Assessing the performance of the Cell Painting assay across different imaging systems.

Quantitative microscopy is a powerful method for performing phenotypic screens from which image-based profiling can extract a wealth of information, termed profiles. These profiles can be used to elucidate the changes in cellular phenotypes across cell populations from different patient samples or following genetic or chemical perturbations. One such image-based profiling method is the Cell Painting assay, which provides morphological insight through the imaging of eight cellular compartments.

Assessing the performance of the Cell Painting assay across different imaging systems.

Quantitative microscopy is a powerful method for performing phenotypic screens from which image-based profiling can extract a wealth of information, termed profiles. These profiles can be used to elucidate the changes in cellular phenotypes across cell populations from different patient samples or following genetic or chemical perturbations. One such image-based profiling method is the Cell Painting assay, which provides morphological insight through the imaging of eight cellular compartments.

Factors to consider when interrogating 3D culture models with plate readers or automated microscopes.

Along with the increased use of more physiologically relevant three-dimensional cell culture models comes the responsibility of researchers to validate new assay methods that measure events in structures that are physically larger and more complex compared to monolayers of cells. It should not be assumed that assays designed using monolayers of cells will work for cells cultured as larger three-dimensional masses. The size and barriers for penetration of molecules through the layers of cells result in a different microenvironment for the cells in the outer layer compared to the center of three-dimensional structures.

Assay Guidance Manual: Quantitative Biology and Pharmacology in Preclinical Drug Discovery.

The Assay Guidance Manual (AGM) is an eBook of best practices for the design, development, and implementation of robust assays for early drug discovery. Initiated by pharmaceutical company scientists, the manual provides guidance for designing a "testing funnel" of assays to identify genuine hits using high-throughput screening (HTS) and advancing them through preclinical development. Combined with a workshop/tutorial component, the overall goal of the AGM is to provide a valuable resource for training translational scientists.

Guidelines for Microplate Selection in High Content Imaging.

Since the inception of commercialized automated high content screening (HCS) imaging devices in the mid to late 1990s, the adoption of media vessels typically used to house and contain biological specimens for interrogation has transitioned from microscope slides and petri dishes into multi-well microtiter plates called microplates. The early 96- and 384-well microplates commonly used in other high-throughput screening (HTS) technology applications were often not designed for optical imaging. Since then, modifications and the use of next-generation materials with improved optical clarity have enhanced the quality of captured images, reduced autofocusing failures, and empowered the use of higher power magnification objectives to resolve fine detailed measurements at the subcellular pixel level.

miR-122 Release in Exosomes Precedes Overt Tolvaptan-Induced Necrosis in a Primary Human Hepatocyte Micropatterned Coculture Model.

Idiosyncratic drug-induced liver injury (IDILI) is thought to often result from an adaptive immune attack on the liver. However, it has been proposed that the cascade of events culminating in an adaptive immune response begins with drug-induced hepatocyte stress, release of exosomal danger signals, and innate immune activation, all of which may occur in the absence of significant hepatocelluar death. A micropatterned coculture model (HepatoPac) was used to explore the possibility that changes in exosome content precede overt necrosis in response to the IDILI drug tolvaptan.

Development of an in vitro high content imaging assay for quantitative assessment of CAR-dependent mouse, rat, and human primary hepatocyte proliferation.

Rodent liver tumors promoted by constitutive androstane receptor (CAR) activation are known to be mediated by key events that include CAR-dependent gene expression and hepatocellular proliferation. Here, an in vitro high content imaging based assay was developed for quantitative assessment of nascent DNA synthesis in primary hepatocyte cultures from mouse, rat, and human species. Detection of DNA synthesis was performed using direct DNA labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU).

A cellular genetics approach identifies gene-drug interactions and pinpoints drug toxicity pathway nodes.

New approaches to toxicity testing have incorporated high-throughput screening across a broad-range of in vitro assays to identify potential key events in response to chemical or drug treatment. To date, these approaches have primarily utilized repurposed drug discovery assays. In this study, we describe an approach that combines in vitro screening with genetic approaches for the experimental identification of genes and pathways involved in chemical or drug toxicity.

Profiling dose-dependent activation of p53-mediated signaling pathways by chemicals with distinct mechanisms of DNA damage.

As part of a larger effort to provide proof-of-concept in vitro-only risk assessments, we have developed a suite of high-throughput assays for key readouts in the p53 DNA damage response toxicity pathway: double-strand break DNA damage (p-H2AX), permanent chromosomal damage (micronuclei), p53 activation, p53 transcriptional activity, and cell fate (cell cycle arrest, apoptosis, micronuclei). Dose-response studies were performed with these protein and cell fate assays, together with whole genome transcriptomics, for three prototype chemicals: etoposide, quercetin, and methyl methanesulfonate. Data were collected in a human cell line expressing wild-type p53 (HT1080) and results were confirmed in a second p53 competent cell line (HCT 116).

A micropatterned hepatocyte coculture model for assessment of liver toxicity using high-content imaging analysis.

