The paracrine effect of normoxic and hypoxic cancer secretions on blood-brain barrier endothelial cells

Abstract

Cancer is the most common leading cause of death worldwide. Glioblastoma and breast cancer are the most aggressive solid tumour. The survival rate of these tumours depends on their ability to progress and spread. These cancers use their high proliferative capabilities for survival, increasing their malignancies. Glioblastoma is considered the most aggressive tumour initiated in the brain, whereas breast cancer is the most common metastatic cancer in the brain, both types of cancer are known as high infiltrated cancer and their invasiveness due to their capability to release factors that can alter the neighbouring cells to facilitate their progression. On the other hand, the brain remains a vital organ where the blood-brain barrier (BBB) plays a protective and homeostasis role, thus ensuring optimum brain function. The endothelial cells are the functional site of the BBB; these cells, with assistance from other brain cells such as astrocytes and pericytes, maintain the BBB protective function. The literature revealed perturbations and disruption in the BBB integrity in glioblastoma and metastatic breast cancer. The endothelial cells and other components of the BBB have been morphologically altered, resulting in impacting the BBB integrity. The interaction between cancer cells and brain endothelial cells is a complex scenario that is not entirely illustrated; however, a vital role in the crosstalk between cancer cells and endothelial cells is played by signalling mediated by soluble factors that can further promote cancer progression. This study aimed to investigate whether the secretions from glioblastoma and breast cancer cells could influence the brain endothelial cells. Brain endothelial cells (bEnd.3) were cocultured with glioblastoma (U-87) or breast cancer (MCF7) cells or subjected to their conditioned media. To mimic the heterogeneous physiological conditions of the solid tumour such as glioblastoma and breast cancer, U87 and MCF7 cells were cultured under normoxia (to reflect cancer cells exposed to oxygen levels in the perivascular regions of the tumour) or hypoxia (to reflect the hypoxic tumour area where invasive cancer cells are detached and invade the surrounding tissue forward to the blood vessels). Our findings underlined the involvement of paracrine secretion of cancer cells in modulating brain endothelial cells’ properties. Our data suggested that both cancer secretions derived from normoxia and hypoxia rendered changes in the brain endothelial cells at the level of mitochondrial activity. To investigate whether the exerted effects were associated with the acquisition of the brain endothelial resistance, we screened the transelectrical endothelial resistance (TEER) of brain endothelial cells after exposure to cancer secretions. Our findings revealed a decrease in TEER of the exposed brain endothelial cells. Moreover, gene expression of the tight junction (Claudin-5 and Occludin) were quantified in brain endothelial cells (bEnd.3) after exposure to normoxic and hypoxic cancer secretion using real-time qPCR; results showed upregulation of Claudin-5 after exposure to normoxic and hypoxic cancer secretion. Occludin gene expression was also upregulated after exposure to normoxic cancer secretion; however, the exposure to hypoxic cancer secretion decreased Occludin gene expression. In addition, the proliferation of endothelial cells (bEnd.3) exposed to cancer secretion was measured by cell counting, followed by analyzing the cell cycle; results showed that long term exposure to cancer secretion suppressed the proliferation of brain endothelial cells (bEnd.3), mostly after exposure to hypoxic cancer secretion. Cells were accumulated in the G1 phase. Overall, data suggest that factors secreted from normoxic and hypoxic cancer cells modulate brain endothelial cells, affecting their function.