Cerebrovascular endothelium cell response to tumor-activated astrocytes in the perivascular stage of the brain metastasis process of HER2-overexpressing breast cancer cells

Brain metastasis is an important cause of death in patients with advanced cancer, and its development begins in a functional microenvironment regulated by cerebrovascular endothelium (CVE) cells and astrocytes of the blood-brain barrier (BBB). However, the molecular pathogenesis of brain metastasis is largely unknown, and there are no molecular biomarkers alerting us on the risk of suffering brain metastasis and that might represent good targets for therapy. The aim of the project was to study the neurovascular microenvironment and the growth pattern of experimental and clinical brain metastasis and the transcriptional activity of the CVE cells in response to soluble factors from cultured astrocytes activated by MDA-231 breast cancer (BC) cells with or without HER2 overexpression. Results showed that the initial growth of both experimental and clinical brain metastases occurred in the perivascular space, with the original BBB being split into two new barriers, an internal hemato-tumoral (between blood and metastatic tissue), and another external neuro-tumoral (between metastatic tissue and unaffected brain tissue). Next, by using an in vitro model recapitulating the functional interrelationship operating in the brain metastasis microenvironment among BC cells, perivascular astrocytes and CVE cells we demonstrated an altered gene expression in CVE cells induced by tumor-activated astrocytes. Interestingly, oxygen deprivation (1% hypoxia), oxidative stress (1 nM hydrogen peroxide), mechanical stimulation (induced by fluid flow) and inflammation (10 ng/ml TNFalpha) activated the production of astrocyte-stimulating factors from HER2-overexpressing BC cells. In turn, conditioned medium of such tumor-activated astrocytes induced a CVE cell transition into a neuroinflammatory phenotype, involving increased gene expression of Claudin-1, VCAM-1 and PECAM-1 (involved in the regulation of intercellular relationships), VEGFR-2, RAGE and Collagen- IV (involved in the regulation of endothelial differentiation) and P-Glycoprotein (involved in the regulation of transcellular molecular exchanges). In conclusion, early metastatic growth in the perivascular microenvironment of the brain remarkably altered astrocyte effects on BBB-forming CVE cells. In addition, overexpression of HER2 determined the functional response of BC cells to the perivascular tumor microenvironment, which in turn affected the action mechanism of BC cells on CVE cells via perivascular astrocytes