The current landscape of in vitro models used to identify drug- or chemical-induced hepatotoxicity relies heavily on cell culture models consisting of HepG2, induced pluripotent stem cell-derived, or primary hepatocytes. While these in vitro models offer powerful approaches for predicting toxicity, each system has challenges, including variable metabolic capacity, brief ex vivo life span in culture, and adoption with standard automated microscopy high-content screening (HCS) systems to measure reproducibility data at the single-cell level. In this report we introduce a novel primary hepatocyte coculture model, HepatoPac™, as an alternative to current model systems for evaluation of in vitro hepatotoxicity in 96-well microtiter plate format examined by HCS.

Assessing dose-dependent differences in DNA-damage, p53 response and genotoxicity for quercetin and curcumin.

As part of a longer-term goal to create a quantitative mechanistic model of the p53-Mdm2 DNA-damage pathway, we are studying cellular responses to compounds causing DNA-damage by various modes-of action, including two natural polyphenols: quercetin (QUE) and curcumin (CUR). QUE and CUR are weak mutagens in some in vitro assays and possess both anti- or pro-oxidant effects depending on dose. This study examines the dose-response of DNA-damage pathway to these compounds in HT1080 cells (a human cell line with wild-type p53) at doses relevant to human exposure.

Composite primary neuronal high-content screening assay for Huntington's disease incorporating non-cell-autonomous interactions.

Huntington's disease (HD) is a fatal neurodegenerative disease characterized by progressive cognitive, behavioral, and motor deficits and caused by expansion of a polyglutamine repeat in the Huntingtin protein (Htt). Despite its monogenic nature, HD pathogenesis includes obligatory non-cell-autonomous pathways involving both the cortex and the striatum, and therefore effective recapitulation of relevant HD disease pathways in cell lines and primary neuronal monocultures is intrinsically limited. To address this, the authors developed an automated high-content imaging screen in high-density primary cultures of cortical and striatal neurons together with supporting glial cells.

High-throughput automated confocal microscopy imaging screen of a kinase-focused library to identify p38 mitogen-activated protein kinase inhibitors using the GE InCell 3000 analyzer.

The integration of fluorescent microscopy imaging technologies and image analysis into high-content screening (HCS) has been applied throughout the drug discovery pipeline to identify, evaluate, and advance compounds from early lead generation through preclinical candidate selection. In this chapter we describe the development, validation, and implementation of an HCS assay to screen compounds from a kinase-focused small-molecule library to identify inhibitors of the p38 pathway using the GE InCell 3000 automated imaging platform. The assay utilized a genetically modified HeLa cell line stably expressing mitogen-activated, protein-activating protein kinase-2 fused to enhanced green fluorescent protein (MK2-EGFP) and measured the subcellular distribution of the MK2-EGFP as a direct readout of p38 activation.

Endothelin signaling in osteoblasts: global genome view and implication of the calcineurin/NFAT pathway.

Patients with prostate cancer develop osteoblastic metastases when tumor cells arrive in the bone and stimulate osteoblasts by secreting growth-promoting factors. Endothelin 1 (ET-1) is believed to be a key factor in promoting osteoblastic metastasis. Selective blockade of the ET(A) receptor is an established strategy in the development of cancer therapeutics.

Assay development and case history of a 32K-biased library high-content MK2-EGFP translocation screen to identify p38 mitogen-activated protein kinase inhibitors on the ArrayScan 3.1 imaging platform.

This chapter describes the conversion and assay development of a 96-well MK2-EGFP translocation assay into a higher density 384-well format high-content assay to be screened on the ArrayScan 3.1 imaging platform. The assay takes advantage of the well-substantiated hypothesis that mitogen-activated protein kinase-activating protein kinase-2 (MK2) is a substrate of p38 MAPK kinase and that p38-induced phosphorylation of MK-2 induces a nucleus-to-cytoplasm translocation.

Development and implementation of three mitogen-activated protein kinase (MAPK) signaling pathway imaging assays to provide MAPK module selectivity profiling for kinase inhibitors: MK2-EGFP translocation, c-Jun, and ERK activation.

This chapter describes the development and implementation of three independent imaging assays for the major mitogen-activated protein kinase (MAPK) signaling modules: p38, JNK, and ERK. There are more than 500 protein kinases encoded in the human genome that share an ATP-binding site and catalytic domain conserved in both sequence and structure. The majority of kinase inhibitors have been found to be competitive with ATP, raising concerns regarding kinase selectivity and potency in an environment of millimolar intracellular concentrations of ATP, as well as the potential for off-target effects via the many other cellular proteins that bind and/or utilize ATP.

Generation and characterization of a stable MK2-EGFP cell line and subsequent development of a high-content imaging assay on the Cellomics ArrayScan platform to screen for p38 mitogen-activated protein kinase inhibitors.

This chapter describes the generation and characterization of a stable MK2-EGFP expressing HeLa cell line and the subsequent development of a high-content imaging assay on the Cellomics ArrayScan platform to screen for p38 MAPK inhibitors. Mitogen-activated protein kinase activating protein kinase-2 (MK2) is a substrate of p38 MAPK kinase, and p38-induced phosphorylation of MK-2 induces a nucleus to cytoplasm translocation (Engel et al., 1998; Neininger et al.