High-throughput Drug Screening System Research Articles

A high-throughput system for drug screening based on the movement analysis of zebrafish

Zebrafish is a highly advantageous model animal for drug screening and toxicity evaluation thanks to its amenability to optical imaging (i.e., transparency), possession of organ structures similar to humans, and the ease with which disease models can be established. However, current zebrafish drug screening technologies and devices suffer from limitations such as low level of automation and throughput, and low accuracy caused by the heterogeneity among individual zebrafish specimens. To address these issues, we herein develop a high-throughput zebrafish drug screening system. This system is capable of maintaining optimal culturing conditions and simultaneously monitoring and analyzing the movement of 288 zebrafish larvae under various external conditions, such as drug combinations. Moreover, to eliminate the effect of heterogeneity, locomotion of participating zebrafish is assessed and grouped before experiments. It is demonstrated that in contrast to the experimental results without pre-selection, which shows ∼20 % damaged motor function (i.e., degree of attenuation), the drug-induced variations among zebrafish with equivalent mobility reaches ∼80 %. Overall, our high-throughput zebrafish drug screening system overcomes current limitations by improving automation, throughput, and accuracy, resulting in enhanced detection of drug-induced variations in zebrafish motor function.

Abstract PO5-14-06: Transcriptomics-based drug screening in 3D ex vivo patient-derived breast cancer model and patient biopsy for personalized therapy

Abstract Breast cancer is a leading cause of cancer-related mortality for women worldwide. Hormone receptor-positive (HR+) breast tumors, which represent 70% of all breast cancer cases, are treated with endocrine therapy. However, not all patients benefit from this treatment regimen because of patient-to-patient variation. Therefore, high throughput drug screening system is warranted to enable personalized medicine. Patient-derived organoids, which serve as a screening platform, lose their hormone receptors and response upon ex vivo culturing, and may not be adequate models for HR+ breast tumors. Moreover, unidentified biological components in the widely-used basement membrane matrix, Matrigel, result in high batch-to-batch variations and poor reproducibility in organoid cultures. Here, we propose a hydrogel-based 3D ex vivo model with defined structural and chemical properties to test hormone and drug sensitivity of HR+ breast tumors from patient-derived xenografts (PDXs) and patient tumor biopsies using microfluidics. Our data demonstrate the feasibility of this model to preserve cell proliferation and hormone receptor expression over 7 days. We also demonstrate that responses to hormones and FDA-approved drugs are faithfully maintained in this model. Finally, to establish a high throughput hormone and drug testing workflow with transcriptomic readout, we multiplexed barcoded- and drug-treated tumor samples in a single experiment with bulk RNA-sequencing. Our preliminary data demonstrate patient-specific responses to hormones and drugs that correspond to patient genetic mutation profiles, treatment history, disease stages and subtypes. This platform also enables testing of drugs in clinical trials that shows promising therapeutic outcomes for breast cancer, such as CDK4/6i, AKTi, PARPi, mTORi and their combination with endocrine therapy, thanks to the high throughput of the screening system and the low consumption of patient-derived or patient tissue. Given the capability of combining this physiologically-relevant 3D ex vivo model with RNA-seq for HR+ breast tumors, this platform holds potential for high throughput compound testing and transcriptomic profiling of patient biopsies for personalized medicine. Citation Format: Yueyun Zhang, Carlos Henrique Venturi Ronchi, Giovanna Ambrosini, Yuanlong Liu, Patrick Aouad, Daria Matvienko, Christoph Merten, Cathrin Brisken. Transcriptomics-based drug screening in 3D ex vivo patient-derived breast cancer model and patient biopsy for personalized therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-14-06.

Unveiling Epigenetic Vulnerabilities in Triple-Negative Breast Cancer through 3D Organoid Drug Screening.

Triple-negative breast cancer (TNBC) poses a therapeutic challenge due to its aggressive nature and lack of targeted therapies. Epigenetic modifications contribute to TNBC tumorigenesis and drug resistance, offering potential therapeutic targets. Recent advancements in three-dimensional (3D) organoid cultures, enabling precise drug screening, hold immense promise for identifying novel compounds targeting TNBC. In this study, we established two patient-derived TNBC organoids and implemented a high-throughput drug screening system using these organoids and two TNBC cell lines. Screening a library of 169 epigenetic compounds, we found that organoid-based systems offer remarkable precision in drug response assessment compared to cell-based models. The top 30 compounds showing the highest drug sensitivity in the initial screening were further assessed in a secondary screen. Four compounds, panobinostat, pacritinib, TAK-901, and JIB-04, targeting histone deacetylase, JAK/STAT, histone demethylases, and aurora kinase pathways, respectively, exhibited potent anti-tumor activity in TNBC organoids, surpassing the effect of paclitaxel. Our study highlights the potential of these novel epigenetic drugs as effective therapeutic agents for TNBC and demonstrates the valuable role of patient-derived organoids in advancing drug discovery.

