Neurotransmitter-associated programs shape macrophage states in colorectal cancer

Introduction: Colorectal cancer (CRC) progression is shaped by the tumor microenvironment (TME), where macrophages can promote immune suppression. Although neurotransmitter signaling modulates immune cells, its role in CRC macrophage states remains unclear. We investigated neurotransmitter-associated, particularly GABAergic, programs in CRC. Methods: We combined in silico, tissue-based, and in vitro analyses. Five public CRC single-cell RNA-seq (scRNA-seq) cohorts (516,038 cells; 95 tumors, 62 matched non-tumor samples) were integrated with scVI. Clustering, differential expression, pathway enrichment, neurotransmitter-module scoring, pseudotime, transcription-factor, and ligand-receptor analyses were conducted in silico. Multiplex immunohistochemistry (IHC) quantified proximity of CD20+ B cells and FOLR2+ macrophages in CRC sections. Healthy-donor monocytes were cultured in vitro with 10 μM gamma-aminobutyric acid (GABA), stimulated with lipopolysaccharide, and profiled by multiparametric flow cytometry. An internal bulk RNA-seq cohort and The Cancer Genome Atlas (TCGA) CRC cohorts provided validation. Results: In silico, tumors showed neurotransmitter-pathway remodeling, greatest in macrophages. Monocyte-to-macrophage trajectories were enriched for FOLR2, GAD1, and GRM4. Ligand-receptor inference identified CCL5-CCR1/CCR5, VEGFB-NRP1, and GAS6-AXL/MERTK axes between B-cell/plasma-cell and macrophage populations; IHC confirmed CD20+ B-cell/FOLR2+ macrophage proximity. GABA conditioning was associated with changes in monocyte CD86, HLA-DR, CD64, IL-1β, and TNF-α expression under inflammatory conditions. In bulk RNA-seq, GABAergic programs correlated with TGFB, IL10-IL10RA, and BAFF-TACI axes; high GABAergic and glutamatergic scores were associated with poorer overall survival. Conclusions: GABAergic signaling is a candidate component of macrophage differentiation and B-cell/macrophage communication, with implications for immune remodeling and CRC progression.