MIF-mediated intercellular crosstalk drives antigen-presenting-like cancer-associated fibroblast differentiation in triple-negative breast cancer via TGF-β signaling

Abstract

Background Triple-negative breast cancer (TNBC) is characterized by an aggressive tumor microenvironment (TME) in which cancer-associated fibroblasts (CAFs) play a pivotal role. However, the molecular mechanisms governing the transformation of quiescent fibroblasts into distinct CAF subtypes within TNBC remain poorly understood. This study aimed to elucidate the molecular mechanisms underlying fibroblast-to-CAF subtype transition in TNBC and identify key signaling pathways involved in TNBC-driven CAF activation. Methods Transcriptomic data from The Cancer Genome Atlas (TCGA)-TNBC were analyzed using weighted gene co-expression network analysis (WGCNA). CAF-related scores and TNBC scores were computed via single-sample gene set enrichment analysis (ssGSEA). CAF infiltration was assessed using Estimating the Proportion of Immune and Cancer cells (EPIC). Survival and correlation analyses were validated in the Gene Expression Omnibus (GEO) dataset GSE58812. Single-cell RNA sequencing (scRNA-seq) data (GSE176078) were employed for cell-type annotation, pseudotime trajectory inference, and cell-cell communication analysis. Cell experiments were further performed to explore the mechanisms of fibroblast activation in TNBC. Results TNBC score was significantly associated with poor prognosis and positively correlated with CAF score/infiltration. Single-cell analysis revealed antigen-presenting CAFs (apCAFs) as the predominant CAF subtype in TNBC. Pseudotime trajectory analysis showed that 96 genes uniquely involved in quiescent-to-apCAF-like differentiation were enriched in TGF-β signaling. Cell-cell communication analysis identified macrophage migration inhibitory factor (MIF)-(CD74 + CXCR4) as significantly enhanced during TNBC-apCAF interactions but absent in TNBC-myofibroblastic CAF (myCAF) crosstalk. Four hub genes, including CNTN4, NLGN1, NLGN4X, and SEMA5A, were identified as associated with the differentiation stage: CNTN4/NLGN1 in MIF-mediated signal initiation and NLGN4X/SEMA5A in the intracellular TGF-β execution phase. Cell experiments revealed that the MIF-CD74/CXCR4 axis promotes apCAF-like differentiation via the TGF-β pathway and enhances malignant phenotypes of TNBC cells. Conclusions A TNBC-to-apCAF signaling axis was identified wherein TNBC-secreted MIF activates CD74/CXCR4 on fibroblasts, engaging TGF-β signaling to drive apCAF-like differentiation. These findings offer potential therapeutic targets for disrupting the pro-tumorigenic microenvironment in TNBC.