SCDT-12. IN VITRO STUDY OF ROLE OF ASTROCYTIC S1P3 IN REGULATING BLOOD-BRAIN/TUMOR BARRIER PERMEABILITY

摘要

Incidence of breast cancer brain metastasis is increasing. Prognosis of patients with brain metastases is extremely poor. One of the major impediments in treating brain metastases is the presence of a blood-brain/tumor barrier that limits the permeability of chemotherapeutic drugs into the brain parenchyma. Blood-tumor barrier (BTB) has been shown to be heterogenous, and only at highly permeable lesions do drugs reach the tumor cells. To explore the molecular differences between highly and poorly permeable BTBs, gene profiling was done on laser microdissected brain tissues from mice bearing breast cancer brain metastases. Sphingosine-1-phosphate receptor 3 (S1P3) was overexpressed in astrocytes at highly permeable lesions. To explore the functional role of S1P3 in BTB permeability we established an in vitro blood brain/tumor model. Inhibition of S1P3 using a specific chemical inhibitor TY-52156 (2µM) significantly increased barrier integrity as shown by increase in transendothelial electrical resistance (TEER) in BBB (1.3 folds, p<0.05) and BTB (1.45 folds, p<0.001). Additionally, there was a significant decrease in permeability for doxorubicin in BBB (1.78 folds, p<0.01) and BTB (2.1 folds, p<0.01), with concomitant increase in membranous ZO-1 and VE-cadherin. Increase in TEER was dependent on astrocytes. Similar results were observed when S1P3 was stably knocked-down in astrocytes. As astrocytes were grown away from the endothelial monolayer, we hypothesized that astrocyte-secreted molecules regulated barrier integrity. Analysis of supernatant from astrocytes revealed that various cytokines including IL-6, IL-8, CCL2, CXCL1, and GM-CSF were downmodulated upon S1P3 knockdown. Inhibition of IL-6 and CCL2 individually using neutralizing antibodies recapitulated the effects of S1P3 inhibition. This study provides a proof of concept for role of S1P3 and downstream cytokine signaling in regulating BTB permeability in breast cancer brain metastases which might lead to discovery of novel strategies for augmenting drug efficacy.