SARS-CoV-2 nsp13 Restricts Episomal DNA Transcription without Affecting Chromosomal DNA.

Nonstructural protein 13 (nsp13), the helicase of SARS-CoV-2, has been shown to possess multiple functions that are essential for viral replication, and is considered an attractive target for the development of novel antivirals. We were initially interested in the interplay between nsp13 and interferon (IFN) signaling, and found that nsp13 inhibited reporter signal in an IFN-β promoter assay. Surprisingly, the ectopic expression of different components of the RIG-I/MDA5 pathway, which were used to stimulate IFN-β promoter, was also mitigated by nsp13. However, endogenous expression of these genes was not affected by nsp13. Interestingly, nsp13 restricted the expression of foreign genes originating from plasmid transfection, but failed to inhibit them after chromosome integration. These data, together with results from a runoff transcription assay and RNA sequencing, suggested a specific inhibition of episomal but not chromosomal gene transcription by nsp13. By using different truncated and mutant forms of nsp13, we demonstrated that its NTPase and helicase activities contributed to the inhibition of episomal DNA transcription, and that this restriction required direct interaction with episomal DNA. Based on these findings, we developed an economical and convenient high-throughput drug screening method targeting nsp13. We evaluated the inhibitory effects of various compounds on nsp13 by the expression of reporter gene plasmid after co-transfection with nsp13. In conclusion, we found that nsp13 can specifically inhibit episomal DNA transcription and developed a high-throughput drug screening method targeting nsp13 to facilitate the development of new antiviral drugs. IMPORTANCE To combat COVID-19, we need to understand SARS-CoV-2 and develop effective antiviral drugs. In our study, we serendipitously found that SARS-CoV-2 nsp13 could suppress episomal DNA transcription without affecting chromosomal DNA. Detailed characterization revealed that nsp13 suppresses episomal gene expression through its NTPase and helicase functions following DNA binding. Furthermore, we developed a high-throughput drug screening system targeting SARS-CoV-2 nsp13. Compared to traditional SARS-CoV-2 drug screening methods, our system is more economical and convenient, facilitating the development of more potent and selective nsp13 inhibitors and enabling the discovery of new antiviral therapies.

Salivary gland regeneration: from salivary gland stem cells to three-dimensional bioprinting.

Hyposalivation and severe dry mouth syndrome are the most common complications in patients with head and neck cancer (HNC) after receiving radiation therapy. Conventional treatment for hyposalivation relies on the use of sialogogues such as pilocarpine; however, their efficacy is constrained by the limited number of remnant acinar cells after radiation. After radiotherapy, the salivary gland (SG) secretory parenchyma is largely destroyed, and due to the reduced stem cell niche, this gland has poor regenerative potential. To tackle this, researchers must be able to generate highly complex cellularized 3D constructs for clinical transplantation via technologies, including those that involve bioprinting of cells and biomaterials. A potential stem cell source with promising clinical outcomes to reserve dry mouth is adipose mesenchymal stem cells (AdMSC). MSC-like cells like human dental pulp stem cells (hDPSC) have been tested in novel magnetic bioprinting platforms using nanoparticles that can bind cell membranes by electrostatic interaction, as well as their paracrine signals arising from extracellular vesicles. Both magnetized cells and their secretome cues were found to increase epithelial and neuronal growth of in vitro and ex vivo irradiated SG models. Interestingly, these magnetic bioprinting platforms can be applied as a high-throughput drug screening system due to the consistency in structure and functions of their organoids. Recently, exogenous decellularized porcine ECM was added to this magnetic platform to stimulate an ideal environment for cell tethering, proliferation, and/or differentiation. The combination of these SG tissue biofabrication strategies will promptly allow for in vitro organoid formation and establishment of cellular senescent organoids for aging models, but challenges remain in terms of epithelial polarization and lumen formation for unidirectional fluid flow. Current magnetic bioprinting nanotechnologies can provide promising functional and aging features to in vitro craniofacial exocrine gland organoids, which can be utilized for novel drug discovery and/or clinical transplantation.

Inhibition of CDCP1 by 8-isopentenylnaringenin synergizes with EGFR inhibitors in lung cancer treatment.

