Single-cell RNA sequencing identifies subtypes of cancer-associated fibroblasts in early and late stages of mycosis fungoides.

Mycosis fungoides (MF), the most common cutaneous T-cell lymphoma, is characterized by infiltration of malignant T-cells into the skin. While early-stage disease (IA-IIA) follows an indolent course, approximately 25% of patients progress to late-stage disease (IIB-IVB) with significantly worse prognosis and a median survival of 1-5 years. Identifying which early-stage MF patients are at high risk for disease progression remains difficult. This may be affected by the complex interaction between malignant tumor cells and the tumor microenvironment. Increased numbers of cancer-associated fibroblasts (CAF) are found in early-stage MF and have been shown to support tumor growth, but the subtypes have not yet been classified in lesional MF tissue. We performed single-cell RNA sequencing on skin samples from healthy individuals, early-stage MF patients, and late-stage MF patients to investigate the fibroblast population. Analysis of the highly differentially expressed genes in the fibroblast populations revealed nine distinct subclusters, comprising five major types: ECM/structural, vascular/metabolic, immune-modulatory, antigen-presenting, and developmental fibroblasts. Stage-specific differences revealed that the vascular/metabolic subcluster was enriched in early-stage MF, while the ECM/structural subcluster was enriched in late-stage MF. An antigen-presenting subcluster, a novel and underrecognized subtype, was identified by its high expression of MHC class II genes and pathways essential for antigen processing and presentation of exogenous peptides. These inappropriate antigen-presenting fibroblasts may play a role in chronic T-cell exhaustion seen in late-stage diseases. Further studies with additional patient samples will validate these findings and clarify the role of these subtypes in diagnosing and predicting the outcome of mycosis fungoides. Translational RelevanceOur findings reveal heterogeneity among fibroblasts in mycosis fungoides (MF) skin lesions, highlighting potential prognostic biomarkers and therapeutic targets with direct implications for improving the risk assessment and treatment of advanced disease. Using single-cell RNA sequencing, nine transcriptionally distinct fibroblast subclusters were identified that are involved in ECM remodeling, immune modulation, and metabolic adaptation. Examining both early- and late-stage MF disease, in a racially diverse patient cohort, offers new insights into how fibroblast composition changes during disease progression and across demographics. Additionally, we characterize an antigen-presenting fibroblast population in MF that may play an underappreciated role in the chronically exhausted T cells of advanced-stage disease.