Progressive Hypoxia Exacerbates Breast Cancer Metastasis by Paracrine Modulation of the Blood-brain Barrier Endothelium

) conditions to generate conditioned media (CM). Diluted CM was used to treat brain endothelial cells (BECs: bEnd.5 cell line). Endothelial suppression of cell division was assessed using the trypan blue exclusion assay. Morphological changes and an abnormality index were evaluated using hematoxylin and eosin (H&E) staining. Transendothelial electrical resistance (TEER) measured the BEC monolayer integrity following CM exposure. Comparatively, all oxygen-derived CM suppressed BEC suppression of cellular division; however, hypoxic-derived CM was most severe, reducing cell division by 67.7% at 96 h. H&E staining similarly showed that hypoxic-derived CM induced an abnormality rate of 980 abnormalities per 1 000 cells. TEER measurements demonstrated that the hypoxic-derived CM overwhelmed BEC homeostatic mechanisms, significantly increasing monolayer permeability. A clear pattern of progressive dysfunction correlated with decreasing oxygen tension in MDA-MB-231 cell cultures. While the normoxic and physioxic CM had comparable effects, hypoxic-derived CM elicited exacerbated responses, with substantial impairment detectable as soon as 24 h. These findings suggest hypoxic tumour secretomes overwhelm the endothelial regulatory mechanisms, leading to BEC dysfunction and, ultimately, BBB compromise.