Real-time Evolutionary Landscape of the Bronchial Epithelium and Corresponding Dynamic Immune Cell Alterations in Lung Squamous Cell Carcinogenesis

The molecular mechanism by which tumor cells and their microenvironment evolve during lung squamous cell carcinoma (LUSC) carcinogenesis remains unclear, greatly limiting its early diagnosis and treatment effectiveness in patients. To replicate pathogenic processes and identify the determinants of cell evolution, a rat model is established using tobacco-derived carcinogens. Here, a series of single-cell transcriptome profiles of normal lung epithelium, hyperplasia, metaplasia, dysplasia, and squamous cell carcinoma in situ (CIS) to invasive squamous cell carcinoma (SCC) is presented. A large proportion of canonical copy number variations (CNVs) are detected in the hyperplasia/metaplasia stages, with their frequency increasing as the lesion progressed. Although bronchial epithelial cells exhibit substantial heterogeneity, three distinct cellular states are identified during their evolution into malignant cells. Immune sensing occurs at the earliest stages of cellular transformation. However, persistent exposure to carcinogens induces microenvironmental remodeling, which is characterized by monocyte-derived macrophage infiltration, plasmacytoid dendritic cell expansion, and progressive T-cell exhaustion. These findings depict the evolutionary trajectory of cancer and the immune microenvironment, emphasizing the need for CNV evaluation in early screening and immune-based therapy for lesions at a high risk of progression to LUSC. Female Wistar rats (8 weeks old, 200 ± 20 g) were housed in a pathogen-free environment at the animal center of the First Affiliated Hospital, Sun Yat-sen University. The rats were fasted overnight prior to instillation and were deprived of water on the day of the experiment. For an increased tumorigenic effect, iodized oil was used to dissolve the chemical carcinogens 3-MCA (100 mg/ml, Sigma) and DEN (95 mg/ml, Sigma) at 65–70 °C overnight. Basal anesthesia was induced through the intraperitoneal injection of 150 μl of 3% sodium pentobarbital solution (Sigma), followed by inhalation administration of isoflurane (RWD) to achieve deep anesthesia. Subsequently, the rats were randomly assigned to six groups (n = 8). In each group, six rats were instilled with 100 μl of carcinogen-containing iodized oil into the left lower lobe through a special blunt needle, while the remaining two rats were given 100 μl of iodized oil without carcinogens as a control. Whether the instillation was successful was confirmed via micro-CT (Bruker). To prevent infections, drinking water containing 1 g/l ampicillin (Sangon Biotech) and 1.5 g/l streptomycin (Sangon Biotech) was administered from day 2 to day 15 post-instillation. The rats were euthanized at various time point (on days 0, 7, 14, 21, 28 and 35) following instillation. lung tissues from the left lower lobe were excised, and a portion of the tissue was processed for hematoxylin and eosin (H&E) staining. The remaining tissue was used for the next single-cell suspension. We randomly selected two samples in each group for scRNA sequencing. However, due to technical issues during library preparation, two samples assigned to hyperplasia and metaplasia, respectively, were excluded from further analysis. Therefore, a total of ten samples underwent scRNA sequencing.