Paired single-cell and spatial transcriptomics identify epithelial-state remodeling and an SPP1-rich microenvironment in colorectal cancer brain metastasis

Colorectal cancer brain metastases are difficult to characterize because matched primary and metastatic specimens are scarce. We integrated single-cell RNA sequencing from four matched primary tumor–brain metastasis pairs, bulk RNA sequencing from eight matched pairs, spatial transcriptomics from three additional matched pairs, and multiplex tissue staining from nine matched pairs. Analysis of 54,193 single cells revealed an increase in the mean proliferative/cycling epithelial fraction from 7.8% in primary tumors to 26.4% in brain metastases (paired P = 0.00260). Epithelial pseudobulk and bulk transcriptomes showed concordant enrichment of cell-cycle and metabolic programs and reduced enrichment of epithelial–mesenchymal transition and interferon-response programs. Similar changes within three epithelial states suggested that both state composition and within-state expression contributed to the metastatic phenotype. Spatial analysis localized glycolysis-associated expression to tumor-rich regions, with a tendency toward higher scores in brain metastases. Communication analysis identified macrophage-associated SPP1 and CD44-expressing epithelial and endothelial cells as a candidate signaling relationship. Increased spatial SPP1/CD44 joint detection and neighboring-spot association were accompanied by greater SPP1 protein positivity and shorter distances between CD44-positive and SPP1-positive cells. These findings characterize colorectal cancer brain metastases by proliferative epithelial-state remodeling and an SPP1-rich microenvironment and identify SPP1–CD44 as a candidate for functional investigation.