[The Application of Human Lung Organoids for Assessing the Cyclophosphamide-Induced Pulmonary Toxicity and Potential Mechanisms].

OBJECTIVE

To investigate Cyclophosphamide (CTX)-induced pulmonary toxicity using a human lung organoid model and explore its effects on DNA damage, inflammatory response, and early fibrosis.

METHODS

Human lung organoids derived from induced pluripotent stem cells (iPSCs) were cultured in vitro, and the morphological features and gene/protein expression profiles at distinct induction stages were systematically identified. Mature organoids were treated with CTX at concentration gradients (0, 1, 5, 10, and 20 mmol/L),and morphological changes were observed at 24, 48, and 72 hours. The expression level of DNA damage marker gene CDKN1A was detected by qRT-PCR to identify the CTX treatment conditions exhibiting significant toxicity. Apoptosis rates were further quantified using flow cytometry. In addition, qRT-PCR and immunofluorescence staining were used to comprehensively evaluate lung epithelial function, inflammatory factor expression, and alterations in fibrosis-related markers.

RESULTS

CTX treatment induced concentration- and time-dependent morphological damage in lung organoids, manifested as branch structure retraction, cell detachment, and three-dimensional structural disruption. The most pronounced effects were observed following treatment with 10 mmol/L CTX for 48 hours. At this dosage and duration, CTX significantly upregulated the DNA damage marker CDKN1A and enhanced apoptosis, while concurrently downregulating alveolar epithelial marker SFTPB and airway epithelial marker MUC5AC. Additionally, the gene expression levels of inflammatory factors IL1B and TNFA were significantly increased. Immunofluorescence analysis further demonstrated accumulation of the mesenchymal marker PDGFRB at organoid peripheries, suggesting initiation of early fibrotic processes.

CONCLUSION

CTX mediates pulmonary toxicity through the induction of DNA damage, inflammatory activation, and fibrotic pathway signaling. The human lung organoid platform established herein represents a physiologically relevant in vitro model suitable for drug safety assessment and toxicological evaluation.