Single-cell and spatial transcriptomic analysis reveal distinct tumor microenvironment signatures in primary and...

Hypopharyngeal squamous cell carcinoma (HPSCC) is a rare, highly aggressive malignancy, with recurrence playing a pivotal role in treatment failure and cancer-related mortality. The tumor microenvironment (TME) characteristics and molecular mechanisms driving recurrence in HPSCC remain poorly defined.

Primary HPSCC (P-HPSCC) samples (n = 6) and recurrent HPSCC (R-HPSCC) samples (n = 3) were analyzed. Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics were employed to dissect the cellular composition and molecular characteristics of the TME. Key regulatory networks were identified through gene expression analysis, pathway enrichment, and intercellular communication profiling. Functional validation experiments were performed to confirm the biological relevance of the pathways identified.

ScRNA-seq analysis revealed that malignant epithelial cells (maEpCs) in recurrent tumors exhibited increased stemness, epithelial-mesenchymal transition (EMT), and immune evasion programs. Mechanistically, IGF2BP2 was selectively upregulated in R-HPSCC and functioned as an m⁶A reader to stabilize SMAD3 mRNA, thereby activating transforming growth factor beta (TGF-β) signaling and facilitating malignant progression. Integrated spatial transcriptomics and functional assays demonstrated that cancer-associated fibroblasts in recurrent tumors promote progression via FN1-mediated signaling. Concurrently, the recurrent TME underwent myeloid remodeling toward an immunosuppressive state, characterized by increased infiltration of SPP1⁺ tumor-associated macrophages and further enrichment of LAMP3⁺ dendritic cells with impaired antigen-presenting capacity, alongside aggravated CD8⁺ T-cell exhaustion, marked by upregulation of CTLA4 as a potential immune escape driver.

This study identifies recurrence-associated cellular and molecular features in HPSCC. Recurrent tumors exhibited maEpC reprogramming via IGF2BP2-mediated activation of TGF-β signaling, coupled with FN1-dependent stromal activation and immune evasion marked by myeloid immunosuppressive remodeling and CD8⁺ T-cell exhaustion, characterized by CTLA4 upregulation. Collectively, these findings reveal a pro-recurrent TME and suggest potential therapeutic targets in R-HPSCC.