CUB domain-containing protein 1 (CDCP1) contributes to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance by regulating EGFR signaling pathways and is a potential target in lung cancer treatment. This study aims to identify a CDCP1 reducer that synergistically improves TKI treatment. Utilizing a high-throughput drug screening system, a phytoestrogen 8-isopentenylnaringenin (8PN) was identified. Upon 8PN treatment, CDCP1 protein levels and malignant features were reduced. 8PN exposure caused the accumulation of lung cancer cells in G0/G1 phase and increased the proportion of senescent cells. In EGFR TKI-resistant lung cancer cells, the combination of 8PN and TKI synergistically reduced cell malignance, inhibited downstream EGFR pathway signaling and exerted additive effects on cell death. Moreover, combination therapy effectively reduced tumor growth and enhanced tumor necrosis in tumor xenograft mice models. Mechanistically, 8PN increased interleukin (IL)6 and IL8 expression, induced neutrophil infiltration, and enhanced neutrophil-mediated cytotoxicity to attenuate lung cancer cell growth. In conclusion, 8PN enhances the anti-cancer efficacy of EGFR TKI on lung cancer, and triggers neutrophil-dependent necrosis, highlighting the potential to overcome TKI resistance in lung cancer patients who have EGFR mutation.

Abstract 5322: High throughput chemotherapeutic drug screening system for gastric cancer: cure-GA

Abstract To discover clinically applicable anticancer drugs and predict therapeutic response for advanced gastric cancer, we developed a high-throughput drug (HTD) screening system that could rapidly evaluate drug reactivity using 3D cultured primary cells derived from gastric cancer (GC) patients. Primary cancer cells were isolated from fresh surgical specimens that resected from 143 GC patients using Gentle Max tissue dissociation system. Primary cells were mixed with Matrigel, and placed on a micropillar for three-dimensional (3D) culture. After the primary cells were stabilized in the complete culture medium (CCM) than added various chemotherapeutic drugs containing 5-FU, Oxaliplatin, and Paclitaxel in CCM and incubated for 7 days. Cell viability was determined through calcein staining and quantified scanned images. The IC50 for each drug was calculated by a sigmoidal dose-response curve, using the GraphPad Prism 9 program. The average weight of gastric cancer tissue used in the experiment was 300 mg (75 mg ~ 1930 mg), and the average number of dissociated viable cells for each tissue was 3.9 × 10^6 cells/case. About 2.4 × 10^5 live cell was required per drug, we were able to obtain an average of 6.4 (Min.2, Max 14) drug reactivity data per tissue using the HTD screening system. GC tissues obtained from the operating room were dissociated within 16 hours and then loaded into the HTS system within 3 hours. Cells were stabilized for 1 day in 3D culture plate and exposed to the drug for 7 days, and then data reports were made within 3 days. As a result, it was possible to obtain within 14 days from fresh surgical GC tissue to drug response data. Additionally, we confirmed that 3D cultured primary cells derived from GC tissues consistently preserved primary characters using IHC. Similar to their parental cancer tissue, GC 3D cultured primary cells derived from adenocarcinoma large glandular patterns and retain the expression of some marker proteins. In this study, we evaluated the drug response data for 101 cases (success rate 71%; 101/143) to 5-FU, Oxaliplatin, and Palitaxel, etc. using the HTD screening system and it was confirmed that individual patient had a difference response to each drug. Here we established the HTD screening system using 3D cultured GC patient derived primary cells. The advantages of this system were that it is the first model system that directly used patient-derived primary cells for drug screening, and it can rapidly evaluate drug reactivity to various anticancer drugs within 10 days. The HTD screening system based on patient-derived primary cells can provide that information to predict drug response and allow for finding more appropriate therapy for each patient. Citation Format: Jieun Lee, Jung Eun Kim, Sanjun Lee, Tae-Kyeong Lee, In Hee Kim, So Hee Yoon, Mira Yoo, Eunju Lee, Doo-Young Hwang, So Hyun Kang, Bo Sung Ku, Dong Woo Lee, Young Suk Park, Ji-Won K, Jin Won Kim, Sang-Hoon Ahn, Keun-Wook Lee, Hyung-Ho Kim, Hyun Jung Oh, Yun-Suhk Suh. High throughput chemotherapeutic drug screening system for gastric cancer: cure-GA. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5322.

Development of a High-Throughput Drug Screening System Based on Human Peripheral Blood Derived Microglia-like Cells from Sickle Cell Patients with Chronic Pain

