High-throughput Drug Screening Assays Research Articles

Organoids Research for Colorectal Cancer: Promising Approach for Precision Medicine, their Applications and Future Perspectives

Background: Organoids are three-dimensional (3D) constructs designed to emulate the complexity and functionality of organs in the body. Organoids have recently been used as powerful instruments for modeling and investigating several diseases, including colorectal cancer. Colorectal cancer is caused by altering colonic epithelial cells, which produce adenomas and carcinomas. Objective: The objective of present study was to investigate impact of organoids on colorectal cancer and their therapeutic outcome in cancer research. Organoids can be grown from stem cells in vitro, which closely resemble the structure and function of the organ they are derived from. They have been used in a variety of research applications, including disease modeling, drug screening, and personalized medicine. Organoids have allowed researchers to understand better the mechanisms underlying colorectal cancer initiation, progression, and resistance to therapy. Methods: The literature review was surveyed, and keywords related to cancer management, organoids, modelling, personized medicine, 3D structures were screened for colorectal cancer management were screened in SCI-hub, SCOPUS, WOS, and ABC Journals. Results: The findings of studies suggested that organoids derived from patient tumors can recapitulate the histopathology and genetic alterations of the original tumor, making them a valuable tool for personalized medicine. Conclusion: Organoids have been used to develop high-throughput drug screening assays and investigate the tumor microenvironment's contribution to colorectal cancer progression. In this review, we summarize recent advances in the use of organoids to study colorectal cancer and discuss their potential applications in the clinic.

ETMR-18. ADVANCING ETMR RESEARCH: NOVELIN VITRO ANDIN VIVO PRECLINICAL MODELS FOR DEVELOPING MORE EFFECTIVE TREATMENTS

Abstract BACKGROUND Embryonal Tumor with Multilayered Rosettes (ETMR) is a rare and aggressive tumor that occurs mainly in infants under three years of age. Genetically, ETMRs are characterized by amplification and fusion of a microRNA cluster on chromosome 19 (C19MC) with TTYH1, present in ~90% of the tumors. ETMR patients still have a poor prognosis without any recent advances in treatment. A major limitation to develop more effective treatment strategies is the lack of sufficient preclinical models. METHODS Establishment of ETMR patient-derived xenograft (PDX) models and tumoroid lines, molecular characterization, high throughput in vitro drug screens, targeted drug treatments in vitro and in vivo. RESULTS We have established four new tumoroid and five new PDX ETMR models. These models faithfully recapitulate ETMR patients’ tumors based on their histological, transcriptomic, and methylome signatures. The in vitro models were derived from two patients and two PDX models and included three C19MC-amplified cell lines, of which two were a primary-relapse pair, and one a primary C19MC non-amplified model. In vivo we established the same models as PDX models subcutaneously plus an additional C19MC-amplified relapse model. High-throughput drug screening assays, using drug libraries with 225 clinically approved drugs and other small molecules, were conducted across the panel of in vitro models. Potential therapeutic candidates with promising activity in ETMR tumoroid lines identified from these screens include FGFR, XPO1, MTOR, MDM2 and topoisomerase inhibitors. CONCLUSIONS The establishment of novel preclinical models represents a significant advancement in ETMR research, offering a platform that captures the molecular complexity of the disease. The identification of several inhibitors shows the potential of these models for drug discovery and preclinical testing. Ongoing in vitro and in vivo experiments with these drugs and combinations aim to further validate their activity, moving towards the development of more effective therapies for ETMR patients.

