Spatiotemporal lineage mapping of tumor immune escape with eTRACER

Deciphering spatiotemporal cell lineage dynamics remains a fundamental yet unresolved challenge. Here we introduce eTRACER, a novel CRISPR-Cas9 lineage tracer that targets neutral 3’UTR of high-expression endogenous genes, enabling efficient recovery of static and evolving barcodes from single-cell and spatial transcriptomics. By optimizing gradient editing efficacy and avoiding large disruptive deletions, eTRACER reconstructs high-fidelity and high-resolution single-cell phylogenies. Applied to EGFR-mutant lung adenocarcinoma (LUAD) under CD8 ⁺ T cell cytotoxicity, eTRACER reveals directional state transitions from Hypoxic and Proliferative states to Epithelial-Mesenchymal Transition state during immune evasion. Spatially-resolved lineage mapping unveils layered stratification of distinct tumor states and location-primed cell migration and state transitions. Lineage-coupled single-cell multiomic analysis uncovers cooperative mechanism between tumor cell-intrinsic AP-1 transcriptional program and spatially restricted macrophage-tumor cell interaction leading to immune evasion. Collectively, we develop a powerful spatiotemporal lineage tracer and uncover microenvironment-primed cellular evolution underlying immune evasion of EGFR-mutant LUAD, with important implication for efficient immunotherapy.