Background: Targeting microglia activation is a therapeutic strategy and a clinically promising drug target to treat microglia involved diseases, such as chronic pain, neurodegenerative disorders. Despite the interest in studying microglia as drug targets, drug discovery has been stymied by species related differences and lack of healthy human CNS microglia in the current models, Importantly, the current model systems do not faithfully recapitulate pathological characteristics of patients or disease processes, leading to the failure of many drug candidates in clinical trials. To address these shortcomings, we developed an efficient suspension culture system to generate peripheral blood derived microglia like cells that can express molecular markers, functional characteristics like true microglia and preserved patient-specific phenotype. As a novel, cost-effective, efficient, and patient-specific microglia model, the system can be applied to screen for compounds that reduce microglia hyperactivity, thereby identifying promising pharmacologic agents to be developed to treat chronic pain. Methods: Human peripheral blood mononuclear cells obtained from patients with sickle cell disease (SCD) were cultured in a suspension culture system for 6 days to generate high-throughput assay-ready human microglia-like cells (HPB-MLC) with human GM-CSF (10 ng/ml), human IL-34 (100 ng/ml), TGF-β2 (2 ng/mL). The microglial phagocytic function of HPB-MLC was determined by assessing the uptake of fluorescent-labeled Zymosan particles with imaging flow cytometry. Microglial activation of HPB-MLC was induced with LPS (100 ng/mL) and characterized by increased expression of TNF-alpha and microglial activation marker CD68. To evaluate pharmaceutical blockade of microglial activation, HPB-MLC were treated with 100 ng/ml LPS with or without minocycline (2.5, 5, 10, 25, 50 μg/mL), clopidogrel (1, 2, 4 μM) and MRS2395 (1, 5, 10 μM) for 24 hours. Commercial homogeneous cell-based assay kits based on HTRF (Homogeneous Time Resolved Fluorescence) and AlphaScreen technologies were used to develop the high-throughput screening (HTS) and miniaturizable assay. Results: Peripheral blood mononuclear cells can be efficiently differentiated into microglia-like cells in a suspension culture system. The suspension culture system enables fast and uniform seeding of cells to 384 well microplate for HTS applications. HPB-MLC express microglia specific markers, microglial phagocytic activity, and can be activated by LPS. HPB-MLC retain the patient pain phenotype: HPB-MLC generated from SCD patients with chronic pain exhibited higher phagocytic activity than that from SCD patients without chronic pain (Figure 1). We developed a cell-based, high-throughput screening assay by combination of our patient-specific microglia model and commercial homogeneous, high-throughput assay technologies HTRF and AlphaScreen (Figure 2). Suspension culture allowed 5 times higher cell concentration than adherent culture, which greatly reduced cost of cytokines in culture and produced assay-ready frozen HPB-MLC. To evaluate the possibility of using the HPB-MLC model system to screen compounds to inhibit microglia activation, we tested HPB-MLC cells with the several microglial inhibitors: minocycline, MRS2395, and clopidogrel. All the drugs significantly suppressed the release of proinflammatory cytokine TNF-alpha from LPS-induced activated PB-MLC in a dose-dependent manner. Conclusions: Peripheral blood monocytes can be efficiently differentiated into MLC in a suspension culture system, using for drug screening as a patient-specific microglia model. The differentiation in suspension culture worked very well in higher cell concentration (5 times higher than adherent culture), which greatly reduced cost of cytokines in culture. Assay-ready frozen HPB-MLC as a bank of uniform aliquots can be directly used for HTS applications. HTRF and AlphaScreen technologies were used to replace traditional ELISA assays to detect inflammatory cytokines, providing a highly sensitive, rapid, homogeneous and miniaturizable method to efficiently perform high throughput drug screening. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Establishment of high-throughput drug screening system using human limb bud mesenchymal cells derived from skeletal dysplasia specific iPSC

Establishment of high-throughput drug screening system using human limb bud mesenchymal cells derived from skeletal dysplasia specific iPSC

High-Throughput Drug Screening System Based on Human Induced Pluripotent Stem Cell-Derived Atrial Myocytes ∼ A Novel Platform to Detect Cardiac Toxicity for Atrial Arrhythmias.

Evaluation of proarrhythmic properties is critical for drug discovery. In particular, QT prolongation in electrocardiograms has been utilized as a surrogate marker in many evaluation systems to assess the risk of torsade de pointes and lethal ventricular arrhythmia. Recently, new evaluation systems based on human iPS cell-derived cardiomyocytes have been established. On the other hand, in clinical situations, it has been reported that the incidence of atrial arrhythmias such as atrial fibrillation has been increasing every year, with the prediction of a persistent increase in the near future. As to the increased incidence of atrial arrhythmias, in addition to the increased population of geriatric patients, a wide variety of drug treatments may be related, as an experimental method to detect drug-induced atrial arrhythmia has not been established so far. In the present study, we characterized the atrial-like cardiomyocytes derived from human induced pluripotent stem cells and examined their potential for the evaluation of drug-induced atrial arrhythmia. Atrial-like cardiomyocytes were induced by adding retinoic acid (RA) during the process of myocardial differentiation, and their characteristics were compared to those of RA-free cardiomyocytes. Using gene expression and membrane potential analysis, it was confirmed that the cells with or without RA treatment have atrial or ventricular like cardiomyocytes, respectively. Using the ultra-rapid activating delayed rectifier potassium current (IKur) channel inhibitor, which is specific to atrial cardiomyocytes, Pulse width duration (PWD) 30cF prolongation was confirmed only in atrial-like cardiomyocytes. In addition, ventricular like cardiomyocytes exhibited an early after depolarization by treatment with rapidly activating delayed rectifier potassium current (IKr) channel inhibitor, which induces ventricular arrhythmia in clinical situations. Here, we have established a high-throughput drug evaluation system using human iPS cell-derived atrial-like cardiomyocytes. Based on the obtained data, the system might be a valuable platform to detect potential risks for drug-induced atrial arrhythmias.

