Multifunctional profiling of triple-negative breast cancer patient-derived tumoroids for disease modeling.

3D cell models derived from patient tumors are highly translational tools that can recapitulate the complex genetic and molecular compositions of solid cancers and accelerate identification of drug targets and drug testing. However, the complexity of performing assays with such models remains a hurdle for their wider adoption. In the present study, we describe methods for processing and multi-functional profiling of tumoroid samples to test compound effects using a novel flowchip system in combination with high content imaging and metabolite analysis. Tumoroids were formed from primary cells isolated from a patient-derived tumor explant, TU-BcX-4IC, that represents metaplastic breast cancer with a triple-negative breast cancer subtype. Assays were performed in a microfluidics-based device (Pu⋅MA System) that allows automated exchange of media and treatments of tumoroids in a tissue culture incubator environment. Multi-functional assay profiling was performed on tumoroids treated with anti-cancer drugs. High-content imaging was used to evaluate drug effects on cell viability and expression of E-cadherin and CD44. Lactate secretion was used to measure tumoroid metabolism as a function of time and drug concentration. Observed responses included loss of cell viability, decrease in E-cadherin expression, and increase of lactate production. Importantly, the tumoroids were sensitive to romidepsin and trametinib, while showed significantly reduced sensitivity to paclitaxel and cytarabine, consistent with the primary tumor response. These methods for multi-parametric profiling of drug effects in patient-derived tumoroids provide an in depth understanding of drug sensitivity of individual tumor types, with important implications for the future development of personalized medicine.

Disease Modeling with 3D Cell-Based Assays Using a Novel Flowchip System and High-Content Imaging.

There is an increasing interest in using three-dimensional (3D) cell structures for modeling tumors, organs, and tissue to accelerate translational research. We describe here a novel automated organoid assay system (the Pu·MA System) combined with microfluidic-based flowchips that can facilitate 3D cell-based assays. The flowchip is composed of sample wells, which contain organoids, connected to additional multiple wells that can hold various assay reagents. Organoids are positioned in a protected chamber in sample wells, and fluids are exchanged from side reservoirs using pressure-driven flow. Media exchange, sample staining, wash steps, and other processes can be performed without disruption to or loss of 3D sample. The bottom of the sample chamber is thin, optically clear plastic compatible with high-content imaging (HCI). The whole system can be kept in an incubator, allowing long-term cellular assays to be performed. We present two examples of use of the system for biological research. In the first example, cytotoxicity effects of anticancer drugs were evaluated on HeLa and HepG2 spheroids using HCI and vascular endothelial growth factor expression. In the second application, the flowchip system was used for the functional evaluation of Ca2+ oscillations in neurospheroids. Neurospheres were incubated with neuroactive compounds, and neuronal activity was assessed using Ca2+-sensitive dyes and fast kinetic fluorescence imaging. This novel assay system using microfluidics enables automation of 3D cell-based cultures that mimic in vivo conditions, performs multidosing protocols and multiple media exchanges, provides gentle handling of spheroids and organoids, and allows a wide range of assay detection modalities.

Determination of Hepatotoxicity in iPSC-Derived Hepatocytes by Multiplexed High Content Assays.

We present here methods for assessing hepatotoxicity by high content imaging and image analysis. The assays focus on the characterization of toxic effects using a variety of phenotypic endpoint readouts. Multi-parametric automated image analysis is used in the protocols to increase assay sensitivity and provide important information about possible in vitro toxicity mechanisms. iPSC-derived hepatocytes were used as a model for the hepatotoxicity assays, but the methods would also be suitable for other liver toxicity cell models. The methods contain detailed step-by-step descriptions of the cell treatment, staining, image acquisition, and image analysis.

In vitro cardiotoxicity assessment of environmental chemicals using an organotypic human induced pluripotent stem cell-derived model.

