Cross-Cohort Multi-Omics Analysis Identifies Novel Clusters Driven by EMT Signatures in Colorectal Cancer

Wu Ning¹Wenqing Jia²Jingyuan Ning³Lei Zhou¹Zongze Li¹Lin Zhang¹Xin Song^1*

• ¹Department of General Surgery, China-Japan Friendship Hospital, Beijing, China
• ²Institute of Clinical Medicine, China-Japan Friendship Hospital, Beijing, China
• ³Chinese Academy of Medical Sciences and Peking Union Medical College, Dongcheng, China

The pronounced heterogeneity of colorectal cancer (CRC) significantly impacts patient prognosis and therapeutic response, making elucidation of its molecular mechanisms critical for developing precision treatment strategies. This study integrated five single-cell RNA sequencing cohorts (comprising 70 CRC samples and 164,173 cells) to systematically characterize tumor cell (TC) heterogeneity, identifying five distinct TC subtypes. Notably, the VEGFR+ TC subtype exhibited marked epithelial-mesenchymal transition (EMT) activation signatures and was strongly associated with metastasis and poor clinical outcomes. Unsupervised clustering based on VEGFR+ TC signature genes stratified CRC patients into three subgroups: C1 (metabolically active), C2 (proliferative), and C3 (invasive), with the C3 subtype demonstrating high metastatic potential, stem-like properties, and an immunosuppressive microenvironment, along with a five-year survival rate below 50%. Mechanistic investigations identified HOXC6 as a key driver of the C3 subtype.In vitro experiments confirmed that HOXC6 knockout significantly suppressed CRC cell proliferation, migration, and invasion, while in vivo animal models demonstrated substantial tumor volume reduction upon HOXC6 inhibition. Molecular docking and dynamics simulations revealed that the targeted agent abemaciclib effectively binds HOXC6, with both cellular and animal experiments confirming its ability to inhibit CRC cell proliferation, migration, and invasion, significantly reducing tumor burden in nude mice. This study establishes the first single-cell-resolution molecular classification system for CRC, delineates the mechanistic link between EMT subtypes and metastatic progression, and nominates HOXC6 as a novel therapeutic vulnerability, providing a translational foundation for precision oncology. These findings represent the inaugural demonstration of a single-cell-based molecular taxonomy for CRC, reveal the association between EMT subtypes and metastasis, and identify HOXC6 as a potential therapeutic target, offering new rationale for precision diagnosis and treatment of colorectal cancer.