Bioengineering a Blood-Brain-Barrier on-Chip to Investigate the Effect of Mechanical Shear Stress on Metastatic Invasion of the Brain

Brain metastasis is an important cause of morbidity and mortality in cancer patients. Sadly, once diagnosed with brain metastasis, the median survival of untreated patients is shorter than 2 months. Unfortunately, our understanding of cell and molecular mechanisms responsible for brain invasion of metastatic cancer is poor and such lack of knowledge limits the possible therapeutic interventions. While it is well understood that certain tumor cells have a specific affinity for the microenvironment of the brain tissue, it is unclear what are the factors that determine such preference. Interestingly, several emerging lines of evidence support the possibility that cancer cells could be able to hijack the physiological platelet-function to evade immune cell surveillance and to penetrate target tissues. Although soluble and transcriptional factors have been identified as relevant determinant of the metastatic invasion of the brain, the puzzle is still unsolved. Interestingly, the shear stress, caused by the natural and continuous blood flow through the vessels of the human body, seems to also play a relevant role. Metastatic cells originated from different organs including lung, intestine, and skin, all need to adapt to the highly dynamic environment of the endothelium in order to navigate their way through the blood torrent and adhere to and invade the target tissue. Common in vitro models do not incorporate fluid flow and fail to reconstitute the vascular shear force that is known to regulate both platelet-function as well as cancer cells extravasation. At the same time, animal models typically used in preclinical testing, cannot be always used as translational models for the study of platelet-function. For instance, rodents and humans present remarkable differences in terms of platelet response to inflammatory and pro-thrombotic stimuli, which reflects the inadequate prediction of animal studies around platelet function. Such limitation of existing models represents a major obstacle to the identification of strategic molecular targets and determinants of the metastatic progression to the brain. Human “Organs-on-Chips” are microengineered fluidic systems typically fabricated using microfabrication techniques, such as soft lithography or 3D printing, and populated with human cells in a dynamic microenvironment that emulates tissue-tissue interactions and organ-level function. This project aims to explore the contribution of mechanical shear stress on the arrest and the extravasation of circulating tumor cells in the presence of human platelets. We will use advanced tissue engineering methodologies to reconstitute a functional BBB-on-Chip (Fig.1). To investigate the possible contribution of platelets to the metastatic invasion of the neural compartment, the BBB-on-Chip will be perfused with metastatic cell lines obtained from different organs and in combination with human platelets. We will also assess the impact of different mechanical shear rates on the metastatic cell arrest, adhesion to the endothelium and extravasation into the neural compartment. We will apply fluorescence imaging techniques to assess and quantify the possible formation of cancer-platelets complexes and fibrin deposition as well as cancer cell adhesion to the vascular endothelium and invasion of the neural compartment.

Digram showing Multi-channel human organ-chip model of the Blood Brain Barrier

Multi-channel human organ-chip model of the Blood Brain Barrier. (A) The BBB-on-Chip is made of an optically clear, flexible, silicone rubber polymer the size of a computer memory stick with two tiny hollow channels running parallel along its length separated by a flexible porous membrane of the same material (channels are filled with blue and pink dyes). (B) Diagrammatic cross-sections of the BBB-on-Chip showing how living human glial cells (Astrocytes and Pericytes) is cultured on top of the porous membrane, which is coated with a thin layer of extracellular matrix (blue), while human microvascular endothelium is grown on the opposite side of the same membrane under continuous medium flow (red). (C) Tridimensional rendering of fluorescent images obtained via confocal microscopy showing the hollow vessel-like geometry of the vascular compartment (bottom) while the presence of glial cells is confined to the top compartment. (D) Glial cells, astrocytes and pericytes, grow on flat surface (2D) and maintain tissue-specific markers such as GFAP (red) and αSMA (green). (E) stem cell derived endothelial cells growing on the opposite surface of the porous membrane form a continuous and compact cell monolayer lined with tight junctional markers (such as ZO1, green). Reference: Cell Stem Cell. 2019 Jun 6;24(6):995-1005.e6. doi: 10.1016/j.stem.2019.05.011

Headshot of Riccardo Barrile

Riccardo Barrile

Asst Professor, CEAS - Biomedical Eng

Engineering Research Cntr