Cancer-associated fibroblast promote transmigration through endothelial brain cells in three-dimensional in vitro models

Brain metastases are associated with high morbidity as well as with poor prognosis and survival in breast cancer patients. Despite its clinical importance, metastasis of breast cancer cells through the blood-brain barrier (BBB) is poorly understood. The objective of our study was to investigate whether cancer-associated fibroblasts (CAFs) play crucial roles in breast cancer brain metastasis. Using a cell adhesion assays, in vitro BBB permeability and transmigration assays and soft agar colony formation assays, we investigated the physical roles of CAFs in breast cancer brain metastasis. We also performed immunofluorescence, flow cytometric analysis, Droplet Digital PCR and Simon™ Simple Western System to confirm changes in expression levels. We established two novel three-dimensional (3D) culture systems using a perpendicular slide chamber and applying 3D embedded culture method to reflect brain metastasis conditions. With a newly developed device, CAFs was proven to promote cell adhesion to human brain microvascular endothelial cells, in vitro BBB permeability and transmigration and colony formation of breast cancer cells. Furthermore, CAFs enhanced the invasive migration of breast cancer cells in two kinds of 3D cultures. These 3D models also reliably recapitulate the initial steps of BBB transmigration, micro-metastasis and colonization. Expression of integrin α5β1 and αvβ3, c-MET and α2,6- siayltransferase was increased in breast cancer cells that migrated through the BBB. In conclusion, based on our in vitro BBB and co-culture models, our data suggest that CAFs may play a role in breast cancer brain metastasis. What's new? Metastasis of breast cancer cells through the blood-brain barrier (BBB) remains poorly understood. This study provides mechanistic insight for cancer-associated fibroblasts (CAFs)-directed breast cancer cell metastasis and colonization, based on integration of biological and physical properties of stromal cells using in vitro 3-D models and co-cultures mimicking brain metastasis. The results support the hypothesis that CAFs mediate BBB disruption and transmigration of breast cancer cells via enhanced vascular permeability, thus facilitating brain metastasis. Uncovering the changes of the BBB system during tumor cell invasion may lead to better understanding of the brain tumor microenvironment and development of novel therapeutic targets.