A Tissue-Engineered 3D Microvessel Model Reveals the Dynamics of Mosaic Vessel Formation in Breast Cancer.

In solid tumors, vascular structure and function varies from the core to the periphery. This structural heterogeneity has been proposed to influence the mechanisms by which tumor cells enter the circulation. Blood vessels exhibit regional defects in endothelial coverage, which can result in cancer cells directly exposed to flow and potentially promoting intravasation. Consistent with prior reports, we observed in human breast tumors and in a mouse model of breast cancer that approximately 6% of vessels consisted of both endothelial cells and tumor cells, so-called mosaic vessels. Due, in part, to the challenges associated with observing tumor-vessel interactions deep within tumors in real-time, the mechanisms by which mosaic vessels form remain incompletely understood. We developed a tissue-engineered model containing a physiologically realistic microvessel in coculture with mammary tumor organoids. This approach allows real-time and quantitative assessment of tumor-vessel interactions under conditions that recapitulate many in vivo features. Imaging revealed that tumor organoids integrate into the endothelial cell lining, resulting in mosaic vessels with gaps in the basement membrane. While mosaic vessel formation was the most frequently observed interaction, tumor organoids also actively constricted and displaced vessels. Furthermore, intravasation of cancer cell clusters was observed following the formation of a mosaic vessel. Taken together, our data reveal that cancer cells can rapidly reshape, destroy, or integrate into existing blood vessels, thereby affecting oxygenation, perfusion, and systemic dissemination. Our novel assay also enables future studies to identify targetable mechanisms of vascular recruitment and intravasation. SIGNIFICANCE: A tissue-engineered microdevice that recapitulates the tumor-vascular microenvironment enables real-time imaging of the cellular mechanisms of mosaic vessel formation and vascular defect generation.

Cerebrovascular plasticity: Processes that lead to changes in the architecture of brain microvessels.

The metabolic demands of the brain are met by oxygen and glucose, supplied by a complex hierarchical network of microvessels (arterioles, capillaries, and venules). Transient changes in neural activity are accommodated by local dilation of arterioles or capillaries to increase cerebral blood flow and hence nutrient availability. Transport and communication between the circulation and the brain is regulated by the brain microvascular endothelial cells that form the blood-brain barrier. Under homeostatic conditions, there is very little turnover in brain microvascular endothelial cells, and the cerebrovascular architecture is largely static. However, changes in the brain microenvironment, due to environmental factors, disease, or trauma, can result in additive or subtractive changes in cerebrovascular architecture. Additions occur by angiogenesis or vasculogenesis, whereas subtractions occur by vascular pruning, injury, or endothelial cell death. Here we review the various processes that lead to changes in the cerebrovascular architecture, including sustained changes in the brain microenvironment, development and aging, and injury, disease, and repair.

Chemotherapeutic Drug Delivery and Quantitative Analysis of Proliferation, Apoptosis, and Migration in a Tissue-Engineered Three-Dimensional Microvessel Model of the Tumor Microenvironment.

Numerous approaches have been employed to improve the efficacy of drug and gene delivery systems, but their strategic development is hindered by a lack of mechanistic understanding and assessment of drug transport and action. Optimizing the efficiency of a drug delivery system requires a detailed understanding of the pharmacokinetics, transendothelial transport, distribution at the tumor site, and uptake in target cells. Elucidating transport kinetics and rate-limiting steps in animal models can be extremely challenging, while in vitro platforms often fail to recapitulate the complexities of drug transport in vivo. To recapitulate the critical aspects of delivery of anticancer agents, we have developed a 3D tissue-engineered microvessel model of the tumor microenvironment. Our model consists of single MDA-MB-231 breast cancer cells embedded within a collagen matrix that surrounds a perfusable cylindrical microvessel lined with human endothelial cells. Here we compare transport and action of free doxorubicin and Doxil, a liposomal formulation of doxorubicin. We show that the mode of drug delivery influences uptake in the vessel endothelium and tumor cells. Through quantification of endothelial and tumor cell proliferation, apoptosis, and motility, we profile the kinetics of drug action with mechanisms of drug transport across the vessel lumen and into the surrounding matrix. Our model can be customized to mimic specific tumor microenvironments and disease states within a physiologically relevant microfluidic platform and provides a basis for characterizing and optimizing drug delivery systems.