Establishing a high throughput drug screening system for cerebral ischemia using zebrafish larvae

We previously generated an ischemic stroke in a zebrafish model using N2 gas perfusion; however, this model was an unsuitable drug screening system due to low throughput. In this study, we examined a zebrafish ischemic stroke model using an oxygen absorber to assess drug effects. Hypoxic exposure more than 2 h using the oxygen absorber significantly induced cell death in the brain and damage to the neuronal cells. To confirm the utility of the ischemic model induced by the oxygen absorber, we treated zebrafish with neuroprotective agents. MK-801, an N-methyl-d-aspartate (NMDA) receptor antagonist, significantly suppressed cell death in the brain, and edaravone, a free radical scavenger, significantly reduced the number of dead cells. These results suggest that the activation of NMDA receptors and the production of reactive oxygen species induce neuronal cell damage in accordance with previous mammalian reports. We demonstrate the suitability of an ischemic stroke model in zebrafish larvae using the oxygen absorber, enabling a high throughput drug screening.

The high-throughput screening system for inhibitor Mycobacterium tuberculosis compounds based on ATP hydrolysis activity of recombinant protein ClpC1

The global Tuberculosis (TB) rate continues to increase by 1% per year with the widespread of drug-resistant TB. Therefore, the development and research to find new anti-TB drugs are becoming an extremely urgent mission. To be able to screen lead anti-tuberculosis drugs, currently, researchers have to carry out directly on the cells of Mycobacterium tuberculosis and to be performed in bio-security facilities level 3 or 4, to prevent infection from pathogens. However, our results demonstrated that the screening of anti-TB drug candidates can be implemented in bio-security facilities level 1 laboratory with the Escherichia coli cell extraction and recombinant ClpC1 protein - an integral part of the Mycobacterium tuberculosis genome. We focused on the ATP hydrolysis activity of ClpC1 to create a specific research direction for the high-throughput anti-tuberculosis drug screening system. ClpC1 protein was overexpressed and purified with functionally characterized (93.5 kDa). The steady-state growth of recombinant ClpC1 protein in Luria-Bertani (LB) broth High Salt medium was maintained and stabilized after extraction. The determined ATPase activity of ClpC1 was performed by measuring the released phosphate from the reaction. Ecumicin was chosen to be a control compound with expected ATP hydrolysis activities (Hill coefficient = 1,19 ± 0,217; Kd value = 0,52 ± 0,275). We tested this high throughput screening system with ten anti-TB drugs to evaluate the effectiveness of our screening system. Based on these results, we built a complete high-throughput screening system anti-tuberculosis drug safety and quickly.

Systematic Evaluation of Gastric Tumor Cell Index and Two-Drug Combination Therapy via 3-Dimensional High-Throughput Drug Screening.

Tumor heterogeneity greatly limits personalized treatment of cancer. Patient-derived tumor cell (PDC) models precisely recapitulate the molecular properties and biology of the disease, making them effective preclinical tools for assessing anti-cancer drug activities. Accurate estimation of tumor purity is essential for performing high-throughput drug screening (HTS). In the present study, we measured and predicted the tumor population index in PDC models for two-drug combinational strategies using HTS system. Gastric cancer cell-lines and PDCs were subjected to multi-color immunofluorescence analysis against EpCAM and vimentin to evaluate the tumor cell index based on EpCAM expression levels. We generated a tumor purity prediction model using five different gastric cancer cell-lines (AGS, KATO-III, MKN-45, NCI-N87, SNU-216) with fluorescence intensity-based techniques. Afterwards, stage IV gastric cancer PDC models were evaluated using a micropillar/microwell chip-based HTS system. HER2/CCNE1-amplified PDCs were considerably resistant to an HER2 inhibitor, while combinational treatment consisting of an HER2 inhibitor with anti-WEE1 compound substantially suppressed tumor cellular growth. Moreover, PDCs with BRCA1/2 mutations were synergistically sensitive to HER2 and PARP inhibition therapy. Finally, somatic mutations in TP53 and CDKN2A with MYC amplification rendered PDCs susceptible to the drug combination of WEE1 and HER2. Collectively, our systematic method of high-throughput drug sensitivity screening is an integral pre-clinical platform for evaluating potential two-drug combinational approaches for personalized treatment of cancer.

