Single-cell transcriptomic and immune microenvironment analyses reveal a TIMP1-associated OLFM4⁺ tumor epithelial program linked to colorectal cancer progression and immune evasion

BACKGROUND: Colorectal cancer (CRC) is characterized by marked epithelial heterogeneity and dynamic tumor-microenvironment interactions. However, epithelial states associated with progression across the normal-adenoma-carcinoma sequence remain incompletely defined. METHODS: Single-cell RNA sequencing data from normal colorectal mucosa, adenoma, and tumor tissues were analyzed using inferCNV, Monocle, Slingshot, CytoTRACE, CellChat, and SCENIC. An OLFM4⁺ tumor cell risk score (OTRS) was constructed using Cox and LASSO regression, and TIMP1 function was evaluated in vitro. RESULTS: Eight major cell types and nine tumor epithelial subtypes were identified. C3 OLFM4⁺ cells showed elevated transcriptional activity, stemness, metabolic activity, and greater representation in carcinoma than adenoma tissues. Trajectory analyses positioned these cells in an early-to-intermediate transitional state. CellChat predicted prominent fibroblast-C3 OLFM4⁺ interactions, including a putative WNT2B-FZD/LRP axis, while SCENIC identified XBP1, NFIA, PPARG, HDAC2, and UBE2K as subtype-associated regulators. The C3 OLFM4⁺-derived OTRS stratified prognosis; high OTRS was associated with poorer survival, macrophage-enriched infiltration, higher stromal, immune, and TIDE scores, and differential predicted drug sensitivity. TIMP1 knockdown suppressed malignant behaviors in CRC cells. CONCLUSION: C3 OLFM4⁺ cells represent a stem-like, metabolically active tumor epithelial state associated with CRC progression, fibroblast-derived WNT signaling, immune-evasion-related features, and unfavorable prognosis. The OTRS model may support prognostic and therapeutic assessment, while TIMP1 contributes to malignant CRC cell behavior.