A Simple Colorimetric System for Detecting Target Antigens by a Three-Stage Signal Transformation-Amplification Strategy.

Inexpensive, straightforward, and rapid medical diagnostics are becoming increasingly important for disease identification in time- and resource-limited settings. Previous attempts to link oligonucleotide-based aptamers and hammerhead ribozymes to form ligand-induced ribozymes have been successful in identifying a variety of small molecule and protein targets. Isothermal exponential amplification reactions (EXPAR) amplify minute amounts of nucleic acid templates without requiring special instrumentation. We introduce a colorimetric assay that we engineered using an aptamer, hammerhead ribozyme, EXPAR, and peroxidase activity in conjunction with a 3,3',5,5'-tetramethylbenzidine (TMB) substrate. This is a modular signal enhancer system that can be easily modified to detect virtually any chosen analyte target within 5-10 min with minimal technical requirements. Ligand-aptamer binding causes the ribozyme to change conformation and self-cleave. The cleaved ribozyme triggers exponential amplification of a reporter sequence during EXPAR. The amplification products fold into single-stranded DNA guanine quadruplexes that exhibit peroxidase-like activity and can oxidize a colorless TMB substrate into a colored reaction product for visual detection. As a proof of concept, we examined the bronchodilator theophylline versus its chemical analogue, caffeine. We demonstrate linear changes in absorption readout across a wide range of target concentrations (0.5-1000 µM) and the ability to visually detect theophylline at 0.5 µM with an approximately 35-fold increased specificity versus that of caffeine. This three-stage detection system is a versatile platform that has the potential to improve the rapid identification of target analytes.

Dissemination from a Solid Tumor: Examining the Multiple Parallel Pathways.

Metastasis can be generalized as a linear sequence of events whereby halting one or more steps in the cascade may reduce tumor cell dissemination and ultimately improve patient outcomes. However, metastasis is a complex process with multiple parallel mechanisms of dissemination. Clinical strategies focus on removing the primary tumor and/or treating distant metastases through chemo- or immunotherapies. Successful strategies for blocking metastasis will need to address the parallel mechanisms of dissemination and identify common bottlenecks. Here, we review the current understanding of common dissemination pathways for tumors. Understanding the complexities of metastasis will guide the design of new therapies that halt dissemination.

Mitosis-Mediated Intravasation in a Tissue-Engineered Tumor-Microvessel Platform.

Intravasation involves the migration of tumor cells across the local endothelium and escape into vessel flow. Although tumor cell invasiveness has been correlated to increased intravasation, the details of transendothelial migration and detachment into circulation are still unclear. Here, we analyzed the intravasation of invasive human breast cancer cells within a tissue-engineered microvessel model of the tumor microenvironment. Using live-cell fluorescence microscopy, we captured 2,330 hours of tumor cell interactions with functional microvessels and provide evidence for a mitosis-mediated mechanism where tumor cells located along the vessel periphery are able to disrupt the vessel endothelium through cell division and detach into circulation. This model provides a framework for understanding the physical and biological parameters of the tumor microenvironment that mediate intravasation of tumor cells across an intact endothelium. Cancer Res; 77(22); 6453-61. ©2017 AACR.

Real-time quantification of endothelial response to shear stress and vascular modulators.