Artesunate Inhibits Graft-Versus-Host Disease in Mice Via a Mechanism of Inducing Mitochondrial Calcium Overloading in Activated T Cells

Despite pharmacological prophylaxis using calcineurin (CN) inhibitors (i.e., cyclosporin A and Tacrolimus), graft-versus-host disease (GVHD) remains a major barrier to the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Upon antigen stimulation, activated T cell receptor (TCR) signaling triggers rapid Ca2+ influx. This induces both CN-mediated NFAT activation, and increase of mitochondrial Ca2+ content, a major driver of metabolic activity. However, mitochondrial Ca2+ overload triggers opening of the mitochondrial permeability transition pore and cell death. We hypothesize that pharmacologically increasing mitochondrial Ca2+ load may decrease T cell survival capability, thereby reducing the GVH reaction. If this can be accomplished, it may lead to new strategies for inhibition of GVHD, which is conceptually different from the use of CN inhibitors. To test this hypothesis, we employed a high throughput drug screening system with the proliferation and cytotoxicity of TCR-activated human T cells as the readout, and screened the NPL-800 library (https://www.timtec.net) composed of 800 pure natural compounds. With a stringent criterion in consideration of dose-dependent effect, 26 compounds stood out for reducing the count of activated human T cells with a reduction rate of at least 30% at both 1.0uM and 10.0uM. Positive hits included inhibitors of DNA synthesis, the Na-K-ATPase and mitochondrial metabolism. We were particularly interested in artesunate (ART), which is a derivative of artemisinin that has been used for treating malaria in patients. While artemisinin acts by inhibiting sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in P. falciparum malaria, which causes passive endoplasmic reticulum (ER) Ca2+ depletion and the subsequent cytosolic Ca2+ influx, ART did not inhibit SERCA in T cells. Ex vivo culture assays showed that ART dose-dependently reduced the survival of TCR-activated murine T cells. This inhibitory effect of ART was abrogated by inhibiting Ca2+ influx using BTP2, a potent inhibitor of store-operated Ca2+ channels. Furthermore, treatment of murine CD8 T cells with ART induced significant increases in mitochondrial Ca2+ loading upon TCR activation. These data suggest that inhibition of T cell survival by ART was dependent on TCR activation-induced Ca2+ influx and associated with enhanced mitochondrial Ca2+ uptake. We examined the impact of ART on GVHD in Balb/c mice receiving C57BL/6 (B6) mouse T cell-depleted bone marrow (TCD-BM) and CD4+ T cells. Intraperitoneal injection of ART (10 mg/kg, every other day) from day 1 to day 28 after transplantation reduced clinical signs of GVHD in these recipients and significantly improved their overall survival. Similar inhibition effects of ART on GVHD were observed in miHA-mismatched B6 anti-Balb/b and haplo-identical B6 anti-BDF1 mouse models of GVHD. Further investigations showed that in vivo administration of ART caused significant decreases in the number of host-reactive donor T cells in the spleen and liver of Balb/c mice 7 days after transfer of B6 TCD-BM plus CD4+ T cells. ART treatment did not affect the capacity of donor T cells to produce effector cytokines (e.g., IFN-g and TNF-α) in individual cells. Importantly, in vivo administration of ART preserved anti-leukemia activity of donor T cells and did not impair the reconstitution of hematopoiesis and lymphocytes. Collectively, our findings indicate that pharmacologically increasing mitochondrial Ca2+ loading may have significant implications in the development of novel strategies to prevent GVHD and other T cell-mediated inflammatory disorders in a broad context. Since ART therapy has been clinically approved, this work could be immediately translated into patients. Disclosures No relevant conflicts of interest to declare.

Systematic Evaluation of Tumor Cell Index and Two-Drug Combination Therapy via 3-Dimensional High-Throughput Drug Screening

