In vitro models of molecular and nano-particle transport across the blood-brain barrier

The blood-brain barrier (BBB) is the tightest endothelial barrier in humans. Characterized by the presence of tight endothelial junctions and adherens junctions, the primary function of the BBB is to maintain brain homeostasis through the control of solute transit across the barrier. The specific features of this barrier make for unique modes of transport of solutes, nanoparticles, and cells across the BBB. Understanding the different routes of traffic adopted by each of these is therefore critical in the development of targeted therapies. In an attempt to move towards controlled experimental assays, multiple groups are now opting for the use of microfluidic systems. A comprehensive understanding of bio-transport processes across the BBB in microfluidic devices is therefore necessary to develop targeted and efficient therapies for a host of diseases ranging from neurological disorders to the spread of metastases in the brain

A model for the blood-brain barrier and its application in modeling metastasis to the brain

The blood-brain barrier (BBB) is known to be one of the least permeable portions of the vascular system, serving to protect the central nervous system from agents deleterious to the brain while also preventing or limiting the passage of drugs to treat neurological diseases. Despite this, the brain is recognized to be one of the primary sites of metastasis, especially for lung and breast cancers. While animal experiments have proven useful, realistic models of the BBB using human cells are limited and remain a subject of intense research. Work over the past several years has led to several promising systems, although it has proven difficult to achieve levels of permeability comparable to those observed in vivo. We have recently sought to develop several systems, first with a mixture of primary rat and human cells (Adriani et al., 2017), and more recently, a model based entirely on human cells. This latter system relies on self-assembly of three critical cell types: human iPSC-derived endothelial cells, primary brain pericytes, and primary brain astrocytes. When suspended in a fibrinogen and thrombin gel solution and introduced to a microfluidic platform, the cells organize into a microvascular network in direct physical contact with pericytes and astrocyte end-feet. Over 7 days, the network forms and attains a relatively stable state that permits perfusion from the side channels of the device. Once formed, the vascular network is characterized by immunofluorescent imaging, RT-PCR analysis, and functional measures such as permeability to fluorescent probes of various molecular weights. In comparing the networks as they increase in cellular complexity from endothelial cells alone, to ECs and pericytes, and finally, ECs, PCs and astrocytes, the expression of basement membrane and junctional proteins is seen to increase. Simultaneously, the network permeability is observed to progressively fall, indicating improved barrier function. Permeability values for a 10 kDa FITC-dextran are found to be comparable to those measured in the rat brain. Preliminary experiments have been conducted to determine the rates of extravasation of circulating tumor cells past the model BBB. These experiments introduce breast cancer cells (MDA-MB-231) into the microvascular network with the flow of medium where they adhere to the endothelium or arrest due to physical trapping. Once immobilized, the cells are observed over time to quantify their tendency to undergo transendothelial migration. Results show that as the CTCs are increasingly invasive as the system complexity increases (mono-culture to tri-culture), opposite to what would be expected if tumor cells follow the trend of permeability. Studies are currently underway to understand the cell-cell interactions that give rise to this counterintuitive behavior. Reference: Adriani, G., Ma, D., Pavesi, A., Kamm, R. D., & Goh, E. L. K. (2017). A 3D neurovascular microfluidic model consisting of neurons, astrocytes and cerebral endothelial cells as a blood–brain barrier. Lab on a Chip, 17 (12), 2067-2075 Acknowledgements: This work was supported by an Ermenegildo Zegna Founder’s scholarship, an MIT-POLITO grant (BIOMODE - Compagnia di San Paolo), the Cure Alzheimer\u27s Fund, the Japan Society for the Promotion of Science, the US National Science Foundation (CBET-0939511), the National Cancer Institute (U01 CA202177), and an MIT and POLITO MITOR project (NANOCAB)

3D self-organized microvascular model of the human blood-brain barrier with endothelial cells, pericytes and astrocytes

