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bioRxiv · 10.1101/2025.08.13.669778

Temporal Clonal Tracing and Functional Perturbation Reveal Niche-Adaptive and Tumor-Intrinsic IFNγ Dependencies Driving Ovarian Cancer Metastasis

Aleksandrovic, E.·Fross, S. R.·Golomb, S. M.·Liu, X.·Zhao, Z.·Das, N. M.·Reese, T. C.·Ma, W.·Lopez, J.·Stack, M. S.·Zhao, M.·Zhang, S.
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Abstract

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 (IFN{gamma})-centric transcriptional trajectory selectively enriched among pioneer clones. In vivo CRISPR/Cas9 screening of niche-specific signatures demonstrated that the tumor-intrinsic IFN{gamma} response is functionally required for peritoneal metastasis. Knockout of the IFN{gamma} receptor 1 (Ifngr1) in the initial pioneer DTCs significantly reduced total metastatic burden, revealing a critical window of time in which IFN{gamma} signaling shapes the post-seeding metastatic niche (PSMN) and subsequent metastatic evolution. Mechanistically, the tumor-intrinsic IFN{gamma} 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.

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Aleksandrovic, E., Fross, S. R., Golomb, S. M., Liu, X., Zhao, Z., Das, N. M., Reese, T. C., Ma, W., Lopez, J., Stack, M. S., Zhao, M., Zhang, S.. 2025-08-17. Temporal Clonal Tracing and Functional Perturbation Reveal Niche-Adaptive and Tumor-Intrinsic IFNγ Dependencies Driving Ovarian Cancer Metastasis. https://doi.org/10.1101/2025.08.13.669778

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