Background: Tumour heterogeneity greatly limits personalised treatment of cancer. Patient-derived tumour cell (PDC) models precisely recapitulate the molecular properties and biology of the disease, making them effective preclinical tools for assessing anti-cancer drug activities. Accurately estimating tumor purity is essential for performing high-throughput drug screening (HTS). In the current study, we measured and predicted the tumour population index in PDC models for two-drug combinational strategies using an HTS system. Methods: Gastric cancer cell-lines and PDCs were subjected to multi-colour immunofluorescence analysis against EpCAM and vimentin to evaluate the tumour cell index based on EpCAM expression levels. We generated a tumour purity prediction model using five different gastric cancer cell-lines (AGS, KATO-III, MKN-45, NCI-N87, SNU-216) with fluorescence intensity-based techniques. These stage IV gastric cancer PDC models were evaluated using a micropillar/microwell chip-based HTS system. Findings: HER2/CCNE1-amplified PDCs were considerably resistant to an HER2 inhibitor, while combinational treatment consisting of an HER2 inhibitor with anti-WEE1 compound substantially suppressed tumor cell growth. Moreover, PDCs with BRCA1/2 mutations were synergistically sensitive to HER2 and PARP inhibition therapy. Finally, somatic mutations in TP53 and CDKN2A with MYC amplification rendered PDCs susceptible to the drug combination of WEE1 and HER2. Interpretation: Collectively, our systematic method of high-throughput drug sensitivity screening is an integral pre-clinical platform for evaluating potential two-drug combinational approaches for personalised treatment of cancer. Funding: This work was supported by the AZ-KHIDI (AstraZeneca-Korea Health Industry Development Institute) R & D program. Declaration of Interest: The authors declare no competing interests. Ethical Approval: This investigation was conducted in accordance with the ethical standards of the Declaration of Helsinki and national and international guidelines and was approved by the Institutional Review Board at Samsung Medical Center in Seoul, Korea.

Zelnorm, an agonist of 5-Hydroxytryptamine 4-receptor, acts as a potential antitumor drug by targeting JAK/STAT3 signaling.

The Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway plays central roles in cancer cell growth and survival. Drug repurposing strategies have provided a valuable approach for developing antitumor drugs. Zelnorm (tegaserod maleate) was originally designed as an agonist of 5-hydroxytryptamine 4 receptor (5-HT4R) and approved by the FDA for treating irritable bowel syndrome with constipation (IBS-C). Through the use of a high-throughput drug screening system, Zelnorm was identified as a JAK/STAT3 signaling inhibitor. Moreover, the inhibition of STAT3 phosphorylation by Zelnorm was independent of its original target 5-HT4R. Zelnorm could cause G1 cell cycle arrest, induce cell apoptosis and inhibit the growth of a variety of cancer cells. The present study identifies Zelnorm as a novel JAK/STAT3 signaling inhibitor and reveals a new clinical application of Zelnorm upon market reintroduction.

1409 High-throughput drug screening system targeting tissue fibrosis: An application of PDMS stretch platform

Fibrosis is a process of abnormal accumulation of extracellular matrix (ECM) substrates, which have been a key driver of progressive organ dysfunction. Keloids are representative fibro-proliferative disease of skin with abnormal fibroblast activity. However, the treatment options are limited and current drug development platforms cannot fully reflect mechanosensitive property of the disease. Strain devices with an expandable silicone such as polydimethylsiloxane (PDMS) culture membranes can mimic mechanically dynamic tissue environments. Here, we established a PDMS based stretchable culture system to evaluate as a drug screening platform. Patient-derived keloid fibroblasts were cultured on the PDMS stretch platform. In response to cyclic stretch, the cellular proliferation reached the optimal activity level. Afterwards, the cells were treated with corticosteroids as a positive control and drugs with anti-fibrosis activity were selected. Expression of fibrosis-related proteins was evaluated; TGF-β was significantly reduced and type 1 collagen was subsequently reduced too (p < 0.05). It is notable that the PDMS platform were sufficient to demonstrate a drug response as in physiologic conditions owing to its own mechanical property of cyclic stretch. For future treatment approaches, our model may facilitate high-throughput screening under a physiologic condition.

Development of a semi-automated image-based high-throughput drug screening system.

We previously reported that the innate sensing of the endosymbiont Leishmania RNA virus 1 (LRV1) within Leishmania (Viannia) guyanensis through Toll-like receptor 3, worsens the pathogenesis of parasite infection in mice. The presence of LRV1 has been associated with the failure of first-line treatment in patients infected with LRV1 containing -L. guyanensis and -L. braziliensis parasites. Here, we established a semi-automated image-based high-throughput drug screening (HTDS) protocol to measure parasiticidal activity of the Prestwick chemical library in primary murine macrophages infected with LRV1-containing L. guyanensis. The two-independent screens generated 14 hit compounds with over sixty-nine percent reduction in parasite growth compared to control, at a single dose in both screens. Our screening strategy offers great potential in the search for new drugs and accelerates the discovery rate in the field of drug repurposing against Leishmania. Moreover, this technique allows the concomitant assessment of the effect of drug toxicity on host cell number.

Skeletal muscle generated from induced pluripotent stem cells - induction and application.

