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.