RBP1-driven retinol metabolism in tumor-associated lymphatic endothelial cells orchestrates triple-negative breast cancer progression and lymphatic metastasis

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and is characterized by a high metastatic potential and limited treatment options. TNBC frequently metastasizes to lymphatic vessels and the presence of breast cancer cells in tumor-draining lymph nodes (LNs) is an important prognostic factor. Nevertheless, the molecular and metabolic reprogramming of TNBC-associated lymphatic vasculature remains poorly understood. A comprehensive single-cell RNA sequencing analysis of 44,000 endothelial cells from human breast tumor datasets was performed. RBP1, a retinol-binding protein, was identified as a metabolic gene highly and specifically upregulated in tumor-associated lymphatic endothelial cells (LECs). Then, RBP1 expression in human TNBC biopsies was assessed, and its association with LN status and overall patient survival was investigated in a cohort of 1,474 breast cancer patients. We further evaluated the in vivo relevance of RBP1 by CRISPR/Cas9-mediated Rbp1 knockout in a pre-clinical TNBC mouse model. In vitro functional studies were performed using co-culture systems and RBP1-silencing in LECs. Finally, RBP1 mechanism of action in the lymphatic vasculature was determined by bulk-RNA sequencing and validated by applying different cellular and molecular approaches. Single-cell profiling revealed that the metabolic gene RBP1 was upregulated in LECs within the human breast tumor microenvironment. Further analysis showed that lymphatic RBP1 expression was associated with LN metastases, and correlated with poor prognosis and reduced survival in TNBC patients. In the pre-clinical TNBC mouse model, CRISPR-Cas9-mediated Rbp1 knockout reduced lymphangiogenesis, tumor growth and metastatic burden, and increased necrosis. Moreover, functional assays demonstrated that direct co-culture of LECs with TNBC cells, or exposure to their secretome, induced lymphatic RBP1 upregulation and enhanced pro-lymphangiogenic activities, whereas RBP1 silencing compromised their biological, redox and metabolic properties. Mechanistically, bulk RNA sequencing of RBP1-silenced LECs combined with experimental validation revealed that RBP1, through its retinol-binding domain, modulates the VEGFR3 and NOTCH pathways, while also involving lipid mobilization. We define lymphatic RBP1 as a key “switch” that couples LEC metabolism to molecular programs, thereby driving tumor-associated lymphangiogenesis, breast tumor growth and lymphatic dissemination in TNBC. Our findings underscore the potential of RBP1 as both a novel prognostic biomarker and a tractable therapeutic target for blocking TNBC progression and lymphatic metastasis.