Agua Sobrino

3D microtumors in vitro supported by perfused vascular networks.

There is a growing interest in developing microphysiological systems that can be used to model both normal and pathological human organs in vitro. This “organs-on-chips” approach aims to capture key structural and physiological characteristics of the target tissue. Here we describe in vitro vascularized microtumors (VMTs). This “tumor-on-a-chip” platform incorporates human tumor and stromal cells that grow in a 3D extracellular matrix and that depend for survival on nutrient delivery through living, perfused microvessels. Both colorectal and breast cancer cells grow vigorously in the platform and respond to standard-of-care therapies, showing reduced growth and/or regression. Vascular-targeting agents with different mechanisms of action can also be distinguished, and we find that drugs targeting only VEGFRs (Apatinib and Vandetanib) are not effective, whereas drugs that target VEGFRs, PDGFR and Tie2 (Linifanib and Cabozantinib) do regress the vasculature. Tumors in the VMT show strong metabolic heterogeneity when imaged using NADH Fluorescent Lifetime Imaging Microscopy and, compared to their surrounding stroma, many show a higher free/bound NADH ratio consistent with their known preference for aerobic glycolysis. The VMT platform provides a unique model for studying vascularized solid tumors in vitro.

A strategy for integrating essential three-dimensional microphysiological systems of human organs for realistic anticancer drug screening.

Cancer is one of the leading causes of morbidity and mortality around the world. Despite some success, traditional anticancer drugs developed to reduce tumor growth face important limitations primarily due to undesirable bone marrow and cardiovascular toxicity. Many drugs fail in clinical development after showing promise in preclinical trials, suggesting that the available in vitro and animal models are poor predictors of drug efficacy and toxicity in humans. Thus, novel models that more accurately mimic the biology of human organs are necessary for high-throughput drug screening. Three-dimensional (3D) microphysiological systems can utilize induced pluripotent stem cell technology, tissue engineering, and microfabrication techniques to develop tissue models of human tumors, cardiac muscle, and bone marrow on the order of 1 mm3 in size. A functional network of human capillaries and microvessels to overcome diffusion limitations in nutrient delivery and waste removal can also nourish the 3D microphysiological tissues. Importantly, the 3D microphysiological tissues are grown on optically clear platforms that offer non-invasive and non-destructive image acquisition with subcellular resolution in real time. Such systems offer a new paradigm for high-throughput drug screening and will significantly improve the efficiency of identifying new drugs for cancer treatment that minimize cardiac and bone marrow toxicity.

Blood–brain barrier-on-a-chip: Microphysiological systems that capture the complexity of the blood–central nervous system interface:

The blood–brain barrier is a dynamic and highly organized structure that strictly regulates the molecules allowed to cross the brain vasculature into the central nervous system. The blood–brain barrier pathology has been associated with a number of central nervous system diseases, including vascular malformations, stroke/vascular dementia, Alzheimer’s disease, multiple sclerosis, and various neurological tumors including glioblastoma multiforme. There is a compelling need for representative models of this critical interface. Current research relies heavily on animal models (mostly mice) or on two-dimensional (2D) in vitro models, neither of which fully capture the complexities of the human blood–brain barrier. Physiological differences between humans and mice make translation to the clinic problematic, while monolayer cultures cannot capture the inherently three-dimensional (3D) nature of the blood–brain barrier, which includes close association of the abluminal side of the endothelium with astrocyte foot-processes and pericytes. Here we discuss the central nervous system diseases associated with blood–brain barrier pathology, recent advances in the development of novel 3D blood–brain barrier -on-a-chip systems that better mimic the physiological complexity and structure of human blood–brain barrier, and provide an outlook on how these blood–brain barrier-on-a-chip systems can be used for central nervous system disease modeling. Impact statement The field of microphysiological systems is rapidly evolving as new technologies are introduced and our understanding of organ physiology develops. In this review, we focus on Blood–Brain Barrier (BBB) models, with a particular emphasis on how they relate to neurological disorders such as Alzheimer’s disease, multiple sclerosis, stroke, cancer, and vascular malformations. We emphasize the importance of capturing the three-dimensional nature of the brain and the unique architecture of the BBB – something that until recently had not been well modeled by in vitro systems. Our hope is that this review will provide a launch pad for new ideas and methodologies that can provide us with truly physiological BBB models capable of yielding new insights into the function of this critical interface.

