Changes in the blood-brain barrier properties in vitro after treatment with sera from breast cancer patients with primary cancer and visceral, bone or cerebral metastases.

Abstract

2026 Background: The progression of brain metastases during breast cancer correlates with poor overall survival, but also with a reduced quality of life. During the metastatic progression of breast cancer, the key event for entry into the brain is the migration of cancer cells across the blood-brain barrier (BBB). To date, it is still controversial which serum factors play a role in cerebral expansion and what effects they have on the BBB. We hypothesize that sera from breast cancer patients with cerebral metastases contain unique factors that can affect BBB integrity. Methods: We used the CD34+ cells-derived human in vitro BBB model (brain-like endothelial cells, BLECs) in co-culture with brain pericytes, which was validated in our previous studies, to analyse the BBB properties after patient sera treatment. We used paracellular permeability measurements for fluorescein (400 Da), immunofluorescence staining, Western blot and mRNA analysis to examine the effects of patient sera on the properties of BBB in vitro. We collected serum samples from five patient cohorts (30 patient per group planned/150 in total/ current recruited patients 130): 1) healthy donors (current recruited patients- 23), 2) breast cancer patients with primary cancer (recruited patients- 30), 3) breast cancer patients with bone metastases (recruited patients- 29), 4) visceral metastases (recruited patients- 30), or 5) cerebral metastases (current recruited patients- 18). We then used 2% patient sera in cell culture medium to treat the cells for 24 hours. Results: Sera from breast cancer patients with cerebral and bone metastases led to a significant increase in the paracellular permeability for fluorescein. This could also be visualized by immunostaining cells with anti-claudin-5 antibody. Tight junction protein claudin-5 and occludin mRNA was reduced in BLECs, while mRNA of efflux pumps Breast cancer resistance protein (BCRP) and P-glycoprotein (P-GP) was induced in BLECs treated with serum from breast cancer patients with primary cancer, cerebral and visceral metastases. At the protein level, efflux pumps BCRP and P-GP were induced in cells treated with sera from breast cancer patients with cerebral metastases, while they were reduced in cells treated with sera from breast cancer patients with bone metastases. Conclusions: Sera from breast cancer patients with primary cancer and breast cancer metastases have an effect on the integrity of BBB in vitro. Reduced barrier properties of brain endothelial cells can contribute to the formation of cerebral metastases in advanced stages of breast cancer. Keywords: metastatic breast cancer, blood-brain barrier, in vitro models