The integrity of the BBB is a key feature responsible for protecting the brain from harmful substances, regulating entry and efflux of macromolecules and immune cells to and from the brain, and maintaining homeostasis of the CNS [
32,
33]. Inflammation can affect the barrier properties of the BBB. However, clinical and in vitro data on inflammation and underlying pathomechanisms at the BBB in breast cancer patients with cerebral metastases are limited. Therefore, we designed the present study to analyse serum-derived chemokines in breast cancer patients with different cancer characteristics and to test the effects of patient sera on the BBB properties in vitro.
We utilized the CD34
+ cells-derived in vitro BBB model first published by Cecchelli et al. in 2014 [
12]. Similar to the first report on this model, we observed the induction of TJ proteins, multiple transporters and cellular receptors either at the protein or mRNA level when the cells were co-cultured together with pericytes. Pericytes have been chosen, because they induce BBB characteristics in CD34
+ cells-derived hematopoietic stem cells [
12] and they are known to play a role in the maturation and stabilization of the BBB [
34,
35]. Induction of TJ-protein expression was accompanied by low paracellular permeability for fluorescein. The permeability values were similar to previously published values for this [
12] and also for other in vitro BBB models [
36]. However, fluorescein is a transport substrate for organic anion transporter 3 (OAT3) and multidrug resistance protein 2 (MRP2) [
37]. These two transporters act in the mammalian BBB in the basolateral-to-apical direction, therefore they limit the apical-to-basolateral flux of fluorescein. This could lead to an underestimation of the paracellular fluorescein diffusion in our model system. However, the differences between the control and the treated cells should still be clearly visible. It can be assumed that a 6 day-co-culture with brain pericytes is sufficient to induce BBB properties in CD34
+ cells-derived hematopoietic stem cells. Differentiated BLECs can be used to study molecular mechanisms underlying brain disorders, such as brain metastases.
In general, inflammatory mediators are mainly expressed by macrophages such as microglia, but also by astrocytes, oligodendrocytes and vascular endothelial cells [
38]. Numerous mediators play a role in this out-balanced process, including anti-inflammatory cytokines, pro-inflammatory cytokines, chemokine-ligands and receptors. A correlation between cancer metastases and cytokine expression is discussed for various tumour entities [
39,
40], including breast cancer [
41,
42]. Therefore, we examined chemokines in the sera of breast cancer patients with and without cerebral metastases in comparison to healthy donors. Among the chemokines tested, CX3CL1 and CXCL13 were selectively and significantly increased in patients with cerebral metastases of breast cancer. The chemokines levels correlated with the tumour properties and the presence of other chemokines in patient sera. We identified a statistically significant correlation between RANTES and MCP-1 as well as BCA-1 and CCL20. RANTES and MCP-1 co-expression in breast cancer was associated with more advanced stages of the disease [
43]. High CCL20 levels contribute to the formation of bone metastases in breast cancer [
44]. CX3CL1 (also known as fractalkine or neurotactin) is a membrane-bound chemokine that can facilitate intercellular interactions, interacts with the TNFα-converting enzyme ADAM17 and is released in its shed form by apoptotic cells to recruit professional phagocytes to the site of cell death [
45,
46]. Fractalkine serum concentrations were higher in patients with the ER/PR negative tumours, which is in line with the literature. Andre et al. showed in a study with 142 patients that a high CX3CL1 expression in the primary cancer correlates with brain metastases in a 13-year median-follow up [
47]. Similarly, Tsang et al. postulated that CX3CL1 expression is associated with poor outcome in breast cancer patients [
48]. High levels of CX3CL1 in cells can attract those cancer cells expressing its receptor CX3CR1 and trigger them to invade the tissue and form metastases as seen e.g. in breast cancer spinal metastases [
49]. In our study, we showed that CX3CL1 is selectively elevated in the serum of breast cancer patients with cerebral metastases. CX3CL1 may therefore be involved in the formation of metastases in the brain, but further investigations are needed to fully elucidate the underlying mechanisms. Another elevated chemokine in the serum of breast cancer patients with brain metastases was CXCL13 (also known as B cell-attracting chemokine 1, BCA-1). Our results are consistent with other reports showing elevated BCA-1 serum concentrations in patients with metastatic disease [
50]. A negative correlation between BCA-1 and the histological grading in patients with brain metastases indicates that high BCA-1 serum concentrations can lead to brain metastases from moderately differentiated tumours with low histological grading. However, our analysis is limited by a small number of samples. In breast cancer cell lines, CXCL13 induced changes of epithelial-to-mesenchymal transition marker expression. It upregulated vimentin, Snail, Slug, N-cadherin, MMP9 and RANKL and downregulated E-cadherin [
51]. The endothelial-to-mesenchymal transition of brain endothelial cells has been described [
52] and could also play a role in our in vitro model. We detected the mRNA of CXCR5 and CX3CR1 in BLECs, which are chemokine receptors for BCA-1 and fractalkine, respectively. The presence of mRNA expression of these receptors also indicates protein expression in BLECs. The receptors can bind BCA-1 and fractalkine present in sera or cell culture medium and transmit their signals. However, the cells treated with recombinant BCA-1 and fractalkine showed an increase in paracellular permeability only at high chemokine concentrations, with no effects in the concentration range observed in patient sera. This suggests that the BCA-1 and fractalkine require other synergistic factors in serum to affect the barrier. Induction of CXCL13 and CX3CL1 accompanied by compromised barrier integrity, has been reported in brain endothelial cells that overexpress claudin-1 and during ischemia/reperfusion injury in animal models [
53]. It appears that CXCL13 and CX3CL1 are part of the endothelial inflammatory phenotype that plays a role in various cellular processes.
One of the limitations of our study may be the moderate number of serum samples taken from breast cancer patients, especially those with cerebral metastases. It will be interesting to increase the number of samples for chemokine analyses and to further evaluate serum effects on BBB properties. In vitro BBB models are valuable tools for studying cellular responses to factors such as patient sera. However, we need to consider that these are model systems that do not fully restore the microenvironment present in the human body.