Human induced pluripotent stem cells (hiPS cells or hiPSCs) can be derived from cells of patients with severe muscle disease. If skeletal muscle induced from patient-iPSCs shows disease-specific phenotypes, it can be useful for studying the disease pathogenesis and for drug development. On the other hand, human iPSCs from healthy donors or hereditary muscle disease-iPSCs whose genomes are edited to express normal protein are expected to be a cell source for cell therapy. Several protocols for the derivation of skeletal muscle from human iPSCs have been reported to allow the development of efficient treatments for devastating muscle diseases. In 2017, the focus of research is shifting to another stage: (1) the establishment of mature myofibers that are suitable for study of the pathogenesis of muscle disease; (2) setting up a high-throughput drug screening system; and (3) the preparation of highly regenerative, non-oncogenic cells in large quantities for cell transplantation, etc.

YM155 Induces Apoptosis through Proteasome-Dependent Degradation of MCL-1 in Primary Effusion Lymphoma

Primary effusion lymphoma (PEL) is a subtype of non-Hodgkin lymphoma caused by human herpes virus 8 (HHV-8), which mainly occurs in patients with acquired immunodeficiency. It is highly refractory to conventional chemotherapies, and has a very poor prognosis. We recently developed patient-derived xenograft (PDX) screening, a novel high-throughput drug screening system using PDX cells that were established by transplantations of primary tumor cells into immunodeficient mice and maintained primary cell phenotype. PDX screening is expected to discover anti-tumor drugs that have been overlooked by conventional screenings using cell lines. Here, we performed a PDX screening to develop a new therapeutic agent for PEL.We previously established a PDX and a cell line designated as GTO from the same primary cells of PEL. We performed screenings of a library containing 3518 known pharmacologically active substance and off-patent drugs using the PDX cells (PDX screening) and GTO (Cell-line screening). We compared the results of both screenings and found that PDX cells and cell lines had quite different drug sensitivity profiles. The correlation coefficient between them was 0.67. Twenty-six drugs (0.7%) were at least 2 times more effective for PDX cells than for GTO and designated as PDX-preferred drugs (Figure A). The opposites were named as cell line-preferred drugs and existed 80 (2.2%). We found that PDX-preferred drugs significantly higher activity to induce reactive oxygen species (ROS) production (P<0.001), indicating the sensitivity of PDX cells to oxidative stress.We examined the reproducibility of anti-tumor effect of top 10 compounds of PDX screening in different system including in vivo mouse model and finally selected YM155, a possible survivin inhibitor, as the best candidate for an anti-tumor drug for PEL. It showed strong and dose-dependent anti-tumor effect on both PDX cells and cell lines of PEL. Its GI50 was 7.8 nM in the PDX cells, and 1.2 - 7.9 nM in three kinds of PEL cell lines.YM155 treatment increased the cleavage of caspase-3, caspase-7, and PARP and caused apoptosis of GTO, which was inhibited by a caspase inhibitor, Z-VAD-FMK. Although YM155 was discovered as a survivin inhibitor, we observed that YM155 reduced myeloid cell leukemia-1 (MCL-1) protein prior to survivin reduction by time course experiments. Observed MCL-1 reduction by YM155 was attenuated by a proteasome inhibitor, MG132, suggesting that MCL-1 reduction was due to proteasome-dependent degradation. Furthermore, we confirmed the importance of MCL-1 for survival by its knockdown by siRNA in PEL cell line.Finally, we assessed the in vivo effect of YM155. NOD/SCID/IL-2Rgnull mice were injected intraperitoneally with PEL-PDX cells and were treated with vehicle or YM155 (5mg/kg) from day 1 to 21. YM155 was administered by continuous subcutaneous injection using osmotic pumps. Treatment with YM155 significantly inhibited progression of ascites compared with control mice (Figure B). These results suggested that YM155 was a promising anti-cancer agent for PEL. [Display omitted] DisclosuresSugimoto:Otsuka Pharmaceutical Co., Ltd.: Employment. Kiyoi:Chugai Pharmaceutical Co. LTD.: Research Funding; Alexion Pharmaceuticals: Research Funding; MSD K.K.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Astellas Pharma Inc.: Consultancy, Research Funding; Yakult Honsha Co.,Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Zenyaku Kogyo Co.LTD.: Research Funding; Phizer Japan Inc.: Research Funding; Novartis Pharma K.K.: Research Funding; Mochida Pharmaceutical Co., Ltd.: Research Funding; Toyama Chemikal Co.,Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; AlexionpharmaLLC.: Research Funding; JCR Pharmaceutlcals Co.,Ltd.: Research Funding; Nippon Boehringer Ingelheim Co., Ltd.: Research Funding; Celgene Corporation: Consultancy; Eisai Co., Ltd.: Research Funding; Kyowa-Hakko Kirin Co.LTD.: Research Funding.