Lung tumor microphysiological system with 3D endothelium to evaluate modulators of T-cell migration.

Lung cancer is a leading cause of death worldwide, with only a fraction of patients responding to immunotherapy. The correlation between increased T-cell infiltration and positive patient outcomes has motivated the search for therapeutics promoting T-cell infiltration. While transwell and spheroid platforms have been employed, these models lack flow and endothelial barriers, and cannot faithfully model T-cell adhesion, extravasation, and migration through 3D tissue. Presented here is a 3D chemotaxis assay, in a lung tumor-on-chip model with 3D endothelium (LToC-Endo), to address this need. The described assay consists of a HUVEC-derived vascular tubule cultured under rocking flow, through which T-cells are added; a collagenous stromal barrier, through which T-cells migrate; and a chemoattractant/tumor (HCC0827 or NCI-H520) compartment. Here, activated T-cells extravasate and migrate in response to gradients of rhCXCL11 and rhCXCL12. Adopting a T-cell activation protocol with a rest period enables proliferative burst prior to introducing T-cells into chips and enhances assay sensitivity. In addition, incorporating this rest recovers endothelial activation in response to rhCXCL12. As a final control, we show that blocking ICAM-1 interferes with T-cell adhesion and chemotaxis. This microphysiological system, which mimics in vivo stromal and vascular barriers, can be used to evaluate potentiation of immune chemotaxis into tumors while probing for vascular responses to potential therapeutics. Finally, we propose translational strategies by which this assay could be linked to preclinical and clinical models to support human dose prediction, personalized medicine, and the reduction, refinement, and replacement of animal models.

High-Throughput Zika Viral Titer Assay for Rapid Screening of Antiviral Drugs.

Zika virus has recently emerged as a worldwide pathogen and public health burden due to its rapid spread and identification as a causative agent for multiple neurological defects, including congenital microcephaly. While there has been a flurry of recent research to address this emerging pathogen, there are currently no approved drug treatments for ZIKV infection. The gold standard for testing antiviral activity is to quantify infectious virion production. However, current infectious viral production assays, such as the plaque-forming or focus-forming unit assay, are tedious and labor intensive with a low-screening throughput. To facilitate drug development, we developed a Zika viral titration assay using an automated imaging system and an image analysis algorithm for viral colony quantification. This assay retained the principle of the classical virus titer assay, while improving workflow and offering higher screening throughput. In addition, this assay can be broadly adapted to quantification of other viruses.

A high-throughput imaging and nuclear segmentation analysis protocol for cleared 3D culture models.

Imaging and subsequent segmentation analysis in three-dimensional (3D) culture models are complicated by the light scattering that occurs when collecting fluorescent signal through multiple cell and extracellular matrix layers. For 3D cell culture models to be usable for drug discovery, effective and efficient imaging and analysis protocols need to be developed that enable high-throughput data acquisition and quantitative analysis of fluorescent signal. Here we report the first high-throughput protocol for optical clearing of spheroids, fluorescent high-content confocal imaging, 3D nuclear segmentation, and post-segmentation analysis. We demonstrate nuclear segmentation in multiple cell types, with accurate identification of fluorescently-labeled subpopulations, and develop a metric to assess the ability of clearing to improve nuclear segmentation deep within the tissue. Ultimately this analysis pipeline allows for previously unattainable segmentation throughput of 3D culture models due to increased sample clarity and optimized batch-processing analysis.

Connecting Neuronal Cell Protective Pathways and Drug Combinations in a Huntington's Disease Model through the Application of Quantitative Systems Pharmacology.