The blood-brain barrier (BBB) regulates molecular trafficking, protects against pathogens, and prevents efficient drug delivery to the brain. Models to date failed to reproduce the human anatomical complexity of brain barriers, contributing to misleading results in clinical trials. To overcome these limitations, a novel 3-dimensional BBB microvascular network model was developed via vasculogenesis to accurately replicate the in vivo neurovascular organization. This microfluidic system includes human induced pluripotent stem cell-derived endothelial cells, brain pericytes, and astrocytes as self-assembled vascular networks in fibrin gel. Gene expression of membrane transporters, tight junction and extracellular matrix proteins, was consistent with computational analysis of geometrical structures and quantitative immunocytochemistry, indicating BBB maturation and microenvironment remodelling. Confocal microscopy validated microvessel-pericyte/astrocyte dynamic contact-interactions. The BBB model exhibited perfusable and selective microvasculature, with permeability lower than conventional in vitro models, and similar to in vivo measurements in rat brain. This robust and physiologically relevant BBB microvascular model offers an innovative and valuable platform for drug discovery to predict neuro-therapeutic transport efficacy in pre-clinical applications as well as recapitulate patient-specific and pathological neurovascular functions in neurodegenerative disease

3D BLOOD-BRAIN BARRIER MICROVASCULAR NETWORK MODEL INCLUDING HUMAN IPS-DERIVED ENDOTHELIAL CELLS, PERICYTES AND ASTROCYTES

The blood-brain barrier (BBB) is a selective barrier that help to maintain brain homeostasis, however it also creates an obstacle to drug delivery. For years, in vivo animal models have been widely used for BBB studies and drug evaluations. Although these techniques are considered the gold standard, 80% of drug candidates that were successful in animal models later failed in clinical trials. For that reason, a cost-effective in vitro BBB model that adequately reflects human in vivo conditions is required. Here we developed a 3D microfluidic model of the BBB by self-organized vascular network including (iPS)-derived endothelial cells, human brain pericytes, and astrocytes

Inflamed neutrophils sequestered at entrapped tumor cells via chemotactic confinement promote tumor cell extravasation

Systemic inflammation occurring around the course of tumor progression and treatment are often correlated with adverse oncological outcomes. As such, it is suspected that neutrophils, the first line of defense against infection, may play important roles in linking inflammation and metastatic seeding. To decipher the dynamic roles of inflamed neutrophils during hematogenous dissemination, we employ a multiplexed microfluidic model of the human microvasculature enabling physiologically relevant transport of circulating cells combined with real-time, high spatial resolution observation of heterotypic cell–cell interactions. LPS-stimulated neutrophils (PMNs) and tumor cells (TCs) form heterotypic aggregates under flow, and arrest due to both mechanical trapping and neutrophil–endothelial adhesions. Surprisingly, PMNs are not static following aggregation, but exhibit a confined migration pattern near TC–PMN clusters. We discover that PMNs are chemotactically confined by self-secreted IL-8 and tumor-derived CXCL-1, which are immobilized by the endothelial glycocalyx. This results in significant neutrophil sequestration with arrested tumor cells, leading to the spatial localization of neutrophil-derived IL-8, which also contributes to increasing the extravasation potential of adjacent tumor cells through modulation of the endothelial barrier. Strikingly similar migration patterns and extravasation behaviors were also observed in an in vivo zebrafish model upon PMN–tumor cell coinjection into the embryo vasculature. These insights into the temporal dynamics of intravascular tumor–PMN interactions elucidate the mechanisms through which inflamed neutrophils can exert proextravasation effects at the distant metastatic site. Keywords: metastasis; inflammation; neutrophils; extravasation; cell migratio

Blood-brain barrier model on a microfluidic chip for the study of tumor cell extravasation

