转移淋巴结（MLN）内成纤维细胞的结缔组织增生是结外扩展（ENE）的指标，导致口腔鳞状细胞癌（OSCC）的死亡率。然而，缺乏对 MLN 中成纤维细胞的系统研究。因此，本研究表征了 OSCC 中原发肿瘤 (PT) 和淋巴结 (LN) 之间成纤维细胞的表型、功能和起源的差异。我们生成了来自三名 OSCC 患者的 PT 和配对 MLN 以及引流 LN 的单细胞图。对单细胞的转录组图谱、伪时间分析、细胞间通讯网络和富集分析进行了表征。体外进一步验证了PTs和MLNs之间成纤维细胞的表型和功能异质性。在 44,052 个成纤维细胞中，我们鉴定了两个不同的癌症相关肌成纤维细胞 (mCAF) 亚群：RGS4 mCAF1 和 COMP mCAF2。值得注意的是，它们表现出不同的分布，mCAF1 主要位于 PT，而 mCAF2 主要位于 MLN。此外，伪时间分析揭示了它们不同的起源：mCAF1起源于PT中固有的正常肌成纤维细胞，而mCAF2起源于LN中的成纤维网状细胞。使用原代成纤维细胞的进一步功能实验表明，与 mCAF1 相比，MLN 中的 mCAF2 与免疫细胞的串扰较弱，但细胞外基质活性增强，这与 OSCC 中 ENE 的形成密切相关。此外，我们还发现了两个处于转化状态的成纤维细胞亚群，表明潜在的上皮-间质转化。我们的研究对 OSCC 中 PT 和 MLN 之间成纤维细胞的异质性提供了深刻的见解，可作为未来药物发现工作的重要资源。版权所有 © 2024。由 Elsevier B.V. 出版。

Desmoplasia in fibroblasts within metastatic lymph nodes (MLNs) serves as an indicator of extranodal extension (ENE), which led mortality in oral squamous cell carcinoma (OSCC). However, systematic studies on fibroblasts in MLNs are lacking. Therefore, this study characterized the differences in phenotype, function, and origin of fibroblasts between primary tumors (PTs) and lymph nodes (LNs) in OSCC. We generated single-cell maps of PTs and paired MLNs and draining LNs from three OSCC patients. The transcriptomic atlas, pseudotime analysis, intercellular communication networks and enrichment analysis of the single cells were characterized. Phenotype and function heterogeneity of fibroblast cells between PTs and MLNs were further verified in vitro. Among 44,052 fibroblasts, we identified two distinct subpopulations of cancer-associated myofibroblastic cells (mCAFs): RGS4+ mCAF1 and COMP+ mCAF2. Notably, they exhibited distinct distributions, with mCAF1 predominantly localized in the PTs and mCAF2 in the MLNs. Moreover, pseudotime analysis revealed their distinct origins: mCAF1 originated from inherent normal myofibroblastic cells in the PT, whereas mCAF2 originated from fibroblastic reticular cells in the LNs. Further functional experiments using primary fibroblasts revealed that, compared to mCAF1, mCAF2 in MLNs exhibited weaker crosstalk with immune cells but enhanced extracellular matrix activity, which is closely linked to ENE formation in OSCC. Additionally, we identified two fibroblast subgroups in a transforming state, indicating a potential epithelial-mesenchymal transition. Our research offers profound insights into the heterogeneity of fibroblasts between the PT and MLN in OSCC, serving as an essential resource for future drug discovery endeavors.Copyright © 2024. Published by Elsevier B.V.