A 3D morphogenetic blueprint for metastatic outgrowth in breast cancer

The tissue-level processes underpinning metastatic outgrowth remain unclear. We combined single-cell RNA sequencing, spatial transcriptomics, and AI-supported 3D imaging in human breast cancer with functional investigations in mice to uncover a 3D morphogenetic process essential for macrometastatic expansion. Macrometastases pervasively activate a metastatic trabecular morphogenesis (MTM) gene-expression program that redeploys developmental branching morphogenesis to build macrometastases as a 3D trabecular lattice of epithelial cords. MTM^HIGH cells pre-exist in primary tumors destined to metastasize, whereas MTM^LOW primaries are non-metastatic and display a compact, expansile growth architecture. Chromatin immunoprecipitation sequencing (ChIP-seq) on metastatic organoids identifies ETV1/4/5 as master regulators of MTM and branching cancer morphogenesis, required for metastatic outgrowth but dispensable for primary tumor take, bulk growth, and initial metastatic dissemination. Spatial and functional analyses reveal stromal fibroblast growth factor (FGF)→fibroblast growth factor receptor (FGFR) signaling as an actionable MTM dependency. Thus, we link metastatic outgrowth to a 3D developmental morphogenetic process, exposing therapeutic vulnerabilities specific to the lethal macrometastatic stage.