Multi-Omics Analysis Reveals the transforming growth factor-β Signaling-Driven Multicellular Interactions with...

While cervical cancer (CC) prognosis depends on tumor staging, the spatiotemporal evolution of tumor microenvironment (TME) heterogeneity during metastatic progression remains poorly characterized at single-cell resolution. We employed an integrative multi-omics approach, combining single-cell RNA sequencing (scRNA-seq; n = 11), spatial transcriptomics (ST), and bulk RNA-seq data from the TCGA-CESC cohort (n = 304), to systematically map TME remodeling across CC progression stages. scRNA-seq was performed on primary lesions from patients with localized (n = 3), regional (n = 4), and metastatic (n = 4) diseases, with in-depth analyses focusing on cellular characteristics, cell type composition alterations, functional changes, differentiation trajectories, and cell-cell interaction networks. These findings were further validated using spatial transcriptomics, bulk RNA-seq data, and multiple immunohistochemistry (mIHC) experiments. ScRNA-seq data revealed that the TME of the metastatic group displayed a distinct immunosuppressive phenotype. Three key subclusters closely linked to TME remodeling in this group were identified. Notably, a novel metastasis-associated epithelial subpopulation (Epi0_AGR2), characterized by both epithelial-mesenchymal transition (EMT) and chemokine secretory phenotypes, was discovered. Gene Set Variation Analysis (GSVA) revealed that transforming growth factor β (TGF-β) signaling activation served as its primary transcriptional driver. Additionally, a neutrophil subset with pro-tumor and immunosuppressive properties, as well as a cancer-associated fibroblasts (CAFs) subset that promoted angiogenesis, were enriched in the metastatic group. Cell-cell interaction analysis and spatial mapping further revealed the formation of coordinated Epi0-neutrophil-CAFs niches, which established TGF-β-CXCL1/2/8-OSM/OSMR feedforward loops. Importantly, a computational model derived from the TME metastatic niche signature demonstrated significant prognostic stratification in the TCGA cohort (HR = 2.5179, p = 0.0144). In all, this study provides the first comprehensive delineation of stage-specific TME dynamics in CC, revealing TGF-β-driven cellular cooperativity as a metastatic switch. The joint framework establishes a potential clinically translatable tool for precision prognosis and therapeutic targeting.