Picturing Breast Cancer Brain Metastasis Development to Unravel Molecular Players and Cellular Crosstalk

SIMPLE SUMMARY: Breast cancer is a devastating disorder affecting millions of women worldwide. With improved therapeutics for the primary tumor, the appearance of metastasis has been increasing. Breast cancer frequently metastasizes to the brain, constituting a major hurdle without cure and with a poor survival. It is imperative to better understand the mechanisms involved in malignant cell transposition of the brain microvasculature and parenchymal colonization by deciphering the alterations occurring in the tumor and microvascular cells, as well as the occurrence of intercellular communication during the process. We aimed to profile the process of the formation of breast cancer brain metastasis and the timeline of events governing it. We used a specific mouse model of the disease to perform extensive microscopic analyses. We identified phenotypic changes and the activation of relevant molecular players in tumorigenesis, together with vascular alterations, and the occurrence of crosstalk. Our findings unravel putative therapeutic targets to tackle breast cancer brain metastasis. ABSTRACT: With breast cancer (BC) therapy improvements, the appearance of brain metastases has been increasing, representing a life-threatening condition. Brain metastasis formation involves BC cell (BCC) extravasation across the blood–brain barrier (BBB) and brain colonization by unclear mechanisms. We aimed to disclose the actors involved in BC brain metastasis formation, focusing on BCCs’ phenotype, growth factor expression, and signaling pathway activation, correlating with BBB alterations and intercellular communication. Hippocampi of female mice inoculated with 4T1 BCCs were examined over time by hematoxylin-eosin, immunohistochemistry and immunofluorescence. Well-established metastases were observed at seven days, increasing thereafter. BCCs entering brain parenchyma presented mesenchymal, migratory, and proliferative features; however, with time, they increasingly expressed epithelial markers, reflecting a mesenchymal–epithelial transition. BCCs also expressed platelet-derived growth factor-B, β(4) integrin, and focal adhesion kinase, suggesting autocrine and/or paracrine regulation with adhesion signaling activation, while balance between Rac1 and RhoA was associated with the motility status. Intercellular communication via gap junctions was clear among BCCs, and between BCCs and endothelial cells. Thrombin accumulation, junctional protein impairment, and vesicular proteins increase reflect BBB alterations related with extravasation. Expression of plasmalemma vesicle-associated protein was increased in BCCs, along with augmented vascularization, whereas pericyte contraction indicated mural cells’ activation. Our results provide further understanding of BC brain metastasis formation, disclosing potential therapeutic targets.