A chronic whole cigarette smoke extract model reveals redox-mitochondrial adaptation in human lung epithelial and organoid models.

Cigarette smoke (CS) imposes continuous oxidative and electrophilic stress that disrupts cellular homeostasis in the lung. While acute smoking exposure induces transient antioxidant responses, how the epithelial system adapts to chronic smoke remains poorly defined. Here we developed a chronic whole CS extract (WCSE) model that integrates both gaseous and particulate fractions to reproduce the complexity of long-term smoking exposure. Using bronchial epithelial cells and human lung organoids, we demonstrate that chronic WCSE exposure induces a coordinated redox-mitochondrial adaptation, supporting survival under persistent oxidative stress. Chronically exposed cells (T-B2B) exhibited reduced apoptosis, enhanced S-phase entry and fragmented but functionally preserved mitochondria characterized by a stable membrane potential and restrained reactive oxygen species accumulation. Whole-exome sequencing revealed oxidative mutational signatures in two-dimensional and organoid models, linking chronic oxidative adaptation with tobacco-associated genomic imprints. Mechanistically, NRF2 activity was sustained through post-translational stabilization and nuclear accumulation independent of KEAP1, accompanied by activated pAKT and suppressed pGSK3β activity. Human lung organoids recapitulated these adaptations, showing enlarged morphology, reduced apoptosis and nuclear NRF2 accumulation, consistent with a stress-tolerant, clonally persistent phenotype. Together, these findings establish a chronic WCSE platform that models early epithelial adaptation to CS and uncovers NRF2-dependent redox remodeling as a key mechanism of long-term survival in smoking-related pulmonary injury.