Quantitative Systems Pharmacology (QSP) is a drug discovery approach that integrates computational and experimental methods in an iterative way to gain a comprehensive, unbiased understanding of disease processes to inform effective therapeutic strategies. We report the implementation of QSP to Huntington's Disease, with the application of a chemogenomics platform to identify strategies to protect neuronal cells from mutant huntingtin induced death. Using the STHdh Q111 cell model, we investigated the protective effects of small molecule probes having diverse canonical modes-of-action to infer pathways of neuronal cell protection connected to drug mechanism. Several mechanistically diverse protective probes were identified, most of which showed less than 50% efficacy. Specific combinations of these probes were synergistic in enhancing efficacy. Computational analysis of these probes revealed a convergence of pathways indicating activation of PKA. Analysis of phospho-PKA levels showed lower cytoplasmic levels in STHdh Q111 cells compared to wild type STHdh Q7 cells, and these levels were increased by several of the protective compounds. Pharmacological inhibition of PKA activity reduced protection supporting the hypothesis that protection may be working, in part, through activation of the PKA network. The systems-level studies described here can be broadly applied to any discovery strategy involving small molecule modulation of disease phenotype.

Assessing inhibitors of mutant isocitrate dehydrogenase using a suite of pre-clinical discovery assays.

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that are mutated in a variety of cancers to confer a gain-of-function activity resulting in the accumulation of an oncometabolite, D-2-hydroxyglutarate (2-HG). Accumulation of 2-HG can result in epigenetic dysregulation and a block in cellular differentiation, suggesting these mutations play a role in neoplasia. Based on its potential as a cancer target, a number of small molecule inhibitors have been developed to specifically inhibit mutant forms of IDH (mIDH1 and mIDH2). We present a comprehensive suite of in vitro preclinical drug development assays that can be used as a tool-box to identify lead compounds for mIDH drug discovery programs, as well as what we believe is the most comprehensive publically available dataset on the top mIDH inhibitors. This involved biochemical, cell-based, and tier-one ADME techniques.

RNAi High-Throughput Screening of Single- and Multi-Cell-Type Tumor Spheroids: A Comprehensive Analysis in Two and Three Dimensions.

The widespread use of two-dimensional (2D) monolayer cultures for high-throughput screening (HTS) to identify targets in drug discovery has led to attrition in the number of drug targets being validated. Solid tumors are complex, aberrantly growing microenvironments that harness structural components from stroma, nutrients fed through vasculature, and immunosuppressive factors. Increasing evidence of stromally-derived signaling broadens the complexity of our understanding of the tumor microenvironment while stressing the importance of developing better models that reflect these interactions. Three-dimensional (3D) models may be more sensitive to certain gene-silencing events than 2D models because of their components of hypoxia, nutrient gradients, and increased dependence on cell-cell interactions and therefore are more representative of in vivo interactions. Colorectal cancer (CRC) and breast cancer (BC) models composed of epithelial cells only, deemed single-cell-type tumor spheroids (SCTS) and multi-cell-type tumor spheroids (MCTS), containing fibroblasts were developed for RNAi HTS in 384-well microplates with flat-bottom wells for 2D screening and round-bottom, ultra-low-attachment wells for 3D screening. We describe the development of a high-throughput assay platform that can assess physiologically relevant phenotypic differences between screening 2D versus 3D SCTS, 3D SCTS, and MCTS in the context of different cancer subtypes. This assay platform represents a paradigm shift in how we approach drug discovery that can reduce the attrition rate of drugs that enter the clinic.

Exploring Drug Dosing Regimens In Vitro Using Real-Time 3D Spheroid Tumor Growth Assays.

Two-dimensional monolayer cell proliferation assays for cancer drug discovery have made the implementation of large-scale screens feasible but only seem to reflect a simplified view that oncogenes or tumor suppressor genes are the genetic drivers of cancer cell proliferation. However, there is now increased evidence that the cellular and physiological context in which these oncogenic events occur play a key role in how they drive tumor growth in vivo and, therefore, in how tumors respond to drug treatments. In vitro 3D spheroid tumor models are being developed to better mimic the physiology of tumors in vivo, in an attempt to improve the predictability and efficiency of drug discovery for the treatment of cancer. Here we describe the establishment of a real-time 3D spheroid growth, 384-well screening assay. The cells used in this study constitutively expressed green fluorescent protein (GFP), which enabled the real-time monitoring of spheroid formation and the effect of chemotherapeutic agents on spheroid size at different time points of sphere growth and drug treatment. This real-time 3D spheroid assay platform represents a first step toward the replication in vitro of drug dosing regimens being investigated in vivo. We hope that further development of this assay platform will allow the investigation of drug dosing regimens, efficacy, and resistance before preclinical and clinical studies.

