AKAP12-LMOD1 signaling defines stromal CAF activation and epithelial junction loss in colorectal cancer

Colorectal cancer (CRC) shows strong heterogeneity in tumor microenvironment (TME) dynamics, but the mechanisms that shape epithelial-stromal crosstalk are still unclear. Here we focus on A-kinase anchor protein 12 (AKAP12) and Leiomodin 1 (LMOD1) and test a compartment-dependent model in which this program aligns with tight-junction features in epithelium but with a fibrotic, immune-suppressive program in stroma. Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) were employed to profile gene expression patterns in CRC tissues. Immunohistochemistry (IHC) and multiplex immunofluorescence (mIF) validated protein expression and localization. Cell-cell communication analysis and trajectory inference were used to dissect spatial interaction networks. Functional experiments were employed to validate the role of the AKAP12-LMOD1 axis in CAFs in regulating ECM remodeling and antitumor immunity. AKAP12-LMOD1 exhibited a compartment-dependent pattern in CRC. In ACTA2⁻ epithelial regions, the epithelial AKAP12-LMOD1 signal was lower in tumors than in matched normal epithelium and showed a positive association with the tight-junction marker CLDN1. In ACTA2⁺ stromal regions, AKAP12-LMOD1 was enriched, positively associated with the gap-junction marker GJA4, and higher in tumor stroma than matched normal stroma. In a CAF-macrophage non-contact co-culture model, AKAP12 overexpression supported CAF activation and collagen deposition, and shifted macrophages toward an M2-like phenotype; LMOD1 knockdown or SB-431542 partially reversed these effects. Stromal AKAP12-LMOD1-enriched regions also aligned with fibrosis- and M2-related features, and these stromal patterns were prominent in mucinous carcinoma. This study defines AKAP12-LMOD1 as a compartment-dependent stromal program in CRC that links ACTA2⁺ stroma to gap-junction features, fibrosis, and M2-like macrophage polarization, while showing a distinct epithelial association with tight-junction features. These findings support a stroma-centered working model for AKAP12-LMOD1 in CRC microenvironmental heterogeneity and suggest that stromal modulation of this program, together with targeting fibrosis and M2-like immune features, may be explored as hypothesis-level, subtype-oriented therapeutic directions in stroma-rich CRC.