The lower the better? Impact of radiation dose reduction on radiation-induced lung fibrosis molecular signature: approach towards preclinical model perfection

Radiation-induced pulmonary fibrosis (RIPF) is the most common bottleneck of radiation therapy (RT) that limits its tumor-killing effect. Up to date, there is no effective therapeutic agent for RIPF treatment due to absence of realistic preclinical models. Lungs of mice exposed to focal, highly localized dose of 75Gy-100Gy represents short time-taking (up to 6 weeks) modality for mimicking recent advanced RT, stereotactic body RT-associated lung fibrosis. However, extensive fibrosis and possibility of animal lethality resulted from exposure to high dose raises a question regarding the appropriate dose threshold for limiting unnecessary responses and obtain clinically relevant results. We observed in preliminary trials that lung tissues of 65Gy-exposed mice are still able to develop inflammation and fibrosis at the same time cap. But the molecular changes associated with dose reduction is still unknown. To examine this, bulk RNA from 65Gy and 75Gy irradiated as well as normal lung tissues of mice were obtained, and analysed using computational approach to uncover the transcriptomal similarities and differences between the differentially irradiated samples. Differentially expressed genes (DEGs) were analysed and 2-week and 6-week intersected DEGs as well as 65 and 75Gy exclusive DEGs at 2-week and 6-week time points were detected using venn diagramme. Expression direction of intersected DEGs between 65Gy and 75Gy was checked and found to be consistent at both inflammation and fibrosis stages proven visually by MDS and heatmap graphs. All-times intersected DEGs are 488 and involved in inflammation, cytokine production, and cell proliferation; 2-week intersected DEGs are 221 and involved in cell cycle and DNA damage; 6-week intersected DEGs are 403 and involved in immunoglobin production, extracellular matrix (ECM) deposition, and ECM-receptor interactions. Interestingly, only 75Gy exclusive DEGs of 2 and 6 weeks presented inflammation and fibrosis-related biological process, respectively. These include anti-inflammatory process, cytokine production, EMT, vasculogenesis, and angiogenesis. These results suggested that 65Gy maybe a better dose for delivering less exaggerated, clinically relevant inflammation and fibrotic events, and thus, it can be utilised in establishing appropriate preclinical models for studying RIPF pathology and therapy.