Abstract:Background and Aims Triple-negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer with limited treatment options. Immune checkpoint blockade (ICB) combined with chemotherapy has emerged as a key neoadjuvant therapeutic strategy for TNBC. However, significant variability in ICB efficacy exists among patients, and the underlying mechanisms related to the tumor immune microenvironment (TME) remain unclear. Cancer-associated fibroblasts (CAFs), as major stromal components, regulate TME and influence immunotherapy responses. Fibroblast activation protein (FAP), a key marker of CAFs, has been associated with poor prognosis in multiple solid tumors, yet its immunological role in TNBC has not been systematically investigated. This study aims to elucidate the expression pattern of FAP in TNBC, its impact on the immune microenvironment and ICB efficacy, and to explore its potential immunosuppressive mechanisms and clinical implications.Methods The data from TCGA and the I-SPY2 clinical trial were integrated to assess the association of FAP expression with prognosis, immune cell infiltration, and immune checkpoint molecule expression in TNBC. Immune landscape profiling was conducted using CIBERSORT, GSEA enrichment analysis, and differential gene expression analysis (DESeq2) to characterize the immune features associated with FAP expression and to identify downstream genes at the transcriptomic level. CAF models with FAP overexpression or knockdown were constructed and co-cultured with CD8? T cells to evaluate FAP's regulatory effects on CD8? T cell activity and apoptosis. The expression of COL1A1, a potential FAP-regulated gene identified from transcriptomic analysis, was validated using qPCR and Western blot. Finally, clinical data and multiplex immunofluorescence pathology samples from TNBC patients at Xiangya Hospital were analyzed alongside I-SPY2 data to determine the predictive value of FAP expression for pathological complete response (pCR) following neoadjuvant immunotherapy.Results FAP was significantly upregulated in TNBC tumor tissues compared to normal tissues and associated with shorter overall survival. Multivariate Cox regression analysis identified FAP as an independent adverse prognostic factor. High FAP expression was correlated with reduced infiltration of CD8? T cells, NK cells, and Tfh cells, as well as upregulation of immune checkpoints including CD276, TIM-3, and PD-L2. In CAF models, FAP overexpression suppressed CD8? T cell activity and promoted apoptosis, while FAP knockdown had the opposite effect. Transcriptomic analysis showed that COL1A1 and other collagen-related genes were significantly upregulated in the FAP-high group and positively correlated with FAP expression; qPCR and Western blot confirmed that FAP positively regulates COL1A1 expression. Analysis of I-SPY2 data revealed that FAP-low patients receiving pembrolizumab plus chemotherapy had significantly higher pCR rates compared to FAP-high patients. Consistently, clinical data from the Xiangya cohort showed reduced CD8? T cell infiltration and lower pCR rates in FAP-high patients, with a ROC AUC of 0.857 for predicting treatment response.Conclusion FAP-high CAFs contribute to the formation of an immunosuppressive TME in TNBC by promoting COL1A1 secretion, inhibiting CD8? T cell function, and upregulating immune checkpoint molecules. High FAP expression is associated with poor prognosis and reduced response to immunotherapy, highlighting FAP as both a prognostic biomarker and a potential therapeutic target for stratified and combination treatment strategies in TNBC.