Myofibroblast‐Like CAFs in Metastatic Lymph Nodes Drive Tumor Stemness and Poor Prognosis in Head and Neck Cancer

ABSTRACT

Lymph node (LN) metastasis is one of the major prognostic factors in head and neck squamous cell carcinoma (HNSCC), and treatment sensitivity often differs between primary tumors and metastatic LNs. Beyond tumor cell–intrinsic differences, heterogeneity of the tumor microenvironment may contribute to these discrepancies. We focused on cancer‐associated fibroblasts (CAFs) and compared CAFs derived from primary tumors and corresponding LN metastases. Bulk tissue analysis showed higher expression of myofibroblast‐related genes ( ACTA2 , MYL9 , MYLK ) and increased COMP expression in metastatic LNs. Thirteen paired CAFs were established and serially passaged. Compared with primary tumor‐derived CAFs (PT‐CAFs), metastatic LN‐derived CAFs (LN‐CAFs) exhibited reduced proliferative and migratory capacities, but enhanced collagen gel contraction, while promoting tumor cell‐sphere formation and cancer stem cell marker expression. These findings support a myofibroblast‐like phenotype in LN‐CAFs. In line with this, PT‐CAFs showed greater induction of myofibroblast‐related genes in response to TGF‐β, but not IL‐6 stimulation, suggesting that LN‐CAFs had already differentiated toward a myofibroblast‐like phenotype. RNA sequencing of five paired CAFs confirmed a myofibroblastic transcriptional profile, with enrichment of fibrosis‐related and malignant trait–associated pathways, including epithelial–mesenchymal transition (EMT), hypoxia, and p53 signaling. Finally, least absolute shrinkage and selection operator‐based prognostic models validated in The Cancer Genome Atlas (TCGA) cohort using genes upregulated in LN‐CAFs were significantly associated with worse progression‐free survival and overall survival. These results demonstrate that CAF heterogeneity depends on tumor localization and is linked to therapeutic resistance and poor clinical outcomes in HNSCC.