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Angiogenesis is an important hallmark of cancer, contributing to tumor formation and metastasis. In vitro angiogenesis models for analyzing tube formation serve as useful tools to study these processes. However, current in vitro co-culture models using primary cells have limitations in usefulness and consistency. Therefore, in the present study, an...
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... addition, most known angiogenesis inhibitors that were approved anticancer and antitumor drugs were identified in our screening, such as topotecan hydrochloride (IC 50 0.03 μM), docetaxel (IC 50 0.0025 μM), and bortezomib (IC 50 0.02 μM), consistent with the previous reports. 9 The known angiogenesis inhibitors and the related pathways are sum- marized in Table 1. Several potential novel inhibitors, like thimerosal and podofilox, were also identified in the screen with an IC 50 of 0.60 and 0.03 μM, respectively. ...
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... in the HIF-1α-NanoLuc reporter gene assay. There were 24 and 29 compounds identified from the NF-κB β-lactamase and lucif- erase reporter gene assays, respectively; 17 compounds were confirmed in both assays. The IC 50 and efficacy for the 50 compounds, as well as cell viability, are summarized for angio- genesis, HIF-1α, and NF-κB assays (Suppl . Table S1). The Figure 1. Establishment of an angiogenesis co-culture assay in response to sunitinib treatment. TeloHAEC-GFPs and hTERT-MSCs were co-cultured for 7 days. The co-cultured cells showed a fluorescent branching structure, which colocalized with the αSMA antibody. There were dose-response effects when the cells were treated with ...
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... Several novel compounds, such as podofilox, thimerosal, and maduramicin ammonium, were identified to be poten- tial angiogenesis inhibitors. Twenty-one compounds with known mechanisms that inhibit angiogenesis have been summarized (Table 1), and their IC 50 measurements were consistent with those of previous studies. Most of the listed compounds inhibited angiogenesis by affecting VEGF and HIF-1α pathways that are main regulators of angiogene- sis. ...
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... On the other hand, some assays are designed for a higher throughput format but can only measure a limited number of targets/pathways, such as the assay that detects chemicals that activate p53-dependent stress response (Ooka et al., 2022). To provide a more informative detection assay in multi-well plate format, we have developed high-content screening assays (Li et al., 2018;Li et al., 2019;Shahane et al., 2014). The high-content screening platform often employs cells expressing fluorescent-labeled proteins so that the kinetics and localization of the target protein can be readily monitored. ...
A number of chemicals in the environment pose a threat to human health. Recent studies indicate estradiol induces DNA damage through the activation of the estrogen receptor alpha (ERα). Given that many environmental chemical compounds act like hormones once they enter the human body, it is possible that they induce DNA damage in the same way as estradiol, which is of great concern to females with the BRCA1 mutation. In this study, we developed an antibody-based high content method measuring γH2AX, a biomarker for DNA damage, to test a subset of 907 chemical compounds in MCF7 cells. The assay was optimized for a 1536 well plate format and had a satisfactory assay performance with Z-factor of 0.67. From the screening, we identified 128 compounds that induce γH2AX expression in the cells. These compounds were further examined for their γH2AX induction in the presence of an ERα inhibitor, tamoxifen. After tamoxifen treatment, four compounds induced less γH2AX expression compared to those without tamoxifen treatment, suggesting these compounds induced γH2AX via ERα activation. These four compounds were chosen for further studies to assess their ERα activating capability and c-MYC induction. Only lestaurtinib, a selective tyrosine kinase inhibitor, induced ERα activation, which was confirmed by both ERα lactamase reporter gene assay and molecular docking analysis. Lestaurtinib also increased c-MYC expression, a target gene of ERα signaling, measured by the quantitative PCR method. This data suggests that lestaurtinib acts as a DNA damage inducer that is related to ERα activation.
... The advantage of this method is that it can mimic physiological conditions due to the capability of culturing different types of cells in one well [132]. For example, to evaluate compounds' developmental toxicity, mesenchymal stem cells and endothelial cells can be co-cultured in an angiogenesis assay [133,134]. Organoids, or 3D culture primary cells, are another future possibility in HTS. While previously mentioned 3D spheroids are derived from cancer cells, organoids are derived from adult organs or pluripotent stem cells [135]. ...