A High-Content Assay Enables the Automated Screening and Identification of Small Molecules with Specific ALDH1A1-Inhibitory Activity.

Aldehyde dehydrogenase enzymes (ALDHs) have a broad spectrum of biological activities through the oxidation of both endogenous and exogenous aldehydes. Increased expression of ALDH1A1 has been identified in a wide-range of human cancer stem cells and is associated with cancer relapse and poor prognosis, raising the potential of ALDH1A1 as a therapeutic target. To facilitate quantitative high-throughput screening (qHTS) campaigns for the discovery, characterization and structure-activity-relationship (SAR) studies of small molecule ALDH1A1 inhibitors with cellular activity, we show herein the miniaturization to 1536-well format and automation of a high-content cell-based ALDEFLUOR assay. We demonstrate the utility of this assay by generating dose-response curves on a comprehensive set of prior art inhibitors as well as hundreds of ALDH1A1 inhibitors synthesized in house. Finally, we established a screening paradigm using a pair of cell lines with low and high ALDH1A1 expression, respectively, to uncover novel cell-active ALDH1A1-specific inhibitors from a collection of over 1,000 small molecules.

Identification of quaternary ammonium compounds as potent inhibitors of hERG potassium channels.

The human ether-a-go-go-related gene (hERG) channel, a member of a family of voltage-gated potassium (K(+)) channels, plays a critical role in the repolarization of the cardiac action potential. The reduction of hERG channel activity as a result of adverse drug effects or genetic mutations may cause QT interval prolongation and potentially leads to acquired long QT syndrome. Thus, screening for hERG channel activity is important in drug development. Cardiotoxicity associated with the inhibition of hERG channels by environmental chemicals is also a public health concern. To assess the inhibitory effects of environmental chemicals on hERG channel function, we screened the National Toxicology Program (NTP) collection of 1408 compounds by measuring thallium influx into cells through hERG channels. Seventeen compounds with hERG channel inhibition were identified with IC(50) potencies ranging from 0.26 to 22µM. Twelve of these compounds were confirmed as hERG channel blockers in an automated whole cell patch clamp experiment. In addition, we investigated the structure-activity relationship of seven compounds belonging to the quaternary ammonium compound (QAC) series on hERG channel inhibition. Among four active QAC compounds, tetra-n-octylammonium bromide was the most potent with an IC(50) value of 260nM in the thallium influx assay and 80nM in the patch clamp assay. The potency of this class of hERG channel inhibitors appears to depend on the number and length of their aliphatic side-chains surrounding the charged nitrogen. Profiling environmental compound libraries for hERG channel inhibition provides information useful in prioritizing these compounds for cardiotoxicity assessment in vivo.

Automated high-content screening for compounds that disassemble the perinucleolar compartment.

All solid malignancies share characteristic traits, including unlimited cellular proliferation, evasion of immune regulation, and the propensity to metastasize. The authors have previously described that a subnuclear structure, the perinucleolar compartment (PNC), is associated with the metastatic phenotype in solid tumor cancer cells. The percentage of cancer cells that contain PNCs (PNC prevalence) is indicative of the malignancy of a tumor both in vitro and in vivo, and thus PNC prevalence is a marker that reflects metastatic capability in a population of tumor cells. Although the function of the PNC remains to be determined, the PNC is highly enriched with small RNAs and RNA binding proteins. The initial chemical biology studies using a set of anticancer drugs that disassemble PNCs revealed a direct association of the structure with DNA. Therefore, PNC prevalence reduction as a phenotypic marker can be used to identify compounds that target cellular processes required for PNC maintenance and hence used to elucidate the nature of the PNC function. Here the authors report the development of an automated high-content screening assay that is capable of detecting PNC prevalence in prostate cancer cells (PC-3M) stably expressing a green fluorescent protein (GFP)-fusion protein that localizes to the PNC. The assay was optimized using known PNC-reducing drugs and non-PNC-reducing cytotoxic drugs. After optimization, the fidelity of the assay was probed with a collection of 8284 compounds and was shown to be robust and capable of detecting known and novel PNC-reducing compounds, making it the first reported high-content phenotypic screen for small changes in nuclear structure.

