Single-cell lineage tracing in tumor organoids identifies expansion states depicting the origin and progression of lung adenocarcinoma.

INTRODUCTION: Only a subset of epithelial cells can initiate progressive lung adenocarcinoma (LUAD), yet the founder cell states that confer this competence remain poorly defined. METHODS: We established a barcoded Kras G12D, Trp53 loss mouse LUAD organoid model that couples 15-nucleotide lineage tracing with single-cell RNA sequencing (scRNA-seq) before and after syngeneic transplantation. Candidate markers for expansion states were further evaluated using wild-type lung organoid, spatial transcriptomics, and independent human scRNA-seq datasets. RESULTS: Barcoded organoids generated LUAD-like tumors. Paired barcode recovery identified expanded, diminished and failed lineages and showed that expanded lineages were enriched for surfaceome genes associated with stemness. Among candidate surface markers, Plxna2 showed the strongest enrichment in baseline organoid cells that expanded after transplantation and marked cells with stronger stemness features. These cells underwent stage-specific reprogramming, with early EMT and inflammatory adaptation followed by late metabolic and MYC programs rebound within an immunosuppressive microenvironment. Wild-type lung organoid analysis showed that Plxna2 marks an alveolar remodeling state sharing expansion signature. Spatial transcriptomics placed Plxna2 high tumor epithelial cells in EMT-rich and stromal-interface niches. Human LUAD scRNA-seq cohorts further showed enrichment of PLXNA2 high malignant cells within expansion and stemness states. DISCUSSION: These findings identify PLXNA2 as a candidate marker of expansion state and provide a lineage-based framework for discovering tumor-initiating programs in LUAD.