Age-related transcriptional and alternative splicing changes in lung progenitor cells predisposing to immune dysfunction

Purpose: Aging is the greatest risk factor for lung cancer, the leading cause of cancer-related deaths worldwide. With advancing age, impaired immune surveillance and cellular senescence create a pro-tumorigenic microenvironment that disrupts epithelial homeostasis and promotes oncogenic remodeling. However, the molecular mechanisms by which age-related transcriptional and splicing changes in lung contribute to immune dysregulation and cancer susceptibility remain poorly understood. We performed integrated transcriptomic profiling of lung progenitor cells to identify age-associated molecular alterations predisposing to immune dysfunction and early tumorigenic changes. Methods: Primary human broncho-epithelial cells from young (age: 18y to 27y, n=5) and old (age: 42y to 67y, n=5) donors were enriched for progenitor cells in 2D culture for seven days following Fulcher et al. We performed Illumina short-read and PacBio long-read RNA-sequencing. Isoform-level analysis on long-reads was performed through IsoSeq3 and SQANTI3. Differential expression was analyzed using DESeq2 (FDR < 0.05) and alternative splicing using rMATS (>10% Delta PSI, FDR < 0.05) with a long-read derived transcriptome. Functional consequences of splicing events were predicted using SpliceDecoder. Results: We identified 47 differentially expressed genes (36 upregulated, 11 downregulated with age). Notably, IL18, MMP25, PGLYRP4, THY1, CDH2, MFAP5, and PLXNC1 were upregulated in older donors, reflecting activation of inflammatory and immune response programs alongside altered epithelial and extracellular matrix remodeling. We also detected 991 age-related differentially spliced events in 632 genes. Among these, intron retention occurred in immune genes including HLA-A, HLA-B, HLA-C, TRIM65, and FOSB, while splicing of cassette exons was observed in IFNAR2-IL10RB, DMKN, HLA-F-AS1, and NOD1. SpliceDecoder predicted that these age-related splicing alterations introduce premature stop codons, alter coding sequences, and modify protein domains, potentially leading to gain- or loss-of-function effects. Finally, long-read RNA-sequencing identified 42,206 full-length spliced isoforms, the majority of which were novel and absent from reference transcriptomes, revealing extensive isoform diversity in lung progenitor cells. Conclusions: Our integrated approach demonstrates that aging reshapes the transcriptomic landscape through changes in gene expression and splicing, that may contribute to immune dysfunction and epithelial remodeling. These changes may create a pro-inflammatory environment that enhances susceptibility to oncogenic transformation and disrupts tissue homeostasis. Together, these findings uncover molecular mechanisms linking aging to lung cancer risk and identify potential biomarkers and therapeutic targets for prevention and intervention.