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

www.nature.com/emm ARTICLE OPEN A chronic whole cigarette smoke extract model reveals redox–mitochondrial adaptation in human lung epithelial and organoid models Joo-Eun Lee1,2,8, Dahye Lee1,8, Jihyun Lee1, Sung-Joon Han3, Sung Hyun Kang4, Ryeo-Eun Go4, Jihyun Kwon4, Younjhin Ahn4, ✉ Mi Jung Lim5, Mahn Jae Lee1,2, Hee Min Yoo 6,7, Da Hyun Kang 1, Jeong Eun Lee1, Dongil Park1,9 and Chaeuk Chung1,9 1234567890();,: © The Author(s) 2026 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. Experimental & Molecular Medicine; https://doi.org/10.1038/s12276-026-01743-x INTRODUCTION Cigarette smoke (CS) is a well-established carcinogenic agent that affects not only respiratory diseases but also multiple organ system disorders1. It contains more than 4,000 identified chemical ingredients, including nicotine, ammonia, nitrogen oxides, hydrogen cyanide and trace metals. These compounds act synergistically to induce oxidative stress, DNA damage, chronic inflammation and cellular senescence, leading to malignant transformation2–4. Recent studies have demonstrated that longterm exposure to CS can promote abnormal cell proliferation, disrupt DNA repair systems and alter the tumor microenvironment, leading to enhanced cancer progression4–6. Moreover, CS has been reported to promote colorectal cancer progression by modulating gut microbial metabolites7. In lung cancer, CS has been shown to promote cell cycle progression and contribute to the aggressiveness of smoking-related non-small cell lung cancer8. Among the components of CS, reactive oxygen species (ROS) play a crucial role in its pathogenic effects. Prolonged CS exposure leads to sustained ROS accumulation, overwhelming endogenous antioxidant systems and inducing chronic oxidative stress. Mitochondria, as the primary site of cellular respiration, are both a major source and a primary target of this ROS-induced damage. Consequently, alterations in mitochondrial dynamics and membrane potential are increasingly recognized as a key feature of cancer, contributing to metabolic reprogramming, resistance to apoptosis and enhanced cell survival9. Smoking also leaves a genomic footprint. Large-scale sequencing has connected tobacco exposure with a single-base substitution (SBS) signature indicative of oxidative injury and DNA-repair imbalance10. To counteract oxidative damage, cells activate the transcription factor NRF2 (nuclear factor erythroid related factor 2), which regulates antioxidant and detoxifying genes, such as NQO1 and HO-11. Under basal conditions, NRF2 is rapidly degraded; however, when subject to oxidative stress, the protein is stabilized and promoted to accumulate in the nucleus, maintaining antioxidant defense under chronic stress11. 1 Division of Pulmonology and Critical Care Medicine, Department of Internal Medicine, College of Medicine, Chungnam National University, Daejeon, Republic of Korea. 2Biomedical Convergence Research Institute, Chungnam National University Hospital, Daejeon, Republic of Korea. 3Division of Thoracic and Cardiovascular Surgery, College of Medicine, Chungnam National University, Daejeon, Republic of Korea. 4Division of Climate Change and Health Hazard, Department of Health Hazard Response, Korea Disease Control and Prevention Agency, Cheongju, Republic of Korea. 5Genomics Department, Keyomics Co. Ltd., Yuseong-gu, Republic of Korea. 6Biometrology Group, Korea Research Institute of Standards and Science (KRISS), Daejeon, Republic of Korea. 7Department of Precision Measurement, University of Science and Technology (UST), Daejeon, Republic of Korea. 8These authors contributed equally: Joo-Eun Lee, Dahye Lee. 9These authors jointly supervised this work: Dongil Park, and Chaeuk Chung. ✉email: Received: 5 December 2025 Revised: 10 March 2026 Accepted: 18 March 2026 J.-E. Lee et al. 2 While transient activation of NRF2 during acute oxidative stress is well established, how NRF2 activity is maintained under chronic cigarette exposure remains poorly understood. Previous experimental models investigating CS-induced cellular responses have primarily relied on either particulate-phase cigarette condensate or gas-phase CS extract, typically applied in short-term exposure settings. However, these models capture only a limited fraction of the whole CS and therefore fail to represent the integrated chemical complexity and sustained oxidative burden associated with chronic smoking12,13. Furthermore, most studies have been conducted in two-dimensional culture systems or animal models, which cannot fully recapitulate the structural and cellular complexity of the human airway epithelium. As a result, these approaches predominantly model acute cytotoxic or inflammatory responses rather than the long-term adaptive remodeling induced by chronic smoke exposure. Although three-dimensional lung organoid systems have recently emerged as promising platforms for studying human airway biology14, their application to sustained CS exposure remains limited. To overcome these limitations, we developed a whole CS extract (WCSE) model that combines both particulate and gaseous components to more accurately represent the physiological complexity of a chronic exposure platform. Furthermore, we established a human lung organoid-based chronic exposure platform, enabling the investigation of cigarette-induced adaptati (...truncated)