Abstract 3234: A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2-deficient pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States, with the lowest five-year survival rate of all cancers. Around 10% of pancreatic cancer diagnoses are thought to be familial pancreatic cancer (FPC). BRCA2 pathogenic variants have been identified to be closely associated with FPC. Personalized medicine approaches that are tailored to FPC patients based on their mutation profiles could improve patient outcomes and minimize adverse effects. This study aims to identify novel potential drug candidates for BRCA2-deficient PDAC and dissect its underlying molecular mechanisms. To discover new vulnerabilities for BRCA2-deficient pancreatic cancer, we performed high-throughput drug screening (HTS) using isogenic murine Brca2 knockout (KO) and control PDAC cells. The HTS assay revealed that JQ1, a small molecule inhibitor of the bromodomain and extra-terminal domain protein (BET) family, selectively induced cytotoxicity in Brca2 KO cells. JQ1 significantly decreased the cell viability of Brca2 KO cells in vitro and suppressed the growth of Brca2 KO tumors in vivo compared to the controls. The transcriptomic analyses showed that JQ1 treatment resulted in upregulation of the gene sets associated with macroautophagy. Multi-orthogonal autophagy assays, including live cell imaging of fluorescence-based autophagy reporter, also supported that Brca2 KO cells had a constitutively higher basal level of autophagic activities compared to the controls, and JQ1 further induced autophagic flux in Brca2 KO cells. Moreover, blocking the autophagy process by pharmacological inhibition or knocking down essential autophagy genes rescued JQ1-induced cell death in Brca2 KO cells, indicating that the increased autophagy is responsible for JQ1-mediated cell death in Brca2 KO cells. In conclusion, we found that BRCA2 deficiency elevated autophagic flux, and extensive autophagy was further activated by BET inhibition, culminating in autophagy-dependent cell death in Brca2-deficient pancreatic cancer cells. Our findings suggest that BET inhibition could be a promising therapeutic strategy for treating BRCA2-deficient pancreatic cancer. Citation Format: Suyakarn Archasappawat, EunJung Lee, Keely Ji, Jocelyn Pena, Virneliz Fernandez Vega, Ritika Gangaraju, Nitin Sai Beesabathuni, Martin Jean Kim, Qi Tian, Priya Shah, Louis Scampavia, Timothy Spicer, Chang-Il Hwang. A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2-deficient pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3234.

Beyond the Nucleus: Plastic Chemicals Activate G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) are central mediators of cell signaling and physiological function. Despite their biological significance, GPCRs have not been widely studied in the field of toxicology. Herein, we investigated these receptors as novel targets of plastic chemicals using a high-throughput drug screening assay with 126 human non-olfactory GPCRs. In a first-pass screen, we tested the activity of triphenol phosphate, bisphenol A, and diethyl phthalate, as well as three real-world mixtures of chemicals extracted from plastic food packaging covering all major polymer types. We found 11 GPCR-chemical interactions, of which the chemical mixtures exhibited the most robust activity at adenosine receptor 1 (ADORA1) and melatonin receptor 1 (MTNR1A). We further confirm that polyvinyl chloride and polyurethane products contain ADORA1 or MTNRA1 agonists using a confirmatory secondary screen and pharmacological knockdown experiments. Finally, an analysis of the associated gene ontology terms suggests that ADORA1 and MTNR1A activation may be linked to downstream effects on circadian and metabolic processes. This work highlights that signaling disruption caused by plastic chemicals is broader than that previously believed and demonstrates the relevance of nongenomic pathways, which have, thus far, remained unexplored.

Abstract A079: A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2-deficient pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States, with the lowest five-year survival rate of all cancers. Around 10% of pancreatic cancer diagnoses are thought to be familial pancreatic cancer (FPC). BRCA2 pathogenic variants have been identified to be closely associated with FPC. Personalized medicine approaches that are tailored to FPC patients based on their mutation profiles could improve patient outcomes and minimize adverse effects. This study aims to identify novel potential drug candidates for BRCA2-deficient PDAC and dissect its underlying molecular mechanisms. To discover new vulnerabilities for BRCA2-deficient pancreatic cancer, we performed high-throughput drug screening (HTS) using isogenic murine Brca2 knockout (KO) and control PDAC cells. The HTS assay revealed that JQ1, a small molecule inhibitor of the bromodomain and extra-terminal domain protein (BET) family, selectively induced cytotoxicity in Brca2 KO cells. JQ1 significantly decreased the cell viability of Brca2 KO cells in vitro and suppressed the growth of Brca2 KO tumors in vivo compared to the controls. The transcriptomic analyses showed that JQ1 treatment resulted in upregulation of the gene sets associated with macroautophagy. Multi-orthogonal autophagy assays, including live cell imaging of fluorescence-based autophagy reporter, also supported that Brca2 KO cells had a constitutively higher basal level of autophagic activities compared to the controls, and JQ1 further induced autophagic flux in Brca2 KO cells. Moreover, blocking the autophagy process by pharmacological inhibition or knocking down essential autophagy genes rescued JQ1-induced cell death in Brca2 KO cells, indicating that the increased autophagy is responsible for JQ1-mediated cell death in Brca2 KO cells. In conclusion, we found that BRCA2 deficiency elevated autophagic flux, and extensive autophagy was further activated by BET inhibition, culminating in autophagy-dependent cell death in Brca2-deficient pancreatic cancer cells. Our findings suggest that BET inhibition could be a promising therapeutic strategy for treating BRCA2-deficient pancreatic cancer. Citation Format: Suyakarn Archasappawat, EunJung Lee, Keely Ji, Jocelyn Pena, Virneliz Fernandez-Vega, Ritika Gangaraju, Nitin Sai Beesabathuni, Martin Jean Kim, Qi Tian, Priya Shah, Louis Scampavia, Timothy Spicer, Chang-Il Hwang. A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2-deficient pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A079.

