YAP-activated Lymph Node Fibroblasts Contribute to Extracellular Matrix Remodeling Associated with Extranodal Extension of Oral Squamous Cell Carcinoma.

Desmoplasia, characterized by excessive expansion of cancer-associated fibroblasts (CAFs) and aberrant extracellular matrix (ECM) deposition, is widely present especially in invasive cancers. The extranodal extension (ENE) of nodal metastasis involves the extension of invasive tumor cells through the lymph node capsule into the perinodal adipose tissue, leading to systemic metastasis. Thus, the specific roles of desmoplasia in ENE formation need to be identified. This study aimed to investigate the association between desmoplasia and its role in ENE formation of oral squamous cell carcinoma, with a focus on Yes-associated protein (YAP)-mediated fibroblast activation and ECM remodeling. Transcriptome profiling and histopathology study based on tissue microarrays were performed to explore the association of metastasis-associated CAFs (MAFs) in ECM remodeling with ENE formation. Primary MAFs were isolated from ENE-positive (ENE+) and ENE-negative (ENE-) lymph nodes for functional characterization. The regulatory role of YAP in MAF-mediated ECM remodeling was investigated through immunohistochemistry, gene knockdown, and co-culture assays with HSC-3 tumor cells. A high degree of desmoplasia and rich collagen remodeling in lymph nodes was associated with higher ENE risk. ENE+ MAFs exhibited superior proliferative capacity, enhanced migration, and increased ECM remodeling activity compared to ENE- MAFs. Co-culture experiments demonstrated that ENE+ MAFs significantly promoted HSC-3 cell invasion through Matrigel. Exploratory RNA sequencing revealed enrichment of ECM receptor interaction and Hippo-YAP pathway in ENE+ lymph nodes. Nuclear YAP localization in MAFs correlated with elevated expression of ECM components (collagen type I alpha 1 [COL1A1], fibronectin 1 [FN1], Tenascin C) and matrix metalloproteinase-2. YAP knockdown in ENE+ MAFs significantly attenuated both ECM remodeling capacity and pro-invasion ability in vitro. These findings suggest that YAP-activated MAFs contribute to ECM remodeling that may create a stromal environment permissive for ENE formation, though direct in vivo validation is needed to establish causality.