Temporal Clonal Tracing Reveals Tumor-Intrinsic IFNγ-Dependencies Driving Niche Adaptation and Early Metastatic Colonization

SUMMARY Metastasis is an emergent continuum driven by evolving reciprocal adaptations between disseminating tumor cells (DTCs) and specialized niches of different organs. The interplay between intrinsic and niche-driven mechanisms that enable DTCs to survive and home to distant organs in peritoneal ovarian cancer metastasis remains incompletely understood. Here, we present MetTag, a single-cell barcoding and transcriptome profiling approach with single cell clonality barcodes and time-stamped batch identifiers (BC.IDs) to resolve metastasis clonality and temporal dynamics of DTC colonization. Deep sequencing of MetTag barcodes revealed enrichment of early-disseminated clones across metastatic sites, and targeted depletion of pioneer DTCs diminished the outgrowth of subsequent arriving DTCs. Subsequent MetTag-coupled single cell RNA sequencing (scRNA-seq) on ascites and metastasis-bearing omenta revealed a distinct interferon-gamma (IFNγ)-centric transcriptional trajectory selectively enriched among pioneer clones. In vivo CRISPR/Cas9 screening of niche-specific signatures demonstrated that the tumor-intrinsic IFNγ response is functionally required for peritoneal metastasis. Knockout of the IFNγ receptor 1 ( Ifngr1 ) in the initial pioneer DTCs significantly reduced total metastatic burden, revealing a critical window of time in which IFNγ signaling shapes the post-seeding metastatic niche (PSMN) and subsequent metastatic evolution. Mechanistically, the tumor-intrinsic IFNγ response induced Poly(ADP-Ribose) polymerase family member 14 (Parp14) and peritoneal macrophages cooperatively shield DTCs from anoikis by promoting pro-survival signaling. Our study defines the temporal clonal architecture of peritoneal niches and reveals a “first come, first served” adaptation principle, where pioneer colonizer fitness determines the success of subsequent colonizers.