Abstract
Breast cancer (BC) brain metastases is a life-threatening condition to which accounts the poor understanding of BC cells’ (BCCs) extravasation into the brain, precluding the development of preventive strategies. Thus, we aimed to unravel the players involved in the interaction between BCCs and blood–brain barrier (BBB) endothelial cells underlying BBB alterations and the transendothelial migration of malignant cells. We used brain microvascular endothelial cells (BMECs) as a BBB in vitro model, under conditions mimicking shear stress to improve in vivo-like BBB features. Mixed cultures were performed by the addition of fluorescently labelled BCCs to distinguish individual cell populations. BCC–BMEC interaction compromised BBB integrity, as revealed by junctional proteins (β-catenin and zonula occludens-1) disruption and caveolae (caveolin-1) increase, reflecting paracellular and transcellular hyperpermeability, respectively. Both BMECs and BCCs presented alterations in the expression pattern of connexin 43, suggesting the involvement of the gap junction protein. Myosin light chain kinase and phosphorylated myosin light chain were upregulated, revealing the involvement of the endothelial cytoskeleton in the extravasation process. β4-Integrin and focal adhesion kinase were colocalised in malignant cells, reflecting molecular interaction. Moreover, BCCs exhibited invadopodia, attesting migratory properties. Collectively, hub players involved in BC brain metastases formation were unveiled, disclosing possible therapeutic targets for metastases prevention.
Highlights
Breast cancer (BC) represents the most commonly diagnosed and leading cause of neoplastic disease in woman, with more than 2.2 million new cases and 684,996 deaths in 2020 [1]
We took advantage of an in-house improved blood–brain barrier (BBB) in vitro model, which better mimics the in vivo features by encompassing the effect of SS [17], where fluorescently labelled triple negative BC cells (BCCs) were added. Using this cell model of BC brain metastasis formation, we provide evidence regarding signalling molecules involved in BCCs’ migratory phenotype, as well as in their adhesion and transendothelial migration (TEM) across the BBB endothelium, essential for BCC extravasation and BC brain metastasis formation
The results obtained allowed a comprehensive understanding of the players and signalling molecules involved in BCC–brain microvascular endothelial cells (BMECs) interaction, differentiating the molecular alterations occurring in malignant cells from those in BBB endothelial cells’ (ECs)
Summary
Breast cancer (BC) represents the most commonly diagnosed and leading cause of neoplastic disease in woman, with more than 2.2 million new cases and 684,996 deaths in 2020 [1]. BC brain metastases occur in triple negative BC, and represent a poor prognosis condition, with a low rate of patient survival [5,6]. It is known that BC brain metastases formation occurs through a process named the metastatic cascade [7] that involves the exit of BC cells (BCCs) from the mammary ducts (invasion), the entrance of malignant cells in circulation (intravasation), their survival in the bloodstream and their arrival to the target organ, where they can transmigrate (extravasation) through the blood–brain barrier (BBB) and colonise the brain [7]. The extravasation process comprises three major sequential steps, rolling, adhesion and transendothelial migration (TEM) [8], involving several receptors and ligands such as selectins, integrins and members of the immunoglobulin superfamily of cell adhesion molecules [9,10]
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