Thesis: S.M., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2018.Cataloged from PDF version of thesis.Includes bibliographical references (pages 50-58).With up to 40% of cancer patients showing metastatic lesions to the brain and a 30% five-year survival rate post-diagnosis, secondary tumors to the brain are a leading cause of cancer-related deaths. Understanding the mechanisms of tumor cell extravasation at the brain is therefore crucial to the development of therapeutic agents targeting this step in cancer metastasis, and to the overall improvement of cancer survival rates . Investigating the interactions between tumor cells and brain stroma is of particular interest due to the site's unique microenvironment. In fact, the interface between brain and blood, known as the blood-brain barrier (BBB), is the tightest endothelial barrier in humans. The presence of tight junctions between brain endothelial cells, coupled with the spatial organization of pericytes and astrocytes around the vasculature, restrict the entry of most solutes and cells into the brain. Yet, the brain constitutes a common metastatic site to many primary cancers originating from the lung, breast and skin. This suggests that tumor cells must employ specific mechanisms to cross the blood-brain barrier. While in vitro models aimed at replicating the human blood-brain barrier exist, most are limited in their physiological relevance. In fact, the majority of these platforms rely on a monolayer of human brain endothelial cells in contact with pencytes, astrocytes and neurons. While this approach focuses on incorporating the relevant cell types of the brain microenvironment, it fails to accurately replicate the geometry of brain capillaries, the barrier tightness of the BBB, and the juxtacrine and paracrine signaling events occurring between brain endothelial cells and stromal cells during vasculogenesis. To integrate these features into a physiologically relevant blood-brain barrier model, we designed an in vitro microvascular network platform formed via vasculogenesis, using endothelial cells derived from human induced pluripotent stem cells, primary human brain pericytes, and primary human brain astrocytes. The vasculatures formed with brain pericytes and astrocytes exhibit decreased cross-section areas, increased endothelial cell-cell tight junction expression and basement membrane deposition, as well as reduced and more physiologically relevant values of vessel permeability, compared to the vasculatures formed with endothelial cells alone. The addition of pericytes and astrocytes in the vascular system was also coupled with increased extravasation efficiencies of different tumor cell subpopulations, despite the lower permeability values measured in this BBB model. Moreover, an increase in the extravasation potential of metastasized breast tumor cells collected from the brain was recorded with the addition of pericytes and astrocytes, with respect to the parental breast tumor cell line. These results were not observed in metastasized breast tumor cells collected from the lung, thus validating our BBB model and providing useful insight into the role of pericytes and astrocytes in extravasation. Our microfluidic platform certainly provides advantages over the current state-of-the-art in vitro blood-brain barrier models. While being more physiologically relevant than most in vitro platforms when it comes to geometry, barrier function and juxtacrine/paracrine signaling between the relevant cell types, our model provides a robust platform to understand tumor cell-brain stromal cell interactions during extravasation.by Cynthia Hajal.S.M

Pericytes Contribute to Dysfunction in a Human 3D Model of Placental Microvasculature through VEGF‐Ang‐Tie2 Signaling

© 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Placental vasculopathies are associated with a number of pregnancy-related diseases, including pre-eclampsia (PE)—a leading cause of maternal–fetal morbidity and mortality worldwide. Placental presentations of PE are associated with endothelial dysfunction, reduced vessel perfusion, white blood cell infiltration, and altered production of angiogenic factors within the placenta (a candidate mechanism). Despite maintaining vascular quiescence in other tissues, how pericytes contribute to vascular growth and signaling in the placenta remains unknown. Here, pericytes are hypothesized to play a detrimental role in the pathogenesis of placental vascular growth. A perfusable triculture model is developed, consisting of human endothelial cells, fibroblasts, and pericytes, capable of recapitulating growth and remodeling in a system that mimics inflamed placental microvessels. Placental pericytes are shown to contribute to growth restriction of microvessels over time, an effect that is strongly regulated by vascular endothelial growth factor and Angiopoietin/Tie2 signaling. Furthermore, this model is capable of recapitulating essential processes including tumor necrosis factor alpha (TNFα)-mediated vascular leakage and leukocyte infiltration, both important aspects associated with placental PE. This placental vascular model highlights that an imbalance in endothelial–pericyte crosstalk can play a critical role in the development of vascular pathology and associated diseases

