Deciphering Precursor Cell Dynamics in Esophageal Preneoplasia via Genetic Barcoding and Single-Cell Transcriptomics

Cancer cell lineage plasticity complicates tumor initiation and progression. Using genetic barcoding and single-cell RNA sequencing (scRNA-seq), we mapped esophageal preneoplastic cell lineages and developed the eXamined Ridge (XR) score to identify high-plasticity cells. We discovered preneoplastic precursor cells (PNPCs) as key progenitors and identified Nfib and Qk as conserved markers, highlighting potential biomarkers for early cancer detection and intervention. Overall design: For scRNA-seq analysis, esophageal organoids were derived from Trp53^floxed/floxed mice, with Ad-Cre-EGFP introducing Trp53 knockout, followed by CRISPR/Cas9-mediated Cdkn2a or Notch1 knockout to generate PC and PCN organoids. Organoid cells were dissociated, cultured in DMEM + 10% FBS with Y-27632, and maintained without Y-27632 after three passages before single-cell analysis.