Single-cell transcriptomic landscape highlights epithelial-fibroblast interactions and CD44-mediated malignant traits...

Laryngeal squamous cell carcinoma (LSCC) frequently presents with lymph node metastasis and poor prognosis. Cancer-associated fibroblasts (CAFs) are key stromal components that modulate tumor progression. We performed single-cell RNA sequencing on nine matched samples, including primary tumors (PT), adjacent non-tumor tissues (NT), and metastatic lymph nodes (LN), from three LSCC patients to profile epithelial and fibroblast heterogeneity. Three malignant epithelial subtypes (EP0-EP2) were identified, with distinct trajectories and functional phenotypes. EP0 was enriched in EMT-related signatures and dominant in metastatic lesions, while EP2 displayed high stemness and proliferative potential. Five fibroblast subtypes (F0-F4) were characterized, showing a pseudotemporal transition from immune-regulatory (F1, F3) and matrix-remodeling (F2) states toward a contractile, pro-invasive F0 subtype, which was enriched in LN and highly expressed ACTA2 and CAV1. Cell-cell communication analysis revealed increased fibroblast-epithelial interactions in LN, primarily via FN1 and COLLAGEN ligands targeting CD44. Functional assays confirmed that CD44 knockdown in vitro reduced proliferation, migration, invasion, and in vivo tumorigenesis, supporting its role in metastasis. These findings reveal the dynamic remodeling of epithelial and fibroblast compartments in LSCC and identify CD44 as a key driver of metastasis through fibroblast-epithelial interactions.