Single-cell transcriptome analysis reveals SOX9 mutation-driven tumor stemness and microenvironment remodeling through the COL1A1–CD44 axis in colorectal cancer

Abstract Colorectal cancer (CRC) exhibits substantial interpatient heterogeneity and is driven by intricate crosstalk between tumor-intrinsic mutations and the tumor microenvironment (TME), yet the mechanisms by which specific genetic alterations remodel the TME and regulate tumor stemness remain elusive. The SRY-box transcription factor 9 (SOX9) is a key regulator of intestinal homeostasis, with mutations implicated in CRC progression, but its single-cell level effects on TME dynamics have not been fully elucidated. To dissect the heterogeneity and cell–cell interactions underlying SOX9 mutation-driven CRC progression, we integrated single-cell RNA sequencing (scRNA-seq) data of 23 CRC tumor samples with bulk sequencing and mutation data from The Cancer Genome Atlas (TCGA) using the Scissor algorithm. We identified a malignant epithelial subpopulation (Scissor + ) specifically associated with SOX9 mutations, which exhibits enhanced stemness signatures. Further, we predicted a significant upregulation of the COL1A1–CD44 axis in SOX9-mutant tumors via CellChat, which was supported by immunohistochemical analyses in patient tissues and mouse xenograft models. By integrating stemness-related genes and fibroblast-derived ligands, we established a 172-gene fibroblast–stemness signature that was associated with overall survival, recurrence-free survival, and disease-specific survival across multiple independent CRC cohorts. Functional validation via CD44 knockdown and in vivo xenograft assays confirmed that the COL1A1–CD44 axis mediates SOX9 mutation-driven stemness maintenance and tumor progression. Collectively, our data enhance understanding of how SOX9 mutations remodel the TME, pinpoint the COL1A1–CD44 axis as a promising therapeutic candidate for SOX9-mutant CRC, and support precise patient stratification and targeted interventions.