Quiescence is commonly used to describe the inactive state of endothelial cells (ECs) in monolayers that have reached homeostasis. Experimentally quiescence is usually described in terms of the relative change in cell activity (e.g. turnover, speed, etc.) in response to a perturbation (e.g. solute, shear stress, etc.). The objective of this study is to provide new insight into EC quiescence by quantitatively defining the morphology and activity of confluent cell monolayers in response to shear stress and vascular modulators. Confluent monolayers of human umbilical vein ECs (HUVECs) were subjected to a range of shear stresses (4-16 dyne cm-2) under steady flow. Using phase contrast, time-lapse microscopy and image analysis, we quantified EC morphology, speed, proliferation, and apoptosis rates over time and detected differences in monolayer responses under various media conditions: basal media supplemented with growth factors, interleukin-8, or cyclic AMP. In all conditions, we observed a transition from cobblestone to spindle-like morphology in a dose-dependent manner due to shear stress. Cyclic AMP enhanced the elongation and alignment of HUVECs due to shear stress and reduced steady state cell speed. We observed the lowest proliferation rates below 8 dyne cm-2 and found that growth factors and cyclic AMP reduced proliferation and apoptosis; interleukin-8 similarly decreased proliferation, but increased apoptosis. We have quantified the response of ECs in confluent monolayers to shear stress and vascular modulators in terms of morphology, speed, proliferation and apoptosis and have established quantifiable metrics of cell activity to define vascular quiescence under shear stress.

Utility of microfluidic devices to study the platelet-endothelium interface.

The integration of biomaterials and understanding of vascular biology has led to the development of perfusable endothelialized flow models, which have been used as valuable tools to study the platelet-endothelium interface under shear. In these models, the parameters of geometry, compliance, biorheology, and cellular complexity are varied to recapitulate the physical biology of platelet recruitment and activation under physiologically relevant conditions of blood flow. In this review, we summarize the mechanistic insights learned from perfusable microvessel models and discuss the potential utility as well as challenges of endothelialized microfluidic devices to study platelet function in the bloodstream in vitro.

Tissue-engineered 3D microvessel and capillary network models for the study of vascular phenomena.

Advances in tissue engineering, cell biology, microfabrication, and microfluidics have led to the development of a wide range of vascular models. Here, we review platforms based on templated microvessel fabrication to generate increasingly complex vascular models of (i) the tumor microenvironment, (ii) occluded microvessels, and (iii) perfused capillary networks. We outline fabrication guidelines and demonstrate a number of experimental methods for probing vascular function such as permeability measurements, tumor cell intravasation, flow characterization, and endothelial cell morphology and proliferation.

In Vitro Tumor Models: Advantages, Disadvantages, Variables, and Selecting the Right Platform.

In vitro tumor models have provided important tools for cancer research and serve as low-cost screening platforms for drug therapies; however, cancer recurrence remains largely unchecked due to metastasis, which is the cause of the majority of cancer-related deaths. The need for an improved understanding of the progression and treatment of cancer has pushed for increased accuracy and physiological relevance of in vitro tumor models. As a result, in vitro tumor models have concurrently increased in complexity and their output parameters further diversified, since these models have progressed beyond simple proliferation, invasion, and cytotoxicity screens and have begun recapitulating critical steps in the metastatic cascade, such as intravasation, extravasation, angiogenesis, matrix remodeling, and tumor cell dormancy. Advances in tumor cell biology, 3D cell culture, tissue engineering, biomaterials, microfabrication, and microfluidics have enabled rapid development of new in vitro tumor models that often incorporate multiple cell types, extracellular matrix materials, and spatial and temporal introduction of soluble factors. Other innovations include the incorporation of perfusable microvessels to simulate the tumor vasculature and model intravasation and extravasation. The drive toward precision medicine has increased interest in adapting in vitro tumor models for patient-specific therapies, clinical management, and assessment of metastatic potential. Here, we review the wide range of current in vitro tumor models and summarize their advantages, disadvantages, and suitability in modeling specific aspects of the metastatic cascade and drug treatment.

Review: in vitro microvessel models.

A wide range of perfusable microvessel models have been developed, exploiting advances in microfabrication, microfluidics, biomaterials, stem cell technology, and tissue engineering. These models vary in complexity and physiological relevance, but provide a diverse tool kit for the study of vascular phenomena and methods to vascularize artificial organs. Here we review the state-of-the-art in perfusable microvessel models, summarizing the different fabrication methods and highlighting advantages and limitations.

Quantitative Analysis of the Enhanced Permeation and Retention (EPR) Effect.