In vitro methods which incorporate metabolic capability into the assays allow us to assess the activity of metabolites from their parent compounds. These methods can be applied into high-throughput screening (HTS) platforms, thereby increasing the speed to identify compounds that become active via the metabolism process. HTS was originally used in the pharmaceutical industry and now is also used in academic settings to evaluate biological activity and/or toxicity of chemicals. Although most chemicals are metabolized in our body, many HTS assays lack the capability to determine compound activity via metabolism. To overcome this problem, several in vitro metabolic methods have been applied to an HTS format. In this review, we describe in vitro metabolism methods and their application in HTS assays, as well as discuss the future perspectives of HTS with metabolic activity. Each in vitro metabolism method has advantages and disadvantages. For instance, the S9 mix has a full set of liver metabolic enzymes, but it displays high cytotoxicity in cell-based assays. In vitro metabolism requires liver fractions or the use of other metabolically capable systems, including primary hepatocytes or recombinant enzymes. Several newly developed in vitro metabolic methods, including HepaRG cells, three-dimensional (3D) cell models, and organ-on-a-chip technology, will also be discussed. These newly developed in vitro metabolism approaches offer significant progress in dissecting biological processes, developing drugs, and making toxicology studies quicker and more efficient.
... While previous tubulogenesis and tubule length assays have demonstrated chemical specific effects on these endpoints [38,39,67], this study aimed to incorporate endothelial cell migration as an important endpoint of vascular formation. This was accomplished through direct measurement of wound area following a scratch assay and through CTNB translocation to the nucleus during migration. ...
Development of the neurovascular unit (NVU) is a complex, multistage process that requires orchestrated cell signaling mechanisms across several cell types and ultimately results in formation of the blood-brain barrier. Typical high-throughput screening (HTS) assays investigate single biochemical or single cell responses following chemical insult. As the NVU comprises multiple cell types interacting at various stages of development, a methodology combining high-throughput results across pertinent cell-based assays is needed to investigate potential chemical-induced disruption to the development of this complex cell system. To this end, we implemented a novel method for screening putative NVU disruptors across diverse assay platforms to predict chemical perturbation of the developing NVU. HTS assay results measuring chemical-induced perturbations to cellular key events across angiogenic and neurogenic outcomes in vitro were combined to create a cell-based prioritization of NVU hazard. Chemicals were grouped according to similar modes of action to train a logistic regression literature model on a training set of 38 chemicals. This model utilizes the chemical-specific pairwise mutual information score for PubMed MeSH annotations to represent a quantitative measure of previously published results. Taken together, this study presents a methodology to investigate NVU developmental hazard using cell-based HTS assays and literature evidence to prioritize screening of putative NVU disruptors towards a knowledge-driven characterization of neurovascular developmental toxicity. The results from these screening efforts demonstrate that chemicals representing a range of putative vascular disrupting compound (pVDC) scores can also produce effects on neurogenic outcomes and characterizes possible modes of action for disrupting the developing NVU.
... Many cellbased HTS assays were used to screen compounds with validation done in 384-well or 1536-well plate format. Readouts for these assays include angiogenesis (Li et al. 2018;Saili et al. 2019), micronucleus (Shahane et al. 2016), phospholipdosis (Shahane et al. 2014), and neurite outgrowth assays ). These HCS assays will be incorporated into the Tox21 screening program. ...
High-content screening (HCS) technology combining automated microscopy and quantitative image analysis can address biological questions in academia and the pharmaceutical industry. Various HCS experimental applications have been utilized in the research field of in vitro toxicology. In this review, we describe several HCS application approaches used for studying the mechanism of compound toxicity, highlight some challenges faced in the toxicological community, and discuss the future directions of HCS in regards to new models, new reagents, data management, and informatics. Many specialized areas of toxicology including developmental toxicity, genotoxicity, developmental neurotoxicity/neurotoxicity, hepatotoxicity, cardiotoxicity, and nephrotoxicity will be examined. In addition, several newly developed cellular assay models including induced pluripotent stem cells (iPSCs), three-dimensional (3D) cell models, and tissues-on-a-chip will be discussed. New genome-editing technologies (e.g., CRISPR/Cas9), data analyzing tools for imaging, and coupling with high-content assays will be reviewed. Finally, the applications of machine learning to image processing will be explored. These new HCS approaches offer a huge step forward in dissecting biological processes, developing drugs, and making toxicology studies easier.
... 4,6,10,11 In addition to technologies that improve the isolation and propagation of desired cells, this year's SLAS Technology Ten also includes work that improves ways to analyze these cells through both improved hardware and software solutions. 8,9,13 Staying close to the roots of SLAS Technology, additional articles highlight improved automation of a wide range of assays, with implications in everything from drug development to forensic science. 5,7,12 We would like to thank the 63 authors of the 2019 SLAS Technology Ten for these groundbreaking contributions and the hundreds of other researchers who also chose to share their impressive achievements in SLAS Technology in 2018. ...
