Abstract
Introduction: Incidence of breast cancer brain metastasis is increasing owing to prolonged life-span and better detection techniques. Prognosis of patients with brain metastases is extremely poor with median survival time of one year. One of the major impediments in treating brain metastases is presence of blood-brain/tumor barrier that limits the permeability of chemotherapeutic drugs into the brain parenchyma. Understanding the mechanisms that regulate blood-brain/tumor barrier permeability in context of brain metastases is imperative to developing successful therapy.
Methods: Mouse models with brain-tropic sublines of MDA-MB-231 (231), JIMT-1, and SUM190 were used to generate breast cancer brain metastases. Using laser capture microscopy, the permeable and non-permeable lesions from mouse brains were isolated and profiled for gene expression using microarray. Immortalized human brain endothelial cells, astrocytes, and pericytes were used for developing in vitro blood-brain/tumor barrier model along with spheres generated with 231-BR6 or JIMT-1-BR3 cells. Secreted cytokines were evaluated using human cytokine profiler. Transendothelial electrical resistance (TEER) was measured using EVOM2 volt/ohm meter.
Results: Gene expression profiling and immunostaining of mouse brains, harboring breast cancer metastases showed that astrocytes at permeable regions express elevated S1P3. Pharmacological inhibition of S1P3 using antagonist TY-52156 (10mg/kg) in mice bearing 231-BR6 brain metastases showed reduction in 3KDa Texas red dextran (TRD) uptake. To investigate the role of S1P3 in regulating barrier permeability, we established in vitro blood-brain/tumor barrier models. Treatment of astrocytes with TY-52156 (2μM) significantly increased mean TEER values (33.9 to 55.8 Ω.cm2; p<0.001, after 24 hrs), while there was a decrease in permeability for TRD (1.9 fold; p<0.0001) and doxorubicin (1.3 folds; p<0.05). Immunostaining on endothelial monolayer showed increased membranous ZO-1 and VE-cadherin expression. The dynamics of increase in TEER was faster when 231-BR6 spheres were included. We observed similar results when S1P3 was knocked down using shRNA. Astrocytes with down-modulated S1P3 showed decreased secretion of various cytokines including IL-6, IL-8, CCL2, CXCL1, and GM-CSF. Inhibition of these cytokines individually using neutralizing antibodies recapitulated the effects of S1P3 inhibition, while treatment of endothelial monolayer with activated cytokines increased the permeability. This study provides a proof of concept for role of S1P3 and downstream cytokine signaling in regulating blood-brain/tumor barrier permeability in breast cancer brain metastases.
Conclusion: Our study shows that astrocytic S1P3 regulates blood-brain/tumor barrier permeability in breast cancer brain metastases by modulating cytokine secretion. This observation might lead to discovery of novel strategies for augmenting drug efficacy.
Citation Format: Anurag N. Paranjape, Brunilde Gril, Stephan Woditschka, Emily Hua, Jeffrey C. Hanson, Xiaolin Wu, Renata Duchnowska, Priscilla K. Brastianos, David L. Liewehr, Seth M. Steinberg, Cody Peer, William D. Figg, Gary T. Pauly, Christina Robinson, Joel P. Schneider, Patricia S. Steeg. Astrocytic S1P3 regulates blood-brain/tumor barrier permeability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4330. doi:10.1158/1538-7445.AM2017-4330
- ©2017 American Association for Cancer Research.
Volume 77, Issue 13 Supplement