Ultralight 3D Graphene Oxide Aerogel Decorated with Pd-Fe Nanoparticles for the Simultaneous Detection of Eight Biomolecules.

In this proof-of-concept study, an ultralight graphene oxide aerogel (GOx-Aero) decorated with bimetallic palladium-iron nanoparticles (Pd-Fe) was synthesized and immobilized on a glassy carbon electrode (GCE) for electrochemical sensor applications. The main objective of this work was to develop a sensitive electrochemical sensor capable of simultaneously detecting eight biomolecules, including ascorbic acid (AA), dopamine (DA), uric acid (UA), 8-hydroxyguanine (8HG), guanine (G), adenine (A), thymine (T), and cytosine (C). To the best of our knowledge, this is the first time that an electrochemical sensor has been able to detect eight biomolecules simultaneously. The bimetallic GOx aerogel significantly enhanced the performance of the sensor by increasing the electroactive area, conductivity, and anodic peak current response. The sensor demonstrated sharp, well-defined, and continuous oxidation peaks for all eight analytes of interest and wide linear ranges of 5.0-1750, 0.25-100.0, 0.5-500.0, 0.5-375.0, 0.5-500.0, 0.5-500.0, 5.0-1500.0, and 5.0-1500.0 μM for AA, DA, UA, 8HG, G, A, T, and C, respectively. The prepared sensor also exhibited excellent stability, reproducibility, and sensitivity with a very low limit of detection (LOD) of 553.7, 1.8, 69.6, 43.2, 42.9, 72.3, 57.2, and 318.4 nM for AA, DA, UA, 8HG, G, A, T, and C, respectively. The Pd-Fe-GOx-Aero-GCE was also tested in various real samples such as artificial saliva, artificial cerebrospinal fluid (CSF), salmon sperm DNA, and genomic DNA from calf thymus, where it demonstrated good recovery values. Additionally, the novel developed sensor was used to monitor the interaction between the anticancer drug, cisplatin, which has well-described binding affinity with the G and A bases in DNA. Overall, Pd-Fe-GOx-Aero-GCE displayed an extremely promising platform not only for the simultaneous detection of eight biomolecules in complex biological matrices but also for DNA-drug interaction studies toward the development of electrochemical high-throughput drug screening assays, which is of great importance in the field.

In vitro cardiac modeling of desmin-related myofibrillar myopathy and deep learning-based image analysis to develop a high-content screening assay

Mutations of DES, the gene encoding desmin, the main intermediate filament of muscle cells, are one of the major cause of Myofibrillar myopathy(MFM). This disease is mainly characterized by a muscular dystrophy but also, in many cases, by the progressive emergence of cardiac dysfunctions evolving frequently from dilated cardiomyopathy to heart failure. For the moment, no specific treatment can revert this disease. The following study relied on the development of an automated tool base on deep-learning to discriminate efficiently mutant and control cardiomyocytes in the perspective of drug screening. We use cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) obtained from control or patients carrying a DES mutation (E439K). Phenotyping of hiPSC-CMs was performed by immunocytostaining of the sarcomeres or the desmin network. After imaging, the morphological perturbations were evaluated manually based either on the sarcomeric organization or on the aspect of the desmin network at single cell level. Then an automated classification model was generated by using machine-learning method trained with the previous images annotated with the results of the manual classifications. In parallel, another automated classification model based on cell profiling from sarcomeric, desmin and mitochondrial staining by using the deep learning EfficientNet B0 model was setting up in order to distinguish Control cell and DES mutated cell. Manual classification based on sarcomeric organization was established on 4 classes ranging from cells with disorganized sarcomeres to cells with a well-organized sarcomeric network on 1500 single cells. We observed that the distribution of DES mutated cells is shifted toward the class of disorganized sarcomere compared to control cells. Identically, we also manually classified 1200 single cells based on the organization of the desmin network (4 classes). We also observed a shift of DES mutant cells to be classified as cells with aggregated desmin network compared to control cells. Finally, the various validation tests of the two automated classification methods showed significant correlation with the manual classification methods. Here we demonstrate the efficacy of our automated classification software to be able to discriminates mutant and control cells. This analysis platform will then be used to perform high throughput drug screening assays to discover new therapeutic molecules.