... Nowadays innovations in combinatorial chemistry enable the preparation of large databases of compounds that cover large chemical space from so-called building blocks [4][5][6]. The development of high-throughput screening (HTS) assay makes it possible to assay and optimize thousands of molecules in 96-to 1536-well formats rapidly [7][8][9].However, these advanced techniques are still time-consuming and expensive. In order to expedite the drug development process in a more cost-efficient way and minimize the failure rate, top pharmaceutical companies and other researchers pay much attention to computer-aided drug discovery (CADD) techniques in various stages of drug discovery and development which can screen thousands of compounds instantly and select efficient candidates against the desired target proteins [10][11][12]. ...
Over the past ten years, the number of three-dimensional protein structures identified by advanced science and technology increases, and the gene information becomes more available than ever before as well. The development of computing science becomes another driving force which makes it possible to use computational methods effectively in various phases of the drug design and research. Now Structure-based drug design (SBDD) tools are widely used to help researchers to predict the position of small molecules within a three-dimensional representation of the protein structure and estimate the affinity of ligands to target protein with considerable accuracy and efficiency. They also accelerate discovery speed of potent drug and reduce the cost and times for drug research. Here we present an overview of SBDD used in drug discovery and highlight its recent successes and major challenges to current SBDD methodologies.
... This is an advance in development of biological tools that aid the process of screening, which may aid the identification of novel GPCR ligands and regulators of signaling in this clinically relevant set of targets. Li et al. 2 focus on the nature of cell culture systems used in high-throughput imaging assays and the development of a novel co-culture system of immortalized endothelial and mesenchymal cell lines to evaluate angiogenesis. Such a system allows the evaluation of compounds that may influence angiogenesis, a key process in development and cancer. ...
Hydrogel‐based 3D cell cultures can recapitulate (patho)physiological phenomena ex vivo . However, due to their complex multifactorial regulation, adapting these tissue and disease models for high‐throughput screening workflows remains challenging. In this study, a new Precision Culture Scaling (PCS‐X) methodology combines statistical techniques ( Design of Experiment and Multiple Linear Regression ) with automated, parallelized experiments and analyses to customize hydrogel‐based vasculogenesis cultures using human umbilical vein endothelial cells and retinal microvascular endothelial cells. Variations of cell density, growth factor supplementation and media composition are systematically explored to induce vasculogenesis in endothelial mono‐ and cocultures with mesenchymal stromal cells or retinal microvascular pericytes in 384‐well plate formats. The developed cultures are shown to respond to vasculogenesis inhibitors in a compound‐ and dose‐dependent manner, demonstrating the scope and power of PCS‐X in creating parallelized tissue and disease models for drug discovery and individualized therapies.
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The tumor microenvironment is crucial in the initiation and progression of cancers. The interplay between cancer cells and the surrounding stroma shapes the tumor biology and dictates the response to cancer therapies. Consequently, a better understanding of the interactions between cancer cells and different components of the tumor microenvironment will drive progress in developing novel, effective, treatment strategies. Co-cultures can be used to study various aspects of these interactions in detail. This includes studies of paracrine relationships between cancer cells and stromal cells such as fibroblasts, endothelial cells, and immune cells, as well as the influence of physical and mechanical interactions with the extracellular matrix of the tumor microenvironment. The development of novel co-culture models to study the tumor microenvironment has progressed rapidly over recent years. Many of these models have already been shown to be powerful tools for further understanding of the pathophysiological role of the stroma and provide mechanistic insights into tumor-stromal interactions. Here we give a structured overview of different co-culture models that have been established to study tumor-stromal interactions and what we have learnt from these models. We also introduce a set of guidelines for generating and reporting co-culture experiments to facilitate experimental robustness and reproducibility.
The number of three-dimensional (3D) structures of macromolecule/drug targets determined by experimental techniques has risen over the past years. Besides, the development and improvement of bioinformatics methods become an essential tool for the prediction of a good quality 3D model. Such growth is a regulator that makes it possible to use computational approaches in drug discovery programs. Structure-based drug design (SBDD) is a chemical structure design and optimization to find a drug candidate suitable for clinical research. It is focused on understanding the nature of a small molecule and how it coordinates with its biological target. In comparison to the conventional approach, it is an easier and cost-effective lead identification technique that accelerates the drug discovery process. Here, we provide a summary of the SBDD used in the discovery of drugs and highlight recent developments in this area and their limitations.
