Fibroblast Subtype-Specific Roles In Malignant Transformation Of Oral Leukoplakia

Building on evidence linking oral leukoplakia (OLK) malignant progression to microenvironment remodeling [1], this study aims to define OLK-associated fibroblast (OLKAF) subpopulations and explores their functional dynamics during transformation, extending prior findings on fibroblast-mediated pathogenesis [2]. ([1] Zhang Y, et al. Single-cell RNA sequencing highlights the immunosuppression of IDO1(+) macrophages in the malignant transformation of oral leukoplakia. Theranostics 2024; 14: 4787-4805. [2] Cheng R, et al. Reduced CX3CL1 Secretion Contributes to the Susceptibility of Oral Leukoplakia-Associated Fibroblasts to Candida albicans. Front Cell Infect Microbiol 2016; 6: 150.) Ethically approved (WCHSIRB-CT-2023-262), oral primary fibroblasts were isolated, cultured, and characterized by subtype via RNA-seq and immunofluorescence. Public scRNA-seq data (GSE181919) identified subpopulations; pseudotime trajectory analysis and Ucell scoring defined biological functions of distinct subtypes, further validated by Masson and immunohistochemistry staining in clinical samples. Primary fibroblasts of hyperplastic (HLKAF) and dysplastic (DLKAF) subtypes showed elevated activation markers. scRNA-seq delineated five oral mucosal fibroblast clusters: Normal tissues predominantly contained Fib_C0 (resembling primary normal fibroblasts), while OLK tissues were enriched with Fib_C1/C2 (aligned with HLKAF/DLKAF). Pseudotime analysis revealed a malignant trajectory from normal-enriched Fib_C0 to OLK- and cancer-associated Fib_C2, marked by upregulated proliferation pathways. HLKAF/Fib_C1 displayed immune-regulatory signatures; DLKAF/Fib_C2 exhibited matrix-remodeling profiles, validated by enhanced stromal collagen in malignant compared to OLK tissues. Fibroblasts represent a highly heterogeneous cell population, and their extracellular matrix remodeling capability is progressively enhanced during the malignant progression of OLK.