Spatial and single-cell transcriptomics decipher butyrate metabolism-related transcriptional programs and its impact on colorectal cancer progression and tumor microenvironment remodeling

Colorectal cancer (CRC) is closely linked to dietary and microbial factors. Butyrate, a major gut microbial metabolite with histone deacetylase-inhibitory and immunomodulatory effects, contributes to epithelial homeostasis, but the spatial heterogeneity and clinical relevance of butyrate metabolism-related transcriptional programs in CRC remain unclear. Multi-algorithm single-cell gene-set scoring was used to calculate a butyrate metabolism-related transcriptional activity (BTA) score and stratify tumor epithelial cells into BTA score-defined epithelial subgroups, which were subsequently mapped spatially using cell2location. CellChat, Monocle, pySCENIC, inferCNVpy, PROGENy, and stLearn were applied to characterize lineage states, transcriptional regulation, signaling activity, and intercellular crosstalk. Ensemble machine learning was used for prognostic modeling, followed by molecular and functional validation. We identified malignant epithelial cells with low transcriptome-derived BTA scores (LowBTAepis), which were associated with poor prognosis, preferential localization at earlier positions along the inferred pseudotime trajectory, increased stem-cell-like features and proliferation, and distinct copy-number variation (CNV) patterns. Across three independent bulk CRC cohorts, low-BTA tumors were consistently enriched for CMS4, linking the BTA-associated transcriptional state with mesenchymal and stromal molecular characteristics. Spatial analysis revealed spatial co-occurrence between LowBTAepis and fibroblasts and identified candidate ligand–receptor relationships involving these populations. Integrated regulon, spatial transcriptomic, and independent butyrate-perturbation analyses prioritized HOXB8 for functional validation. Sodium butyrate (NaB) suppressed CRC cell proliferation, migration, and invasion in a dose-dependent manner, accompanied by reduced HOXB8 expression, inhibition of AKT/mTOR signaling, and changes in apoptosis- and metastasis-related proteins. A prognostic model derived from LowBTAepis marker genes showed strong prognostic performance and was associated with an immunosuppressive tumor microenvironment. LowBTAepis represents an aggressive CRC epithelial state characterized by BTA scores, stromal crosstalk, and malignant transcriptional features. Complementary experiments suggest that exogenous sodium butyrate may suppress CRC cell proliferation, migration, and invasion partly through the HOXB8/AKT/mTOR axis.