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

Abstract

Cancer remains a major global concern, with metastasis responsible for most cancer-related deaths. Disseminated cancer cells become circulating tumour cells (CTCs) that travel until arresting at preferential vascular sites such as the blood-brain barrier (BBB), where they cross into the central nervous system (CNS). Given that the BBB robustly maintains CNS homeostasis, the mechanism whereby CTCs metastatically enter the brain is largely unknown. The triple-negative breast cancer (TNBC) (MDA-MB-231 cell line) cells were cultured under normoxic (21% O₂), physioxic (5% O₂), and hypoxic (1.5-2% O₂) 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.

Graphical Abstract

The alternative text for this image may have been generated using AI.

Illustration of breast cancer circulating tumour cells (CTCs) origin and metastasis to the blood-brain barrier (BBB). Illustrations from Server Medical Art (https://smart.servier.com), used under CC BY 4.0 license (https://creativecommons.org/licenses/by.4.0/).