CA9+ cancer-associated fibroblasts cooperate with SPP1+ tumor-associated macrophages driving immune resistance in triple-negative breast cancer

BACKGROUND: Triple-negative breast cancer (TNBC) is a highly invasive and refractory subtype of breast cancer. Despite the promise of immune checkpoint blockade (ICB) therapy, response rates remain limited. The immune resistance driven by the tumor microenvironment has not yet been understood entirely, which hinders the personalized precision treatment of TNBC. METHODS: We integrated single-cell RNA data from 12 cohorts with TNBC and performed a multi-omics analysis combining spatial transcriptomics (ST), bulk RNA sequencing, and multiplex immunofluorescence (mIF) staining to identify immune-resistant subpopulations. Cell-to-cell communication was explored based on NicheNet and CellChat, and the function of CAF was verified by gene knockdown and overexpression in human mammary fibroblasts, followed by co-culture experiments with TNBC cell lines. ST and mIF data were used to analyze and verify cellular co-localization, while deconvolution was used to examine the relationship between two-cell characteristics and immunotherapy or antibody-drug conjugates (ADC) agent benefit. RESULTS: We identified CA9+cancer-associated fibroblasts (CA9+CAF) as a key subset enriched in non-responders to ICB that promotes immune resistance by establishing a hypoxic and immunosuppressive microenvironment via abnormal angiogenesis and glycolysis. ST and mIF analyses revealed a strong co-localization and interaction between CA9+CAF and SPP1+tumor-associated macrophages (SPP1+TAM), forming a stroma-myeloid axis that promotes immune escape through VEGFA/NRP2 axis in co-localization core region compared to the boundary. In vitro experiments demonstrated that the over-expression of CA9 in fibroblasts enhanced the proliferation, invasion, and migration of TNBC cells, while CA9 knockdown inhibited the tumorigenic effects. The high CA9+CAF/SPP1+TAM profile indicated a poor prognosis, reduced effector T cell infiltration, and attenuated response to immunotherapy, may benefit from TROP2, MUC1, and NECTIN4-based ADC agents. The result was validated in TNBC samples treated with neoadjuvant immunotherapy from our center. CONCLUSION: This study unveils the critical immunosuppressive axis orchestrated by CA9+CAF and SPP1+TAM in TNBC, offering novel insights into the stromal regulatory mechanisms driving immune resistance. The cell-to-cell interaction signature holds promise as predictor of immunotherapy response and potential therapeutic target.