Molecular Analysis of Breast Cancer Metastasis to the Brain

Division Of Basic Sciences - Nci

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Abstract

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 asked whether the BTB is a chaotic breakdown of the BBB or alternatively has consistent features: Quantitative immunofluorescence of the BTB in three model systems was performed. The relative expression of known 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 consistent 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 presented the first description of the BTB. These data are published in Clin. Cancer Res. 22: 5287-5299, 2016. A subsequent series of experiments identified molecular alterations correlated with BTB permeability using gene expression analysis. Permeable and impermeable brain metastases were laser capture microdissected from mouse brains and both human (tumor cell) and mouse (brain microenvironment) gene expression determined on microarrays. Most of the gene expression changes correlated with metastasis permeability were from the microenvironment, rather than the tumor cells. The sphingosine-1-phosphate receptor 3 (S1P3) was identified as overexpressed in more permeable metastases. This trend was confirmed at the protein level, and S1P3 expression was localized to astrocytes in the neuro-inflammatory response. Using a S1P3 antagonist and S1PR3 gene knockdown, in vitro TEER assays (modeling the BBB and BTB) have shown that reduced S1P3 expression and function causes altered BTB permeability. In vivo a S1P3 antagonist functionally modulated the permeability of the BTB. This research is published in Nature Comm. 9:2705, 2018. Rather than drugs and markers, we addressed the uptake of antibody based therapeutics in experimental brain metastases, using an antibody-drug conjugate (ADC) under development by MedImmune/AZ. The ADC significantly prevented the development of HER2+ brain metastases in one model system and produced a strong trend for prevention in an independent HER2+ model. Uptake of ADC was heterogeneous and low overall, with occasional high expression. Remarkably, ADC uptake was observed in the absence of paracellular permeability, suggesting that a new type of BTB permeability is operative. This work is published in NeuroOncology, 2020. Current work is extending our research past paracellular permeability to transcytotic pathways in brain metastasis. Five transcytotic ligands of potential translational importance were compared in vivo and Far red labeled albumin exhibited BTB distribution without significant BBB uptake. BBB uptake is thought to results in adverse effects on neuronal function. Further investigation of the albumin transcytotic pathway revealed that it is widespread in both large and micrometastases, and uses a clathrin-independent endocytosis mechanism. Future experiments will investigate this and other clathrin-independent ligands for possible linkage to drugs. 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 (Clin. Cancer Res. 20: 2727-2739, 2014). 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 I trial has been conductedin the WMB to test the effect of TMZ on preventing brain metastases in patients with 1-10 brain metastases from HER2+ metastatic breast cancer, having only local brain metastasis treatment (SRS or neurosurgery). Patients received T-DM1 + temozolomide with a primary endpoint of ssafety (ClinicalTrials.gov Identifier: NCT03190967). Current work is investigating molecular correlates of brain metastasis prevention from serum and CSF. An affiliated project on leptomeningeal metastases was partially funded by a DOD grant. Three cell lines have been injected into the ventricle of the brain and complete leptomeningeal spread through the spinal cord. The most interesting line is derived from lobular breast cancer, a subtype with a high level of leptomeningeal spread. Drug screening is ongoing.