(72h) Evaluating the Role of Endothelial to Mesenchymal Transition in Breast Cancer Extravasation into the Brain

The brain is a common site for breast cancer metastasis, despite the fact that the blood-brain barrier (BBB), comprised of brain-specific microvascular endothelial cells (BMECs) linked together tight junction and adherens junction proteins, highly restricts the transport of molecules and cells into the brain. The mechanisms by which breast cancer cells are able to overcome this barrier and transmigrate into the brain remain poorly understood. It has been hypothesized that one mechanism through which cancer cells are able to extravasate across endothelial barriers is by inducing an endothelial-to-mesenchymal transition (EndoMT), a process that involves transient de-differentiation of endothelial cells to a mesenchymal phenotype. The main inducers of EndoMT are inflammatory cytokines such as TGFβ, IL-1β, and TNF-α. Thus, we sought to evaluate the ability of inflammatory cytokines to induce EndoMT in human induced pluripotent stem cell-derived BMECs, which appropriately mimic the high barrier properties of the in vivo BBB, and the consequent extravasation of breast cancer cells. Our results demonstrated that inflammatory cytokines that are secreted by a brain-seeking metastatic variant of the MDA-MB-231 human breast cancer cell line (231BR) were able to downregulate expression and localization of tight junction proteins claudin-5 and ZO-1, resulting in a decrease in trans-endothelial electrical resistance (TEER) after 6 hours of treatment. After 3 days of treatment with inflammatory cytokines, expression of endothelial markers vWF and CD31 was downregulated, while expression of mesenchymal marker SM22-α was upregulated, which are hallmarks of EndoMT. Interestingly, when astrocytes were placed on the brain side of the BMEC monolayer (i.e. below it), addition of inflammatory cytokines above the BMEC monolayer (i.e. on the blood side) resulted in activation of the astrocytes, as evidenced by upregulated expression of GFAP as well as several chemokines and cytokines (IL-1β, TNF-α, CCL5, and CX3CL1). EndoMT marker expression in the BMECs was also affected, indicating that astrocyte activation may influence EndoMT. Next, using a BBB-on-a-chip platform, we showed that MDA-MB-231 and 231BR cells were able to adhere and transmigrate across the BMEC barrier. Addition of astrocytes to the device in a hydrogel below the BMEC monolayer increased the rate of transmigration of the breast cancer cells to the brain, which could be a consequence of both increased chemotaxis of the breast cancer cells and further promotion of EndoMT due to the chemokines and cytokines secreted by the activated astrocytes. Taken together, these results demonstrate that inflammatory cytokines associated with EndoMT that are secreted by breast cancer cells may aid their transmigration into the brain through both induction of EndoMT in the endothelial cells and activation of astrocytes within the brain.