Eugenol attenuates ischemia-mediated oxidative stress in cardiomyocytes via acetylation of histone at H3K27.

Despite clinical advances, ischemia-induced cardiac diseases remain an underlying cause of death worldwide. Epigenetic modifications, especially alterations in the acetylation of histone proteins play a pivotal role in counteracting stressful conditions, including ischemia. In our study, we found that histone active mark H3K27ac was significantly reduced and histone repressive mark H3K27me3 was significantly upregulated in the cardiomyocytes exposed to the ischemic condition. Then, we performed a high throughput drug screening assay using rat ventricular cardiomyocytes during the ischemic condition and screened an antioxidant compound library comprising of 84 drugs for H3K27ac by fluorescence microscopy. Our data revealed that most of the phenolic compounds like eugenol, apigenin, resveratrol, bis-demethoxy curcumin, D-gamma-tocopherol, ambroxol, and non-phenolic compounds like l-Ergothioneine, ciclopirox ethanolamine, and Tanshinone IIA have a crucial role in maintaining the cellular H3K27ac histone marks during the ischemic condition. Further, we tested the role of eugenol on cellular protection during ischemia. Our study shows that ischemia significantly reduces cellular viability and increases total reactive oxygen species (ROS), and mitochondrial ROS in the cells. Interestingly, eugenol treatment significantly restores the cellular acetylation at H3K27, decreases cellular ROS, and improves cellular viability. To explore the mechanism of eugenol-medicated inhibition of deacetylation, we performed a RNAseq experiment. Analysis of transcriptome data using IPA indicated that eugenol regulates several cellular functions associated with cardiovascular diseases, and metabolic processes. Further, we found that eugenol regulates the expression of HMGN1, CD151 and Ppp2ca genes during ischemia. Furthermore, we found that eugenol might protect the cells from ischemia through modulation of HMGN1 protein expression, which plays an active role in regulation of histone acetylation and cellular protection during stress. Thus, our study indicated that eugenol can be exploited as an agent to protect the ischemic cells and also could be used to develop a novel drug for treating cardiac disease.

Cryopreservation of assay-ready hepatocyte monolayers by chemically-induced ice nucleation: preservation of hepatic function and hepatotoxicity screening capabilities.

Cell culture plays a critical role in biomedical discovery and drug development. Primary hepatocytes and hepatocyte-derived cell lines are especially important cellular models for drug discovery and development. To enable high-throughput screening and ensure consistent cell phenotypes, there is a need for practical and efficient cryopreservation methods for hepatocyte-derived cell lines and primary hepatocytes in an assay-ready format. Cryopreservation of cells as adherent monolayers in 96-well plates presents unique challenges due to low volumes being susceptible to supercooling, leading to low recovery and well-to-well variation. Primary cell cryopreservation is also particularly challenging due to the loss of cell viability and function. In this study, we demonstrate the use of soluble ice nucleator materials (IN) to cryopreserve a hepatic-derived cell line (HepG2) and primary mouse hepatocytes, as adherent monolayers. HepG2 cell recovery was near 100% and ∼75% of primary hepatocytes were recovered 24 hours post-thaw compared to just 10% and 50% with standard 10% DMSO, respectively. Post-thaw assessment showed that cryopreserved HepG2 cells retain membrane integrity, metabolic activity, proliferative capacity and differentiated hepatic functions including urea secretion, cytochrome P450 levels and lipid droplet accumulation. Cryopreserved primary hepatocytes exhibited reduced hepatic functions compared to fresh hepatocytes, but functional levels were similar to commercial suspension-cryopreserved hepatocytes, with the added benefit of being stored in an assay-ready format. In addition, normal cuboidal morphology and minimal membrane damage were observed 24 hours post-thaw. Cryopreserved HepG2 and mouse hepatocytes treated with a panel of pharmaceutically active compounds produced near-identical dose-response curves and EC50 values compared to fresh hepatocytes, confirming the utility of cryopreserved bankable cells in drug metabolism and hepatotoxicity studies. Cryopreserved adherent HepG2 cells and primary hepatocytes in 96 well plates can significantly reduce the time and resource burden associated with routine cell culture and increases the efficiency and productivity of high-throughput drug screening assays.

Organic electronic transmembrane device for hosting and monitoring 3D cell cultures.