Tumor vasculature is characterized by a variety of abnormalities including irregular architecture, poor lymphatic drainage, and the upregulation of factors that increase the paracellular permeability. The increased permeability is important in mediating the uptake of an intravenously administered drug in a solid tumor and is known as the enhanced permeation and retention (EPR) effect. Studies in animal models have demonstrated a cut-off size of 500 nm - 1 µm for molecules or nanoparticles to extravasate into a tumor, however, surprisingly little is known about the kinetics of the EPR effect. Here we present a pharmacokinetic model to quantitatively assess the influence of the EPR effect on the uptake of a drug into a solid tumor. We use pharmacokinetic data for Doxil and doxorubicin from human clinical trials to illustrate how the EPR effect influences tumor uptake. This model provides a quantitative framework to guide preclinical trials of new chemotherapies and ultimately to develop design rules that can increase targeting efficiency and decrease unwanted side effects in normal tissue.

Human brain microvascular endothelial cells resist elongation due to shear stress.

Endothelial cells in straight sections of vessels are known to elongate and align in the direction of flow. This phenotype has been replicated in confluent monolayers of bovine aortic endothelial cells and human umbilical vein endothelial cells (HUVECs) in cell culture under physiological shear stress. Here we report on the morphological response of human brain microvascular endothelial cells (HBMECs) in confluent monolayers in response to shear stress. Using a microfluidic platform we image confluent monolayers of HBMECs and HUVECs under shear stresses up to 16 dyne cm(-2). From live-cell imaging we quantitatively analyze the cell morphology and cell speed as a function of time. We show that HBMECs do not undergo a classical transition from cobblestone to spindle-like morphology in response to shear stress. We further show that under shear stress, actin fibers are randomly oriented in the cells indicating that there is no cytoskeletal remodeling. These results suggest that HBMECs are programmed to resist elongation and alignment under shear stress, a phenotype that may be associated with the unique properties of the blood-brain barrier.

Polyglyoxylates: a versatile class of triggerable self-immolative polymers from readily accessible monomers.

Self-immolative polymers, which degrade by an end-to-end depolymerization mechanism in response to the cleavage of a stabilizing end-cap from the polymer terminus, are of increasing interest for a wide variety of applications ranging from sensors to controlled release. However, the preparation of these materials often requires expensive, multistep monomer syntheses, and the degradation products such as quinone methides or phthalaldehydes are potentially toxic to humans and the environment. We demonstrate here that polyglyxoylates can serve as a new and versatile class of self-immolative polymers. Polymerization of the commercially available monomer ethyl glyoxylate, followed by end-capping with a 6-nitroveratryl carbonate, provides a poly(ethyl glyoxylate) that depolymerizes selectively upon irradiation with UV light, ultimately generating ethanol and the metabolic intermediate glyoxylic acid hydrate. To access polyglyoxylates with different properties, the polymerization and end-capping approach can also be extended to other glyoxylate monomers including methyl glyoxylate, n-butyl glyoxylate, and benzyl glyoxylate, which can be easily prepared from their corresponding fumaric or maleic acid derivatives. Random copolymers of these monomers with ethyl glyoxylate can also be prepared. Furthermore, using a multifunctional end-cap that is UV-responsive and also enables the conjugation of another polymer block via an azide-alkyne "click" cycloaddition, amphiphilic self-immolative block copolymers are also prepared. These block copolymers self-assemble into micelles in aqueous solution, and their poly(ethyl glyoxylate) blocks rapidly depolymerize upon UV irradiation. Overall, these strategies are expected to greatly expand the utility of self-immolative polymers by providing access for the first time to self-immolative polymers with tunable properties that can be readily obtained from simple monomers and can be designed to depolymerize into nontoxic products.

Live-cell imaging of invasion and intravasation in an artificial microvessel platform.

