Unveiling the intricate dynamics of the interplay between triple-negative breast cancer cells and the blood-brain barrier endothelium

Brain metastases (BM) critically reduce breast cancer (BC) patients' survival. Extravasation is pivotal for BM development, but the underlying events at the blood-brain barrier (BBB) remain elusive. We aimed to unravel the players and mechanisms governing BC cells (BCCs)-BBB interaction. For that, mixed cultures of human brain microvascular endothelial cells (HBMECs), mimicking the BBB, and brain-tropic triple-negative BCCs (MDA-MB-231 Br4), or non-brain-tropic (MDA-MB-231) or non-metastatic cells (MCF-7) were established. Temporal and spatial analysis of BCCs-BBB interactions (live-cell imaging automated microscopy), and assessments of endothelial-to-mesenchymal transition (EndMT) markers, transcription factors, cytoskeletal proteins, and morphology (immunocytochemistry) were performed. BBB integrity (permeability, transendothelial electrical resistance) and endothelial migration (wound-healing) were also assessed. Our results revealed that contrasting with non-metastatic and non-brain-tropic cells, BCCs quickly developed an invasive, migratory phenotype, characterized by invadopodium formation and reduced roundness. Spatial analysis showed different positioning of BCCs relative to the BBB endothelium over time, with 14% of BCCs transmigrated after 3 h, compromising BBB integrity through endothelial holes, reduced tightness, and increased permeability. Prior to transmigration, alterations in adhesion markers (E-selectin, ICAM-1, CD24, CD34, β3-integrin, Sialyl-Lewis X) were observed. EndMT was also evident by decreased endothelial (β-catenin and pan cytokeratin) and increased mesenchymal (vimentin, neuronal-cadherin, Slug, ZEB1) markers, elongation (RhoA, α-SMA), nuclear deformation, and migratory capacity. Caveolin-1 silencing in HBMEC decreased BCCs transmigration. This study reveals significant BBB phenotypic and structural changes, facilitating both paracellular and transcellular BCCs transmigration. These findings provide advanced understanding of BCCs trafficking across the BBB, aiding strategy development to prevent extravasation and BM.