226 - Neutrophil Extracellular Traps Formation Mediated by Senescent Fibroblast Drives Radiation-Induced Lung Fibrosis
Presenter(s)
Y. Dong1, J. Yuan2, Z. Zhang1, Y. Zhou3, B. Tian2, J. Yu4, and D. Chen5; 1Shandong Cancer Hospital and Institute, Jinan, Shandong, China, 2Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, Shandong, China, 3Tianjin Medical University Cancer Institute & Hospital, National Clinical Research Center for Cancer,Tianjin’s Clinical Research Center for Cancer,Key Laboratory of Cancer Prevention and Therapy, Tianjin, Tianjin, China, 4Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences Department of Radiation Oncology, Jinan, Shandong, China, 5Department of Radiation Oncology and Shandong Provincial Key Laboratory of Precision Oncology, Shandong Cancer Hospital and Institute, Shandong First Medical University and Shandong Academy of Medical Sciences, Jinan, China
Purpose/Objective(s): Radiation-induced pulmonary fibrosis (RIPF) is a severe late-stage complication in lung cancer patients undergoing radiotherapy, characterized by progressive and irreversible lung tissue fibrosis. RIPF not only impairs the quality of patients’ life but also poses a serious threat to their survival. However, there are few of effective therapeutic interventions because the pathogenesis and mechanisms remain largely unknown. Several studies have reported the role of neutrophil extracellular traps (NETs) in chronic fibrosis. Meanwhile, our previous work published in Int J Radiat Oncol Biol Phys revealed that the accumulation of senescent fibroblasts may drives RIPF. Therefore, this study aims to elucidate the pivotal roles of interaction between senescent fibroblasts and NETs in the development and progression of RIPF.
Materials/Methods: A RIPF murine model was established using C57BL/6 mice through precision thoracic irradiation with a single fraction of 20 Gy delivered by the Small Animal Radiation Research Platform. Disease progression was longitudinally monitored at 2, 4, and 6 months post-radiation using the Animal micro-CT Imaging System. Lung tissue specimens were obtained for comprehensive analysis, including Single-Cell RNA-seq, flow cytometry, and histological staining. NETs formation was validated through ELISA, multiplex immunofluorescence (mIF) and IF.
Results: Our findings reveal that neutrophil infiltration and NETs formation increase in lung tissue after radiation, clearance of neutrophil and NETs alleviates RIPF significantly. Further mechanistic exploration indicates that NETs activate the ILK/PI3K/AKT pathway by interacting with the CCDC25 receptor on fibroblasts. This activation of CCDC25 triggers a persistent epithelial-mesenchymal transition (EMT) in fibroblasts, leading to excessive collagen deposition, ultimately resulting in RIPF. Furthermore, we investigated the mechanisms of neutrophil recruitment and NETs formation. Through Single-Cell RNA-seq and mIF of irradiated lung tissue, we identified that senescent fibroblasts have the most interaction signal with neutrophils and the minimum spatial distance with NETs. Additionally, through cytokine arrays of human and mice serum , we found that senescent fibroblasts upregulated neutrophil migration pathway and focused on chemokine CXCL1 . We validated CXCL1 the key to recruit neutrophils and format NETs. Meanwhile, clearance of senescent fibroblasts, p21 inhibition and anti-CXCL1 alleviates RIPF by decreasing neutrophil infiltration and NETs formation in the lung.
Conclusion: Our study reveals that senescent fibroblasts exacerbate RIPF by secreting CXCL1 to recruit neutrophils and drive NETs formation. NETs promote EMT and excessive collagen deposition of fibroblasts by binding to CCDC25. Targeting fibroblast senescence represents a promising therapeutic strategy to disrupt the senescence-inflammation axis and attenuate RIPF progression.