持续性高级别癌前病变中p21+细胞的积聚及生长停滞特征的富集

Introduction: Lung squamous cell carcinoma is preceded by premalignant lesions (PML) or dysplasia that may persist, progress, or regress. Individuals with persistent PMLs have a significantly higher risk of incident cancer compared to those whose lesions regress. Mechanisms that control dysplasia fate, therefore, could be important for early detection and prevention. Epithelial basal progenitors are critical for maintaining a healthy airway, but their role in determining PML fate has not been studied before. Methods: Basal progenitors grown from endobronchial biopsies collected from high-risk individuals were assayed for their cardinal features of self-renewal and multipotent differentiation. A combination of cellular, functional, and molecular analyses, including mutations and single-cell (sc) RNAseq, was used to investigate progenitor function and their roles in determining PML fate. Results: Progenitor function decreased with worsening dysplasia grade, and poor progenitor self-renewal predicted dysplasia persistence and progression to cancer. Mutational analysis of high- and low-self-renewing progenitors did not detect any meaningful difference in overall mutational burden, including oncogenic mutations. Interestingly, there were increased numbers of cells expressing cyclin-dependent kinase inhibitor (CDKN1A) or p21 in the progenitors with low self-renewal compared to those with high self-renewal. Single-cell (sc) RNA-seq of biopsies revealed enrichment of p21-expressing basal cells in lesions with low progenitor self-renewal. VECTRA multiplexed immunofluorescence staining of PMLs confirmed significantly greater numbers of p21+ cells in persistent lesions, and the abundance of p21+ cells increased over time. Integration of the multiplexed immunostaining images with the VISIUM spatial transcriptomic profiles generated from serial sections of the same biopsies allowed for direct alignment of p21 protein expression and the corresponding transcriptomic features. A spot-level differential analysis between six p21 high and p21 low regions from four PMLs revealed the enrichment of specific pathways (primary cilium, cell-cycle arrest, DNA damage responses, metabolic rewiring, and immune engagement) in p21 high spots. Conclusions: p21 is a key determinant of cell cycle progression, and increased expression of this molecule indicates growth arrest by induction of a transient G0 state. The discovery of p21+ cells in PMLs containing low self-renewing progenitors and spatial analyses confirming the presence of a cell-cycle arrest signature in the p21 high areas in multiple high-grade PMLs indicated a role of these pathways in determining PML fate. Future studies will focus on analyzing these signatures in independent PMLs with a known natural history of persistence, progression, and regression.