Three-Dimensional Adult Cardiac Extracellular Matrix Promotes Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Pluripotent stem cell-derived cardiomyocytes (CMs) have great potential in the development of new therapies for cardiovascular disease. In particular, human induced pluripotent stem cells (iPSCs) may prove especially advantageous due to their pluripotency, their self-renewal potential, and their ability to create patient-specific cell lines. Unfortunately, pluripotent stem cell-derived CMs are immature, with characteristics more closely resembling fetal CMs than adult CMs, and this immaturity has limited their use in drug screening and cell-based therapies. Extracellular matrix (ECM) influences cellular behavior and maturation, as does the geometry of the environment-two-dimensional (2D) versus three-dimensional (3D). We therefore tested the hypothesis that native cardiac ECM and 3D cultures might enhance the maturation of iPSC-derived CMs in vitro. We demonstrate that maturation of iPSC-derived CMs was enhanced when cells were seeded into a 3D cardiac ECM scaffold, compared with 2D culture. 3D cardiac ECM promoted increased expression of calcium-handling genes, Junctin, CaV1.2, NCX1, HCN4, SERCA2a, Triadin, and CASQ2. Consistent with this, we find that iPSC-derived CMs in 3D adult cardiac ECM show increased calcium signaling (amplitude) and kinetics (maximum upstroke and downstroke) compared with cells in 2D. Cells in 3D culture were also more responsive to caffeine, likely reflecting an increased availability of calcium in the sarcoplasmic reticulum. Taken together, these studies provide novel strategies for maturing iPSC-derived CMs that may have applications in drug screening and transplantation therapies to treat heart disease.

Supervised Machine Learning for Classification of the Electrophysiological Effects of Chronotropic Drugs on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Supervised machine learning can be used to predict which drugs human cardiomyocytes have been exposed to. Using electrophysiological data collected from human cardiomyocytes with known exposure to different drugs, a supervised machine learning algorithm can be trained to recognize and classify cells that have been exposed to an unknown drug. Furthermore, the learning algorithm provides information on the relative contribution of each data parameter to the overall classification. Probabilities and confidence in the accuracy of each classification may also be determined by the algorithm. In this study, the electrophysiological effects of β–adrenergic drugs, propranolol and isoproterenol, on cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CM) were assessed. The electrophysiological data were collected using high temporal resolution 2-photon microscopy of voltage sensitive dyes as a reporter of membrane voltage. The results demonstrate the ability of our algorithm to accurately assess, classify, and predict hiPS-CM membrane depolarization following exposure to chronotropic drugs.

Fluorescence Lifetime Imaging Microscopy to study Metabolism in a Microfluidic Device based Tumor Microenvironment

Here we present 2-photon fluorescence lifetime imaging microscopy of nicotinamide adenine dinucleotide to study metabolism of vascularized “tumor-on-a-chip” consisting of tumor cells, fibroblast and vascular network in an optically clear PDMS device.

Abstract 5500: A 3D tumor tissue microphysiological system for realistic tumor microenvironment mimicry and therapeutic modeling

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Introduction: Although cancer is a heterogeneous disease, current chemotherapy is based on the use of anti-cancer agents that broadly affect cell proliferation. As a result of this non-specific tumor therapy there is often high toxicity and therapeutic resistance, which increases the risk of disease progression. There is thus a critical need to find novel anti-cancer compounds, however, as a result of preliminary screens often performed in 2D systems, and the use of mouse models for secondary screens, many drugs fail during full clinical trials. Replicating a complex, 3D microenvironment incorporating human cells is therefore likely to improve drug screening efficiency. Methods: Here we have developed a system for high throughput screening of drug efficacy. Our system features 3D tumor tissues incorporating human cells co-cultured with stromal cells and connected by human microvessels in a naturally occurring 3D matrix in a PDMS microdevice. Transduced colorectal cancer cells (CRC) such as HCT116, SW480 and SW620s that express green fluorescent protein (GFP) constitutively are introduced into the device in co-culture with human endothelial colony-forming cell-derived EC (ECFC-EC) and fibroblasts. ECFC-EC or fibroblasts that express longer wavelength fluorescent proteins are also used to track both tumor cells and vasculature/fibroblast growth. The tri-culture is resuspended in fibrinogen (10 mg/ml) and mixed with thrombin before injecting into the microchambers. Cells are fed with endothelial growth media-2 and drugs are delivered through the microfluidic channels using a hydrostatic pressure gradient. Results: A complete vascular network was formed in the chambers by 7-10 days and this carried medium into the tissue. CRC showed continuous growth, often forming spheroids. After 7-10 days of culture, cells were exposed to various FDA-approved drugs at concentrations reported for plasma levels in patients. Tumor growing in the device responded differently to most drugs compared to cells growing in 2D. We identified cytotoxic and cytostatic drug actions in the device with IC50s close to the in vivo pharmacologic plasma concentration, but higher than those obtained in parallel 2D experiments. Importantly, we also identified drugs that were active in our 3D-device but not in 2D cultures. Conclusions: Preliminary results using our 3D microphysiological system, in which we have created a more complex microenvironment than used in 2D cultures, support an improved strategy for drug validation. In addition, future experiments will include the use of non-invasive optical imaging techniques to better understand the human 3D tumor microenvironment, including the study of different tumor behaviors, tumor metabolism and pathological angiogenesis. This proof-of-concept study validates the use of 3D microphysiological systems in place of more simplistic 2D systems. Citation Format: Agua Sobrino, Duc Phan, Steven C George, Christopher C.W Hughes. A 3D tumor tissue microphysiological system for realistic tumor microenvironment mimicry and therapeutic modeling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5500. doi:10.1158/1538-7445.AM2015-5500