Single-cell and spatial transcriptomics define a C3-centred multicellular ecology in colorectal liver metastasis

Abstract Colorectal liver metastasis (CRLM) is shaped by malignant-cell plasticity and microenvironmental remodeling, but how malignant, stromal, vascular and myeloid states assemble spatially remains unclear. We sought to define recurrent multicellular programs associated with CRLM. We integrated a 136,995-cell single-cell atlas with copy-number inference, consensus non-negative matrix factorization, regulatory-network and patient-aligned trajectory analyses, intercellular-communication modeling, four Visium sections, bulk-cohort survival analyses, virtual knockout, connectivity-map screening and molecular docking. Three malignant epithelial programs were resolved along a C1-to-C2-to-C3 continuum. The rare C3 state displayed secretory, invasive and epithelial-to-mesenchymal transition features and was associated with shorter recurrence-free survival. Fib- COL10A1 fibroblasts and Endo- GJA5 endothelial cells formed a recurrent stromal–vascular scaffold, whereas CTSL -program macrophages occupied focal interfaces whose geometry varied among metastases. Spatial pathway and multiview analyses implicated MAPK- and VEGF-associated activity. An INHBA – CST6 – LBH – FSTL3 score was associated with recurrence in TCGA and showed partial external validation. Virtual knockout indicated convergent CST6 / LBH network effects, and connectivity-map analysis nominated PD-0325901 as a candidate for reversing the associated state; docking provided exploratory structural support for CST6 but not LBH . These analyses define a spatially flexible C3-centred ecology and a MAPK-linked network vulnerability in CRLM. The framework is computational and requires experimental validation.