An important target area for addressing data gaps through in vitro screening is the detection of potential cardiotoxicants. Despite the fact that current conservative estimates relate at least 23% of all cardiovascular disease cases to environmental exposures, the identities of the causative agents remain largely uncharacterized. Here, we evaluate the feasibility of a combinatorial in vitro/in silico screening approach for functional and mechanistic cardiotoxicity profiling of environmental hazards using a library of 69 representative environmental chemicals and drugs. Human induced pluripotent stem cell-derived cardiomyocytes were exposed in concentration-response for 30min or 24h and effects on cardiomyocyte beating and cellular and mitochondrial toxicity were assessed by kinetic measurements of intracellular Ca2+ flux and high-content imaging using the nuclear dye Hoechst 33342, the cell viability marker Calcein AM, and the mitochondrial depolarization probe JC-10. More than half of the tested chemicals exhibited effects on cardiomyocyte beating after 30min of exposure. In contrast, after 24h, effects on cell beating without concomitant cytotoxicity were observed in about one third of the compounds. Concentration-response data for in vitro bioactivity phenotypes visualized using the Toxicological Prioritization Index (ToxPi) showed chemical class-specific clustering of environmental chemicals, including pesticides, flame retardants, and polycyclic aromatic hydrocarbons. For environmental chemicals with human exposure predictions, the activity-to-exposure ratios between modeled blood concentrations and in vitro bioactivity were between one and five orders of magnitude. These findings not only demonstrate that some ubiquitous environmental pollutants might have the potential at high exposure levels to alter cardiomyocyte function, but also indicate similarities in the mechanism of these effects both within and among chemicals and classes.

Neurite outgrowth in human induced pluripotent stem cell-derived neurons as a high-throughput screen for developmental neurotoxicity or neurotoxicity.

Due to the increasing prevalence of neurological disorders and the large number of untested compounds in the environment, there is a need to develop reliable and efficient screening tools to identify environmental chemicals that could potentially affect neurological development. Herein, we report on a library of 80 compounds screened for their ability to inhibit neurite outgrowth, a process by which compounds may elicit developmental neurotoxicity, in a high-throughput, high-content assay using human neurons derived from induced pluripotent stem cells (iPSC). The library contains a diverse set of compounds including those that have been known to be associated with developmental neurotoxicity (DNT) and/or neurotoxicity (NT), environmental compounds with unknown neurotoxic potential (e.g., polycyclic aromatic hydrocarbons (PAHs) and flame retardants (FRs)), as well as compounds with no documented neurotoxic potential. Neurons were treated for 72h across a 6-point concentration range (∼0.3-100µM) in 384-well plates. Effects on neurite outgrowth were assessed by quantifying total outgrowth, branches, and processes. We also assessed the number ofviable cells per well. Concentration-response profiles were evaluated using a Hill model to derive benchmark concentration (BMC) values. Assay performance was evaluated using positive and negative controls and test replicates. Compounds were ranked by activity and selectivity (i.e., specific effects on neurite outgrowth in the absence of concomitant cytotoxicity) and repeat studies were conducted to confirm selectivity. Among the 80 compounds tested, 38 compounds were active, of which 16 selectively inhibited neurite outgrowth. Of these 16 compounds, 12 were known to cause DNT/NT and the remaining 4 compounds included 3 PAHs and 1 FR. In independent repeat studies, 14/16 selective compounds were reproducibly active in the assay, of which only 6 were selective for inhibition of neurite outgrowth. These 6 compounds were previously shown in the literature to be neurotoxic. These studies shed light on the current status of human iPSCs in DNT/NT screening and their utility in identifying, ranking, and prioritizing compounds with DNT/NT potential for further in vivo testing.

High-content assays for characterizing the viability and morphology of 3D cancer spheroid cultures.