The CCL2-CCR2 astrocyte-cancer cell axis in tumor extravasation at the brain

Although brain metastases are common in cancer patients, little is known about the mechanisms of cancer extravasation across the blood-brain barrier (BBB), a key step in the metastatic cascade that regulates the entry of cancer cells into the brain parenchyma. Here, we show, in a three-dimensional in vitro BBB microvascular model, that astrocytes promote cancer cell transmigration via their secretion of C-C motif chemokine ligand 2 (CCL2). We found that this chemokine, produced primarily by astrocytes, promoted the chemotaxis and chemokinesis of cancer cells via their C-C chemokine receptor type 2 (CCR2), with no notable changes in vascular permeability. These findings were validated in vivo, where CCR2-deficient cancer cells exhibited significantly reduced rates of arrest and transmigration in mouse brain capillaries. Our results reveal that the CCL2-CCR2 astrocyte-cancer cell axis plays a fundamental role in extravasation and, consequently, metastasis to the brain.</jats:p

Epithelial-Mesenchymal Transition Induces Podocalyxin to Promote Extravasation via Ezrin Signaling

The epithelial-mesenchymal transition (EMT) endows carcinoma cells with traits needed to complete many of the steps leading to metastasis formation, but its contributions specifically to the late step of extravasation remain understudied. We find that breast cancer cells that have undergone an EMT extravasate more efficiently from blood vessels both in vitro and in vivo. Analysis of gene expression changes associated with the EMT program led to the identification of an EMT-induced cell-surface protein, podocalyxin (PODXL), as a key mediator of extravasation in mesenchymal breast and pancreatic carcinoma cells. PODXL promotes extravasation through direct interaction of its intracellular domain with the cytoskeletal linker protein ezrin. Ezrin proceeds to establish dorsal cortical polarity, enabling the transition of cancer cells from a non-polarized, rounded cell morphology to an invasive extravasation-competent shape. Hence, the EMT program can directly enhance the efficiency of extravasation and subsequent metastasis formation through a PODXL-ezrin signaling axis. Keyword: metastasis; EMT; extravasation; PODXL; ezrinNational Institutes of Health (U.S.) (Grant R01-CA078461

Inflamed neutrophils sequestered at entrapped tumor cells via chemotactic confinement promote tumor cell extravasation

Systemic inflammation occurring around the course of tumor progression and treatment are often correlated with adverse oncological outcomes. As such, it is suspected that neutrophils, the first line of defense against infection, may play important roles in linking inflammation and metastatic seeding. To decipher the dynamic roles of inflamed neutrophils during hematogenous dissemination, we employ a multiplexed microfluidic model of the human microvasculature enabling physiologically relevant transport of circulating cells combined with real-time, high spatial resolution observation of heterotypic cell–cell interactions. LPS-stimulated neutrophils (PMNs) and tumor cells (TCs) form heterotypic aggregates under flow, and arrest due to both mechanical trapping and neutrophil–endothelial adhesions. Surprisingly, PMNs are not static following aggregation, but exhibit a confined migration pattern near TC–PMN clusters. We discover that PMNs are chemotactically confined by self-secreted IL-8 and tumor-derived CXCL-1, which are immobilized by the endothelial glycocalyx. This results in significant neutrophil sequestration with arrested tumor cells, leading to the spatial localization of neutrophil-derived IL-8, which also contributes to increasing the extravasation potential of adjacent tumor cells through modulation of the endothelial barrier. Strikingly similar migration patterns and extravasation behaviors were also observed in an in vivo zebrafish model upon PMN–tumor cell coinjection into the embryo vasculature. These insights into the temporal dynamics of intravascular tumor–PMN interactions elucidate the mechanisms through which inflamed neutrophils can exert proextravasation effects at the distant metastatic site. Keywords: metastasis; inflammation; neutrophils; extravasation; cell migratio