Methods to visualize metastasis exist, but additional tools to better define the biologic and physical processes underlying invasion and intravasation are still needed. One difficulty in studying metastasis stems from the complexity of the interface between the tumor microenvironment and the vascular system. Here, we report the development of an investigational platform that positions tumor cells next to an artificial vessel embedded in an extracellular matrix. On this platform, we used live-cell fluorescence microscopy to analyze the complex interplay between metastatic cancer cells and a functional artificial microvessel that was lined with endothelial cells. The platform recapitulated known interactions, and its use demonstrated the capabilities for a systematic study of novel physical and biologic parameters involved in invasion and intravasation. In summary, our work offers an important new tool to advance knowledge about metastasis and candidate antimetastatic therapies.

Brain microvascular endothelial cells resist elongation due to curvature and shear stress.

The highly specialized endothelial cells in brain capillaries are a key component of the blood-brain barrier, forming a network of tight junctions that almost completely block paracellular transport. In contrast to vascular endothelial cells in other organs, we show that brain microvascular endothelial cells resist elongation in response to curvature and shear stress. Since the tight junction network is defined by endothelial cell morphology, these results suggest that there may be an evolutionary advantage to resisting elongation by minimizing the total length of cell-cell junctions per unit length of vessel.

Multiresponsive Azobenzene End-Cap for Self-Immolative Polymers.

Azobenzene was introduced as a new multiresponsive end-cap for self-immolative polymers. Using small-molecule model compounds, it was demonstrated that reducing agents including hydrazine and dithiothreitol could reduce the azobenzene to the corresponding hydrazobenzene, resulting in a 1,6-elimination reaction with the potential to initiate the depolymerization of self-immolative polycarbamates. An activated azobenzene derivative was then prepared, allowing for its incorporation as an end-cap for polycarbamates based on alternating N,N'-dimethylethylene diamine and 4-hydroxybenzyl alcohol. Upon treatment with hydrazine, depolymerization proceeded. To demonstrate the versatility of this end-cap, the chemistry was also applied to polycarbamates based on 4-aminobenzyl alcohol. In addition to allowing triggered depolymerization, the azobenzene end-cap also provides a visual signal upon triggering owing to the strong visible absorption of the azobenzene, which shifts to the UV range upon reduction. Furthermore, azobenzene is capable of undergoing trans-cis isomerization in response to UV light, providing multiple functions in a single end-cap.

The blood-brain barrier: an engineering perspective.

It has been more than 100 years since Paul Ehrlich reported that various water-soluble dyes injected into the circulation did not enter the brain. Since Ehrlich's first experiments, only a small number of molecules, such as alcohol and caffeine have been found to cross the blood-brain barrier, and this selective permeability remains the major roadblock to treatment of many central nervous system diseases. At the same time, many central nervous system diseases are associated with disruption of the blood-brain barrier that can lead to changes in permeability, modulation of immune cell transport, and trafficking of pathogens into the brain. Therefore, advances in our understanding of the structure and function of the blood-brain barrier are key to developing effective treatments for a wide range of central nervous system diseases. Over the past 10 years it has become recognized that the blood-brain barrier is a complex, dynamic system that involves biomechanical and biochemical signaling between the vascular system and the brain. Here we reconstruct the structure, function, and transport properties of the blood-brain barrier from an engineering perspective. New insight into the physics of the blood-brain barrier could ultimately lead to clinical advances in the treatment of central nervous system diseases.

Amplified release through the stimulus triggered degradation of self-immolative oligomers, dendrimers, and linear polymers.

In recent years, numerous delivery systems based on polymers, dendrimers, and nano-scale assemblies have been developed to improve the properties of drug molecules. In general, for the drug molecules to be active, they must be released from these delivery systems, ideally in a selective manner at the therapeutic target. As the changes in physiological conditions are relatively subtle from one tissue to another and the concentrations of specific enzymes are often quite low, a release strategy involving the amplification of a biological signal is particularly attractive. This article describes the development of oligomers, dendrimers, and linear polymers based on self-immolative spacers. This new class of molecules is designed to undergo a cascade of intramolecular reactions in response to the cleavage of a trigger moiety, resulting in molecular fragmentation and the release of multiple reporter or drug molecules. Progress in the development of these materials as drug delivery vehicles and sensors will be highlighted.