A scalable Zebrafish Platform for genetic validation and functional discovery of stage-specific breast cancer metastasis regulators

Metastatic disease accounts for over 90% of breast cancer-related deaths, yet scalable in vivo models for functional analysis of distinct metastatic steps remain limited. Here we show that an optimized zebrafish xenotransplantation platform enables quantitative, stage-resolved analysis of primary tumor growth, intravasation, extravasation and metastatic outgrowth at single-cell resolution. Subcutaneous and intravenous transplantation of GFP-labeled breast cancer cells into zebrafish larvae allows systematic functional interrogation of candidate metastasis regulators in vivo. Using this platform, we evaluated eight candidate genes implicated in breast cancer metastasis. ANGPTL4 selectively promoted extravasation, IFI6 and KCNQ1OT1 enhanced early dissemination, whereas LY6E and ITGB4 promoted multiple metastatic steps. GAS5, LRRC75A-AS1 and FST displayed distinct context-dependent effects on tumor growth or metastatic progression. These findings establish zebrafish xenografts as a scalable platform that complements mouse models for quantitative functional analysis of breast cancer metastasis. An optimized zebrafish xenograft platform enables scalable, stage-specific analysis of breast cancer metastasis in vivo. Screening candidate genes identified distinct regulators of dissemination, extravasation, and metastatic growth, providing a powerful tool for studying metastatic mechanisms.