 +cells. However, overall proliferation was reduced.ConclusionNOTCH pathway is needed for stem cell survival and DNA damage response upon irradiation of undifferentiated progenitors. These studies underscore the importance of studying the effect of targeted anti-cancer drugs for normal tissue effects.