Regulatory effects of smoking cessation on the cellular microenvironment and differentially expressed genes in precancerous lesions of pulmonary nodules in mice based on single-cell RNA sequencing and immune repertoire-sequencing.

BACKGROUND: Smoking cessation decreases lung cancer progression; however, its effects on precancerous lesions and the underlying mechanisms remain unclear. This study established a mouse model of precancerous pulmonary nodules and employed single-cell RNA sequencing (scRNA-seq) and immune repertoire sequencing (IR-seq) to elucidate the regulatory mechanisms by which smoking cessation influences the development of lung precancerous lesions. METHODS: A B[a]P-induced mouse model of pulmonary precancerous lesions was established after 6 weeks of B[a]P treatment. Lung nodules were identified via micro-MRI, and histopathological features were assessed by H&E staining. After confirming the establishment of precancerous lesions in smoking-exposed mice, graded B[a]P withdrawal for 4 and 8 weeks was implemented. Nodule size was measured using Generic Medical Imaging software. scRNA-seq and IR-seq analyzed immune cell composition, proportions, signaling, and gene expression. RESULTS: B[a]P withdrawal reduced new lung nodule formation and decreased the diameter, area, and volume of existing nodules. It alleviated cellular atypia, reduced inflammation, and altered the tumor microenvironment by increasing T-, B-, and NK cells while reducing monocytes and neutrophils. Concurrently, B[a]P withdrawal downregulated Vim, GPX1, Atf3, CD44, and Arpc5 and upregulated Hsph1, Hsp90aa, and Hspa1b. B[a]P withdrawal upregulated CD80 and ICOS, suppressed intercellular communication via Icam1-(Itgal+Itgb2), Ppia-Bsg, and Thbs1-Cd47 pathways. The APP-CD74 pathway emerged as a novel regulator in precancerous lesions. IR-seq revealed restored T- and B-cell receptor diversity, CDR3 sequence alterations, and V(D)J gene regulation. CONCLUSIONS: B[a]P withdrawal reduces lung nodules, inhibits progression, potentially by enhancing immune surveillance by modulating immune cells, signaling pathways, and gene expression, while promoting ferroptosis, suppressing inflammation, and preventing tumor stem cell-like traits. These findings may provide mechanistic insights into the beneficial effects associated with smoking cessation.