Fig. 4

BLECSs treated with serum from patients with cerebral metastases show decreased barrier properties. BLECS were co-cultured with brain pericytes for 6 days in a transwell system. a BLECs were treated with the control sera (C) and sera from breast cancer patients with cerebral metastases (CM) for 24 h followed by measuring the paracellular permeability for fluorescein. Data are shown as mean ± SD of three independent experiments, *p < 0.05. b Immunostaining of claudin-5 in BLECs co-cultured with pericytes and treated either with serum from healthy donors (C) or breast cancer patients with cerebral metastases (CM). The arrow indicates loss of claudin-5 staining at the tight junctions of BLECs treated with serum from breast cancer patients with CM. Shown is one representative image of n = 6. Magnification ×400, green: claudin-5, blue: DAPI nuclear staining. c Quantification of cells with intact claudin-5 junctions presented in B. d Messenger RNA expression of chemokines and BBB-markers in BLECs treated with control serum and serum from breast cancer patients with cerebral metastases. The mRNA was quantified by qPCR. The target gene expression was normalized to endogenous control and shown as fold over control, which was arbitrarily set as 1 (control level marked in graph). Data are shown as mean ± SD of n = 4 (control sera) and n = 5 (CM). **p < 0.01 statistically significant versus control. C-X-C Motif Chemokine Receptor 5 (CXCR5), C-X3-C Motif Chemokine Receptor 1 (CX3CR1)