Modeling hyperosmotic blood-brain barrier opening within human tissue-engineered in vitro brain microvessels.

As the majority of therapeutic agents do not cross the blood-brain barrier (BBB), transient BBB opening (BBBO) is one strategy to enable delivery into the brain for effective treatment of CNS disease. Intra-arterial infusion of the hyperosmotic agent mannitol reversibly opens the BBB; however, widespread clinical use has been limited due to the variability in outcomes. The current model for mannitol-induced BBBO assumes a transient but homogeneous increase in permeability; however, the details are poorly understood. To elucidate the mechanism of hyperosmotic opening at the cellular level, we developed a tissue-engineered microvessel model using stem cell-derived human brain microvascular endothelial cells (BMECs) perturbed with clinically relevant mannitol doses. This model recapitulates physiological shear stress, barrier function, microvessel geometry, and cell-matrix interactions. Using live-cell imaging, we show that mannitol results in dose-dependent and spatially heterogeneous increases in paracellular permeability through the formation of transient focal leaks. Additionally, we find that the degree of BBB opening and subsequent recovery is modulated by treatment with basic fibroblast growth factor. These results show that tissue-engineered BBB models can provide insight into the mechanisms of BBBO and hence improve the reproducibility of hyperosmotic therapies for treatment of CNS disease.

Controlled Three-Dimensional Tumor Microenvironments Recapitulate Phenotypic Features and Differential Drug Response in Early vs Advanced Stage Breast Cancer.

Progression to advanced stage metastatic disease, resistance to endocrine therapies, and failure of drug combinations remain major barriers in the breast cancer therapy. Tumor microenvironments play an important role in progression from non-invasive to invasive disease as well as in response to therapies. Development of physiologically relevant, three-dimensional (3D) controlled microenvironments that can reliably recapitulate tumor progression from the early non-invasive to advanced metastatic stage will contribute to our understanding of disease biology and serve as a tool for screening of drug regimens targeting different disease stages. We have recently engineered physicochemical microenvironments by precisely controlling the size of 3D microtumors of non-invasive T47D breast cancer cells. We hypothesized that the precise control over physiochemical microenvironments will generate unique molecular signatures in size-controlled microtumors (small 150 µm vs large 600 µm) leading to differential phenotypic features and drug responses. The results indicated that large (600 µm) T47D microtumors exhibited traits of clinically advanced tumors such as hypoxia, reactive oxygen species, mesenchymal marker upregulation and collective cell migration unlike non-hypoxic, non-migratory small microtumors (150 µm). Interestingly, large microtumors also lost estrogen receptor alpha (ER-α) protein, consequently showing resistance to 4-hydroxytamoxifen (4-OHT). On the other hand, large microtumors showed upregulation of pro-angiogenic marker, vascular endothelial growth factor (VEGF), and hence were more responsive than small microtumors to the growth inhibition by anti-VEGF antibody. Surprisingly, both small and large microtumors exhibited comparable levels of phosphorylated epidermal growth factor receptor (pEGFR) and downstream signaling molecules such as AKT. As a consequence, both small and large microtumors showed comparable growth inhibition in response to gefitinib (inhibitor preferentially targeting EGFR) independent of microtumor size. Thus, precise control over the microenvironmental factors successfully recapitulated molecular characteristics underlying early vs advanced stage disease using the same non-invasive T47D cells. Such unique molecular signatures further resulted in differential response of small and large microtumors to anti-estrogen, and anti-VEGF treatments with comparable response to the EGFR-targeted therapies, underlining the importance of such stage-specific disease progression models in cancer drug discovery.