3D cell models have made strides in the past decades in response to failures of 2D cultures to translate targets during the drug discovery process. Here, we report on a novel multiwell plate bioelectronic platform, namely, the e-transmembrane, capable of supporting and monitoring complex 3D cell architectures. Scaffolds made of PEDOT:PSS [poly(3,4-ethylenedioxythiophene):polystyrene sulfonate] are microengineered to function as separating membranes for compartmentalized cell cultures, as well as electronic components for real-time in situ recordings of cell growth and function. Owing to the high surface area–to–volume ratio, the e-transmembrane allows generation of deep, stratified tissues within the porous bulk and cell polarization at the apico-basal domains. Impedance spectroscopy measurements carried out throughout the tissue growth identified signatures from different cellular systems and allowed extraction of critical functional parameters. This platform has the potential to become a universal tool for biologists for the next generation of high-throughput drug screening assays.

Evaluating the reliability of tumour spheroid-on-chip models for replicating intratumoural drug delivery: considering the role of microfluidic parameters

Several tumour spheroid-on-chip models have already been proposed in the literature to conduct high throughput drug screening assays. The microfluidic configurations in these models generally depend on the strategies adopted for spheroid formation and entrapment. However, it is not clear how successful they are to mimic in vivo transport mechanisms. In this study, drug transport in different tumour spheroid-on-chip models is numerically investigated under static and dynamic conditions using porous media theory. Moreover, the treatment of a solid tumour at the initial stage of development is modelled using bolus injection and continuous infusion methods. Then, the results of tumour spheroid-on-chip, including drug concentration, cell viability, as well as pressure and fluid shear stress distributions, are compared with those of the solid tumour, assuming identical transport properties in all models. Finally, a new configuration of the microfluidic device along with the optimal drug concentrations is proposed, which can well imitate a given in vivo situation.

FC076: Development of a High-Throughput in Vivo Drug Screening Assay Using a FSGS-Like Zebrafish Model

Abstract BACKGROUND AND AIMS Focal segmental sclerosis (FSGS) is a severe histopathological condition which often results in end-stage renal disease. Loss of podocytes and severe changes of podocyte foot process morphology accompanied by proteinuria are the hallmarks of FSGS. Since no therapeutic agents are available until now, further efforts must be made to identify new drugs to treat this disease. The zebrafish larva is a widely used kidney research model as the morphology and function of zebrafish and mammalian glomeruli are very similar. Since our group has already established a FSGS-like zebrafish injury model based on the nitroreductase-metronidazole cell ablation system, we have developed a high-throughput based in vivo drug screening assay. METHOD We used a zebrafish screening strain that expresses mCherry and the bacterial enzyme nitroreductase in podocytes. Additionally, a circulating vitamin-D-binding eGFP fusion protein with a size of 78 kDa is expressed and serves as a read-out for proteinuria. Incubation with 80 µM metronidazole for 24 h at 4 days post-fertilization (dpf) induces an FSGS-like phenotype in these larvae. A library of 138 epigenetic drugs were screened and the results are presented herein. Groups of 12 larvae received injury induction as well as drug treatment and were transferred individually into 96-well plates prepared with custom agarose molds. Larvae were oriented laterally and imaged with a high-content screening microscope (Acquifer Imaging Machine). The eGFP signal in the caudal vasculature as well as the glomerular mCherry signal served as readout systems. At 6 dpf, the same larvae and regions were imaged again. Custom written FIJI codes automatically segmented and measured the fluorescence intensities of both readouts and timepoints. For each larva, intensity ratios were determined and the ratios of each treatment group served as the input for statistical analysis. RESULTS Treatment for 24 hours with 80 µM metronidazole resulted in a measurable decline of the eGFP signal in the vasculature and of the podocyte mCherry signal compared to the vehicle (DMSO) group. Simultaneous treatment of metronidazole with the 138 drugs in the epigenetics library resulted in beneficial outcomes in varying degrees (9 drugs were positive, 36 compounds were lethal). CONCLUSION This new in vivo semi-automated high-throughput compatible drug screening assay allows rapid identification of FSGS protective drugs. Here we screened 138 epigenetic drugs, which led to the identification of 9 potential drugs which have had a beneficial effect on podocytes and/or the glomerular filtration barrier.

