The Role of Breast Cancer-Derived Extracellular Vesicles in Brain Metastasis

Abstract

Breast cancer brain metastasis is a major clinical challenge that is often associated with an extremely poor prognosis. Current diagnostics lack the sensitivity to detect early stages of brain metastasis and available therapeutics have failed to improve the outcome of this disease. Elucidating the mechanisms underlying the early stages of brain metastasis development is, therefore, of high significance in informing the development of efficient diagnostics and therapeutics for breast cancer brain metastasis. Tumor-derived extracellular vesicles (EVs) are known as intercellular communicators that can transfer a variety of proteins and genetic materials between cells within the tumor microenvironment and in distant pre-metastatic organs and promote tumor progression and metastasis. However, the current understanding of the role of EVs in breast cancer brain metastasis is limited. In my dissertation research, I investigated the role of breast cancer-derived EVs in the early stages of brain metastasis development. Comparing the EVs derived from a parental breast cancer cell line (P-EVs) and a brain-seeking variant of these cells (Br-EVs), I demonstrate that Br-EVs, but not P-EVs, promote brain metastasis incidence and growth in a mouse model of brain metastasis. I further demonstrate that Br-EVs have the ability to breach an intact blood brain barrier (BBB) and using state-of-the-art models of the BBB, we identify transcytosis as the mechanism underlying this transport. Moreover, I show that Br-EVs have the ability to circumvent the low rate of transcytosis at the blood brain barrier, through down-regulating the expression of rab7 in brain endothelial cells. Next, I demonstrate that following their transcytosis, Br-EVs are predominantly internalized by astrocytes. Astrocytes exhibit a preference to uptake Br-EVs when compared to P-EVs and this uptake occurs through a noncanonical clathrin-independent carriers/GPI-AP enriched compartments (CLIC/GEEC) pathway. Through quantitative proteomics and validation studies, we demonstrate that Br-EVs are highly enriched in GPI-interacting proteins which can drive their uptake by astrocytes. Next, through a series of in vitro and in vivo studies, I demonstrate that Br-EVs, but not P-EVs, decrease the expression of TIMP-2 in astrocytes. TIMP-2 is an endogenous inhibitor of matrix metalloproteinases and its down-regulation can be associated with the increased activity of these matrix-modulating enzymes, leading to extracellular matrix remodeling, a major component of pre-metastasis niche preparation. I further demonstrate that this down-regulation of TIMP-2 is driven by miR-301a-3p, transferred by Br-EVs to astrocytes. Taken together, these studies identify, for the first time, the mechanisms by which breast cancer-derived EVs breach an intact blood brain barrier and elucidate some of the functional consequences associated with EV entry into the brain. These studies also identify novel targets that can guide the development of early diagnostics and efficient therapeutics for breast cancer brain metastasis.