HKDC1 as a Prognostic and Tumor Microenvironment‑Associated Biomarker in Colorectal Cancer: An Integrated Multi‑omics and Spatial Transcriptomic Study.

Colorectal cancer (CRC) is a leading cause of cancer-related mortality, necessitating the discovery of novel metabolic biomarkers. Hexokinases (HKs) are pivotal in dysregulated glucose metabolism. However, the role of HKDC1, which is the fifth and most recently identified isoform, remains poorly understood in CRC. We employed an integrated multi-omics approach to characterize HKDC1. Candidate glycolysis-related genes were screened using WGCNA and differential expression analysis across TCGA, GEO, and GTEx datasets. Spatial transcriptomics and single-cell RNA sequencing (scRNA-seq) were utilized to map the precise localization and cellular communication patterns of HKDC1. Findings were validated in a clinical cohort (n = 168) via Western blot and immunohistochemistry. Functional roles were assessed through HKDC1 knockdown in CRC cell lines using CCK-8, colony formation, Transwell, and wound healing assays. Finally, the relationship between HKDC1, immune infiltration, and drug sensitivity was explored. HKDC1 was significantly upregulated in CRC across multiple cohorts and pan-cancer datasets. Spatial transcriptomics confirmed HKDC1 enrichment specifically within malignant regions compared to benign zones. High HKDC1 expression was identified as an independent prognostic factor correlated with poor overall survival. Functional assays demonstrated that HKDC1 knockdown significantly inhibited CRC cell proliferation, migration, invasion, and was associated with suppression of epithelial-mesenchymal transition (EMT) related features. Mechanistically, scRNA-seq analysis suggested that HKDC1+ epithelial cells may contribute to tumor progression and potentially interact with fibroblasts through ligand-receptor signaling, including the COL1A2-ITGA2 axis. Furthermore, HKDC1 expression was closely associated with immune cell infiltration and predicted drug sensitivity profiles. Our study identifies HKDC1 may act as a potential metabolic regulator associated with CRC progression, possibly involving EMT and tumor microenvironmental interactions. HKDC1 may serve as a potential prognostic biomarker and a candidate target for further therapeutic investigation.