A new homogeneous high-throughput screening assay for profiling compound activity on the human ether-a-go-go-related gene channel.

Long QT syndrome, either inherited or acquired from drug treatments, can result in ventricular arrhythmia (torsade de pointes) and sudden death. Human ether-a-go-go-related gene (hERG) channel inhibition by drugs is now recognized as a common reason for the acquired form of long QT syndrome. It has been reported that more than 100 known drugs inhibit the activity of the hERG channel. Since 1997, several drugs have been withdrawn from the market due to the long QT syndrome caused by hERG inhibition. Food and Drug Administration regulations now require safety data on hERG channels for investigative new drug (IND) applications. The assessment of compound activity on the hERG channel has now become an important part of the safety evaluation in the process of drug discovery. During the past decade, several in vitro assay methods have been developed and significant resources have been used to characterize hERG channel activities. However, evaluation of compound activities on hERG have not been performed for large compound collections due to technical difficulty, lack of throughput, and/or lack of biological relevance to function. Here we report a modified form of the FluxOR thallium flux assay, capable of measuring hERG activity in a homogeneous 1536-well plate format. To validate the assay, we screened a 7-point dilution series of the LOPAC 1280 library collection and reported rank order potencies of ten common hERG inhibitors. A correlation was also observed for the hERG channel activities of 10 known hERG inhibitors determined in this thallium flux assay and in the patch clamp experiment. Our findings indicate that this thallium flux assay can be used as an alternative method to profile large-volume compound libraries for compound activity on the hERG channel.

A cell-based PDE4 assay in 1536-well plate format for high-throughput screening.

The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3,'5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases, including asthma, cardiovascular disease, attention-deficit hyperactivity disorder, Parkinson's disease, and Alzheimer's disease. Although biochemical assays with purified recombinant PDE enzymes and cAMP or cGMP substrate are commonly used for compound screening, cell-based assays would provide a better assessment of compound activity in a more physiological context. The authors report the development and validation of a new cell-based PDE4 assay using a constitutively active G-protein-coupled receptor as a driving force for cAMP production and a cyclic nucleotide-gated cation channel as a biosensor in 1536-well plates.

Molecular characterization, functional expression, and developmental profile of an ether à-go-go K+ channel in the tobacco hornworm Manduca sexta.

A very large number of evolutionarily conserved potassium channels have been identified but very little is known about their function or modulation in vivo. Metamorphosis of the tobacco hornworm, Manduca sexta, is a compelling model system for such studies because it permits analysis to be conducted at the level of identified neurons whose roles in simple behaviors and endocrine regulation are known. We present here the characterization of the first ion channel to be cloned from this animal. Partial genomic sequence for Manduca sexta ether à-go-go (Mseag) and a cDNA clone encoding the Mseag open reading frame were obtained. Genomic Southern analysis indicates that Manduca contains a single member of the eag subfamily per haploid genome. When expressed in Xenopus oocytes, MsEag channels conduct a voltage-dependent, K+ selective outward current with an inactivating component that closely resembles the Drosophila eag current. Mseag transcripts were restricted to the nervous system, adult antenna, and one set of larval skeletal muscles. Steroid hormonal regulation of Mseag expression is suggested by the temporal correlation of developmental changes in transcript expression with the changing steroid titers that promote metamorphosis. These results provide the foundation for functional and modulatory studies of the Eag family of K+ channels in Manduca, which will complement the genetic analysis in Drosophila.