Effect of EDIL3+ CAFs on lymph node metastasis through disruption of lymphatic endothelial barriers in breast cancer.

e13029 Background: Axillary lymph node (LN) metastasis is the most common metastatic route in breast cancer and a critical prognostic indicator. While previous studies focused on tumor cells, the role of stromal components within the tumor microenvironment (TME) in mediating LN metastasis remains unclear. This study aims to identify key functional stromal cell subsets and molecular mechanisms driving breast cancer LN metastasis. Methods: We analyzed high-LN-metastasis cases from our cohort, TCGA, and METABRIC databases. Immunohistochemistry and transcriptomic deconvolution were used to compare stromal cell abundance, including lymphatic endothelial cells (LECs) and cancer-associated fibroblasts (CAFs). Single-nucleus RNA sequencing (snRNA-seq) was employed to identify metastasis-associated CAF subsets. Functional assays, including siRNA knockdown, lentiviral overexpression, CCK-8, tube formation, wound healing, endothelial permeability, and trans-endothelial migration assays, were conducted to assess the role of EGF-like repeat and discoidin domain-containing protein 3 (EDIL3) on LEC function. In vivo effects were validated using immunofluorescence, H&E staining, and intravital imaging in murine models. Transcriptomic sequencing, western blot, and small-molecule inhibitors were employed to elucidate the molecular mechanisms by which EDIL3 regulates LEC permeability. Results: snRNA-seq of our cohort (n = 9) revealed significant enrichment of EDIL3+ CAFs in primary tumors with high LN metastasis, correlating with axillary LN burden and poor survival. EDIL3 was predominantly expressed by a specific CAF subset within TME. Using immortalized 4T1 breast cancer CAF cell lines, we demonstrated that EDIL3+ CAFs enhance LEC permeability via EDIL3. In vitro, EDIL3 disrupted LEC tight junction integrity without affecting proliferation, migration, or lymphangiogenesis. In murine models, EDIL3 promoted lymphatic metastasis by increasing LEC permeability. Mechanistically, EDIL3 activated the integrin αvβ3/Src/ERK signaling axis to drive LEC barrier dysfunction. Conclusions: This study identifies EDIL3+ CAFs as a novel stromal subset driving breast cancer LN metastasis. EDIL3 enhances lymphatic dissemination by activating integrin αvβ3/Src/ERK signaling to compromise LEC integrity. These findings highlight CAFs as pivotal mediators of lymphatic metastasis and propose EDIL3 as a potential therapeutic target to mitigate LN metastasis in breast cancer.