Testing the plasticity-first theory of tumor evolution with spatiotemporal lineage tracing

The plasticity-first hypothesis posits that phenotypic plasticity emerges first in novel environments, followed by (epi)genetic changes that stabilize adaptive phenotypes. Whether tumor evolution follows this model, and how non-genetic mechanisms sustain long-term therapeutic resistance, remain unclear. Here, we develop eTRACER, an endogenously targeted, high-resolution and spatially-resolved CRISPR-Cas9 lineage-tracing system, to study tumor adaptation during longitudinal CD8+ T cell-mediated immunotherapy. Integrated clonal and single-cell transcriptomic analyses show that tumor immune evasion is governed by clonal state composition rather than clonal identity, arguing against selection from pre-existing genetic heterogeneity. Spatiotemporal lineage tracing reveals highly reversible state transitions prior to therapy, followed by consolidated epithelial-mesenchymal transition (EMT) during therapy. Single-cell multiomics identifies AP-1 as a key regulator of EMT stabilization and its inhibition enhances tumor sensitivity to immune killing. Together, these findings show that tumor plasticity precedes stable immune evasion, revealing an AP-1-centered mechanism underlying plasticity-first tumor evolution.