Integrated single-cell and spatial transcriptomics uncover SPP1⁺ and HLA-DRB5⁺ macrophages as key modulators of the immune microenvironment in colorectal cancer liver metastasis

Liver metastasis is the leading cause of mortality in colorectal cancer (CRC), yet the phenotypic diversity and spatial organization of myeloid cells in this setting remain unclear. Publicly available single-cell RNA sequencing, spatial transcriptomic, and bulk transcriptomic datasets from CRC and liver metastasis were integrated. Single-cell analyses were performed to characterize myeloid cell heterogeneity, differentiation trajectories, and inferred intercellular communication, while spatial transcriptomic analyses were conducted to assess cell-type enrichment and inferred spatial relationships. Differential expression and survival analyses were combined to construct an HLA-DRB5⁺ macrophage-associated prognostic signature, which was validated across multiple independent cohorts. Eleven myeloid cell subsets were identified, among which SPP1⁺ and HLA-DRB5⁺ macrophages were markedly enriched in liver metastases and occupied distinct positions along myeloid differentiation trajectories. Cell–cell communication analysis revealed distinct predicted signaling patterns, with SPP1⁺ macrophages interacting with B cells and regulatory T cells through MIF–(CD74 + CXCR4) signaling, and HLA-DRB5⁺ macrophages communicating with T and NK cells via LGALS9–CD45 signaling. Spatial transcriptomic analyses demonstrated distinct localization patterns of these macrophage subsets across tumor, stromal, and hepatocyte regions, and MISTy-based analysis identified dynamic spatial associations with immune and stromal populations. In addition, a six-gene prognostic signature derived from HLA-DRB5⁺ macrophage-associated genes exhibited robust and independent predictive performance for overall survival across multiple CRC cohorts. This integrative multi-omics analysis reveals distinct transcriptional and spatial features of SPP1⁺ and HLA-DRB5⁺ macrophages in CRC liver metastasis and highlights their potential roles in shaping the metastatic immune microenvironment.