There is an increasing interest in using three-dimensional (3D) spheroids for modeling cancer and tissue biology to accelerate translation research. Development of higher throughput assays to quantify phenotypic changes in spheroids is an active area of investigation. The goal of this study was to develop higher throughput high-content imaging and analysis methods to characterize phenotypic changes in human cancer spheroids in response to compound treatment. We optimized spheroid cell culture protocols using low adhesion U-bottom 96- and 384-well plates for three common cancer cell lines and improved the workflow with a one-step staining procedure that reduces assay time and minimizes variability. We streamlined imaging acquisition by using a maximum projection algorithm that combines cellular information from multiple slices through a 3D object into a single image, enabling efficient comparison of different spheroid phenotypes. A custom image analysis method was implemented to provide multiparametric characterization of single-cell and spheroid phenotypes. We report a number of readouts, including quantification of marker-specific cell numbers, measurement of cell viability and apoptosis, and characterization of spheroid size and shape. Assay performance was assessed using established anticancer cytostatic and cytotoxic drugs. We demonstrated concentration-response effects for different readouts and measured IC50 values, comparing 3D spheroid results to two-dimensional cell cultures. Finally, a library of 119 approved anticancer drugs was screened across a wide range of concentrations using HCT116 colon cancer spheroids. The proposed methods can increase performance and throughput of high-content assays for compound screening and evaluation of anticancer drugs with 3D cell models.

High-content high-throughput assays for characterizing the viability and morphology of human iPSC-derived neuronal cultures.

Abstract Development of quantitative high-throughput in vitro assays that enable assessment of viability and morphological changes in neuronal cells is an active area of investigation in drug discovery and environmental chemical safety assessment. High-content imaging is an emerging and efficient tool for generating multidimensional quantitative cellular readouts; in addition, human induced pluripotent stem cell (iPSC)-derived neurons are a promising in vitro model system that emulates both the functionality and behavior of mature neurons, and they are available in quantities sufficient for screening workflows. The goal of this study was to develop high-content imaging and analysis methods to assess multiple phenotypes in human iPSC-derived neuronal cells. Specifically, we optimized cell culture, staining, and imaging protocols in a 384-well assay format and improved laboratory workflow by designing a one-step procedure to reduce assay time and minimize cell disturbance. Phenotypic readouts include quantitative characterization of neurite outgrowth and branching, cell number and viability, as well as measures of adverse effects on mitochondrial integrity and membrane potential. To verify the robustness of the workflow, we tested a series of compounds that are established toxicants. We report concentration-response effects of selected test compounds on human iPSC-derived neuronal cells and illustrate how the proposed methods may be used for high-content high-throughput compound toxicity screening and safety evaluation of drugs and environmental chemicals.

High-content assays for hepatotoxicity using induced pluripotent stem cell-derived cells.

Development of predictive in vitro assays for early toxicity evaluation is extremely important for improving the drug development process and reducing drug attrition rates during clinical development. High-content imaging-based in vitro toxicity assays are emerging as efficient tools for safety and efficacy testing to improve drug development efficiency. In this report we have used an induced pluripotent stem cell (iPSC)-derived hepatocyte cell model having a primary tissue-like phenotype, unlimited availability, and the potential to compare cells from different individuals. We examined a number of assays and phenotypic markers and developed automated screening methods for assessing multiparameter readouts of general and mechanism-specific hepatotoxicity. Endpoints assessed were cell viability, nuclear shape, average and integrated cell area, mitochondrial membrane potential, phospholipid accumulation, cytoskeleton integrity, and apoptosis. We assayed compounds with known mechanisms of toxicity and also evaluated a diverse hepatotoxicity library of 240 compounds. We conclude that high-content automated screening assays using iPSC-derived hepatocytes are feasible, provide information about mechanisms of toxicity, and can facilitate the safety assessment of drugs and chemicals.

Assessment of beating parameters in human induced pluripotent stem cells enables quantitative in vitro screening for cardiotoxicity.

Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes show promise for screening during early drug development. Here, we tested a hypothesis that in vitro assessment of multiple cardiomyocyte physiological parameters enables predictive and mechanistically-interpretable evaluation of cardiotoxicity in a high-throughput format. Human iPSC-derived cardiomyocytes were exposed for 30 min or 24 h to 131 drugs, positive (107) and negative (24) for in vivo cardiotoxicity, in up to 6 concentrations (3 nM to 30 uM) in 384-well plates. Fast kinetic imaging was used to monitor changes in cardiomyocyte function using intracellular Ca(2+) flux readouts synchronous with beating, and cell viability. A number of physiological parameters of cardiomyocyte beating, such as beat rate, peak shape (amplitude, width, raise, decay, etc.) and regularity were collected using automated data analysis. Concentration-response profiles were evaluated using logistic modeling to derive a benchmark concentration (BMC) point-of-departure value, based on one standard deviation departure from the estimated baseline in vehicle (0.3% dimethyl sulfoxide)-treated cells. BMC values were used for cardiotoxicity classification and ranking of compounds. Beat rate and several peak shape parameters were found to be good predictors, while cell viability had poor classification accuracy. In addition, we applied the Toxicological Prioritization Index (ToxPi) approach to integrate and display data across many collected parameters, to derive "cardiosafety" ranking of tested compounds. Multi-parameter screening of beating profiles allows for cardiotoxicity risk assessment and identification of specific patterns defining mechanism-specific effects. These data and analysis methods may be used widely for compound screening and early safety evaluation in drug development.

Multiparameter in vitro assessment of compound effects on cardiomyocyte physiology using iPSC cells.

A large percentage of drugs fail in clinical studies due to cardiac toxicity; thus, development of sensitive in vitro assays that can evaluate potential adverse effects on cardiomyocytes is extremely important for drug development. Human cardiomyocytes derived from stem cell sources offer more clinically relevant cell-based models than those presently available. Human-induced pluripotent stem cell-derived cardiomyocytes are especially attractive because they express ion channels and demonstrate spontaneous mechanical and electrical activity similar to adult cardiomyocytes. Here we demonstrate techniques for measuring the impact of pharmacologic compounds on the beating rate of cardiomyocytes with ImageXpress Micro and FLIPR Tetra systems. The assays employ calcium-sensitive dyes to monitor changes in Ca(2+) fluxes synchronous with cell beating, which allows monitoring of the beat rate, amplitude, and other parameters. We demonstrate here that the system is able to detect concentration-dependent atypical patterns caused by hERG inhibitors and other ion channel blockers. We also show that both positive and negative chronotropic effects on cardiac rate can be observed and IC(50) values determined. This methodology is well suited for safety testing and can be used to estimate efficacy and dosing of drug candidates prior to clinical studies.

A genome-wide siRNA screen reveals diverse cellular processes and pathways that mediate genome stability.

Signaling pathways that respond to DNA damage are essential for the maintenance of genome stability and are linked to many diseases, including cancer. Here, a genome-wide siRNA screen was employed to identify additional genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. We identified hundreds of genes whose downregulation led to elevated levels of H2AX phosphorylation (gammaH2AX) and revealed links to cellular complexes and to genes with unclassified functions. We demonstrate a widespread role for mRNA-processing factors in preventing DNA damage, which in some cases is caused by aberrant RNA-DNA structures. Furthermore, we connect increased gammaH2AX levels to the neurological disorder Charcot-Marie-Tooth (CMT) syndrome, and we find a role for several CMT proteins in the DNA-damage response. These data indicate that preservation of genome stability is mediated by a larger network of biological processes than previously appreciated.

Development of a homogeneous high-throughput live-cell G-protein-coupled receptor binding assay.

The measurement of ligand receptor binding parameters for G-protein-coupled receptors is indispensable in the drug discovery process. Traditional ligand receptor binding assays require scale-up of cells and membrane preparations, which is an expensive and time-consuming process. In this report, the authors describe the development of a homogeneous live-cell binding assay for GPCRs using a fluorophore-labeled nonpeptide ligand. The model assay used Cy3B-labeled telenzepine and Chinese hamster ovary cells expressing M1 muscarinic acetylcholine receptors. This homogeneous live-cell fluorescence binding assay format is superior to the traditional binding methods because it measures binding of a ligand to intact receptors on living cells. The assay requires no washing or separation steps, thereby allowing a real-time kinetic readout for the determination of ligand association and dissociation from the intact receptors. The results also suggest that miniaturization is feasible without compromising the data quality.