Extracellular Vesicle‑Driven CNS Niche Formation in Triple Negative Breast Cancer Metastasis

Abstract

Triple negative breast cancer (TNBC) frequently metastasizes the central nervous system, where outcomes remain poor, yet how tumor-derived signals condition before tumor arrival is still not well understood. Tumor-derived extracellular vesicles (EVs) are known mediators of long-range communication that can alter distant tissues and contribute to pre-metastatic niche formation, but their impact on the meningeal lymphatic system and glial cells, two systems that regulate fluid balance, immune signaling, and CNS homeostasis, remains unclear. To address this, we examined how TNBC-derived EVs influence lymphatic endothelial cells (LECs), meningeal stromal cells (HMCs), astrocytes, and microglia across early and extended time points. In the meningeal lymphatic model, EV exposure did not change overall cell coverage, but LECs showed clear changes in junctional organization, with increased spacing at early time points followed by reorganization, alongside a consistent increase in EV secretion, indicating an active cellular response rather than passive disruption. HMCs maintained structural stability while showing a more modest increase in EV secretion, suggesting a supportive role within the system. In parallel, glial cells did not exhibit strong morphological or proliferative changes, but both astrocytes and microglia increased EV secretion, and cytokine profiling revealed elevated inflammatory signaling, with astrocytes showing increased IL-6 expression linked to aquaporin-4 regulation and fluid movement within the CNS. These findings suggest that TNBC-derived EVs drive early changes by modulating cellular communication and signaling rather than inducing immediate structural damage, shifting the CNS microenvironment into a more responsive and potentially permissive state. This work provides insight into how early tumor-independent signaling may influence CNS function and highlights EV-mediated interactions as a key mechanism in shaping disease progression before metastasis occurs.