Brain metastases of breast cancer are thought to increasing in incidence, particuarly among metastatic patients with Her-2+ or triple negative tumors, and confer a dismal prognosis. Our goal is to understand the nature of the blood-tumor barrier (BTB), the endothelial-associated structure that results when the formation of a metastasis disturbs the normal blood-brain barrier (BBB). In initial studies the paracellular permeability of the BTB was tested in experimental brain metastasis model systems in mice, using dyes and drugs. Published collaborative research showed that experimental brain metastases of breast cancer were heterogeneous in their permeability, both within and between metastases in the same brain. While most metastases were permeable as compared to the normal BBB, only 10% exhibited sufficient permeability to enable a cytotoxic response to a systemic drug. We have completed a quanitative immunofluorescence study of the BTB in three model systems. The relative expression of BBB components was compared between normal (uninvolved) brain and brain metastases, and also between brain metastases that were either poorly or highly permeable to Texas red dextran. Many alterations were observed between normal brain and the BTB, including endothelial cell size, neuro-inflammation, absence of astrycyte endfeet, increased VEGF, etc. In contrast, few differences were observed between poorly and highly permeable metastases. The relative expression of subpopulations of pericytes was the major difference observed. Highly permeable lesions exhibited an increase in Desmin+ pericytes and a decrease in CD13+ pericytes. This work presents the first description of the BTB and the one of the first markers of higher permeability. To investigate the role of pericyte subpopulations we have set up and validated in vitro assays of the BBB and BTB, using either electrical resistance between the brain endothelial cells (TEER) or passage of drug as endpoints. These models use confluent brain endothelial cells on one side of a coated membrane (luminal) with pericytes on the abluminal side, and astrocytes +/- tumor spheres in the bottom of the wells. Pericytes were derived from normal brain and were 100% CD13+. Twelve different culture conditions were attempted to increase pericyte desmin expression, the best of which was co-culture with astrocytes. When CD13+ and Desmin+ pericyte subpopulations were separated by FACS and applied to TEER assays, they showed distinct effects on permeability. The data suggest that pericyte subpopulations functionally contribute to the permeability of the BTB. Future research will test this hypothesis in vivo, and investigate translational leads to elevate desmin+ pericyte expression. A second series of experiments aimed to identify additional alterations correlated with BTB permeability using gene expression analysis. Permeable and impermeable brain metastasses have also been identified in frozen sections of mouse brains; using the adjacent section the material was laser-capture microdissected. RNA extracted from these lesions was hybridized to both human (tumor cell genes) and mouse (brain microenvironment genes) microarrays. The goal of these experiments are to identify differentially expressed genes between permeable and impermeable lesions.All are from the microenvironment, rather than the tumor cells. This project is expected to provide an agnostic analysis of permeability in brain metastases. the sphingosine-1-phosphate receptor 3 (S1PR3) was identified as being overexpressed in more permeable metastases. This trend was confirmed at the protein level, and S1PR3 expression was localized to astrocytes in the neuro-inflammatory response. Using a S1PR3 antagonist and S1PR3 gene knockdown, TEER assays have shown that reduced S1PR3 expression and function causes alterred BTB permeability (manuscript in preparation). In vivo testing is underway. We published experimental brain metastasis data indicating that temozolomide, a first line treatment for primary brain tumors, was 100% effective at preventing brain metastases of 231-BR cells over a two log dose response. This type of profound prevention has not been previously observed. Given later when brain metastases are already at least partially established, temozolomide was ineffective. Activity was dependent on methylguanine methyltransferase (MGMT). Staining of matched sets of primary breast cancers and resected brain metastases showed poor concordance, but 60% of the brain metastases were low in MGMT. A phase II trial is being developed in the WMB to test the effect of TMZ on preventing brain metastases in patients with 1-5 brain metastases from HER2+ metastatic breast cancer, having only local treatment.
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