EXTH-67. THERAPEUTIC EFFICACY OF GC1118, A NOVEL ANTI-EGFR ANTIBODY, AGAINST GLIOBLASTOMA WITH HIGH EGFR AMPLIFICATION IN PATIENT-DERIVED XENOGRAFTS

Abstract We aimed to evaluate the preclinical efficacy of GC1118, a novel anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb), against glioblastoma (GBM) tumors using patient-derived xenograft (PDX) models. A total of 15 distinct GBM PDX models were used to evaluate the therapeutic efficacy of GC1118. Genomic data derived from PDX models were analyzed to identify potential biomarkers associated with the anti-tumor efficacy of GC1118. A patient-derived cell-based high-throughput drug screening assay was performed to further validate the efficacy of GC1118. Compared to cetuximab, GC1118 exerted comparable growth inhibitory effects on the GBM tumors in the PDX models. We confirmed that GC1118 accumulated within the tumor by crossing the blood–brain barrier in in vivo specimens and observed the survival benefit in GC1118-treated intracranial models. Genomic analysis revealed high EGFR amplification as a potent biomarker for predicting the therapeutic efficacy of GC1118 in GBM tumors. In summary, GC1118 exerted a potent anti-tumor effect on GBM tumors in PDX models, and its therapeutic efficacy was especially pronounced in the tumors with high EGFR amplification. Our study supports the importance of patient stratification based on EGFR copy number variation in clinical trials for GBM. The superiority of GC1118 over other EGFR mAbs in GBM tumors should be assessed in future studies.

Integration of FRET and sequencing to engineer kinase biosensors from mammalian cell libraries

The limited sensitivity of Förster Resonance Energy Transfer (FRET) biosensors hinders their broader applications. Here, we develop an approach integrating high-throughput FRET sorting and next-generation sequencing (FRET-Seq) to identify sensitive biosensors with varying substrate sequences from large-scale libraries directly in mammalian cells, utilizing the design of self-activating FRET (saFRET) biosensor. The resulting biosensors of Fyn and ZAP70 kinases exhibit enhanced performance and enable the dynamic imaging of T-cell activation mediated by T cell receptor (TCR) or chimeric antigen receptor (CAR), revealing a highly organized ZAP70 subcellular activity pattern upon TCR but not CAR engagement. The ZAP70 biosensor elucidates the role of immunoreceptor tyrosine-based activation motif (ITAM) in affecting ZAP70 activation to regulate CAR functions. A saFRET biosensor-based high-throughput drug screening (saFRET-HTDS) assay further enables the identification of an FDA-approved cancer drug, Sunitinib, that can be repurposed to inhibit ZAP70 activity and autoimmune-disease-related T-cell activation.

Multiplex Patient-Based Drug Response Assay in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is an extremely lethal malignancy arising from the pancreas. The treatment of PDA is complicated by ineffective treatments and a lack of biomarkers predictive of treatment success. We have designed a patient-derived organoid (PDO) based high-throughput drug screening assay to model treatment response to a variety of conventional and investigational treatments for PDA. Consecutive patients undergoing endoscopic ultrasound-guided fine-needle biopsy for tissue diagnosis of PDA at Rush University Medical Center were offered to participate in the study. Biopsies were immediately processed to develop organoids. Fifteen PDOs were screened for sensitivity to 18 compounds, including conventional PDA chemotherapies and FDA-approved investigational targeted therapies in cancer using Cell-titer GLO 3D (Promega) cell viability assay. The area under the curve (AUC) was calculated and normalized to the maximum area under the curve to generate a normalized AUC between 0 and 1. Molecular profiling of PDOs was conducted using RNA-seq. Human PDA transcriptomic was extracted from The Cancer Genome Atlas (TCGA). The drug response curves were reproducible. We observed variation in response to conventional therapies overall as well as among individual patients. There were distinct transcriptome signatures associated with response to the conventional chemotherapeutics in PDA. The transcriptomic profile of overall resistance to conventional therapies in our study was associated with poor survival in PDA patients in TCGA. Our pathway analysis for targeted drugs revealed a number of predictors of response associated with the mechanism of action of the tested drug. The multiplex organoid-based drug assay could be used in preclinical to inform patient stratification and therapeutic selection in PDA. When combined with omics data, ex vivo response to treatment could help identify gene signatures associated with response to novel therapies.

Biophysical analysis of drug efficacy on C. elegans models for neurodegenerative and neuromuscular diseases.

Caenorhabditis elegans has emerged as a powerful model organism for drug screening due to its cellular simplicity, genetic amenability and homology to humans combined with its small size and low cost. Currently, high-throughput drug screening assays are mostly based on image-based phenotyping with the focus on morphological-descriptive traits not exploiting key locomotory parameters of this multicellular model with muscles such as its thrashing force, a critical biophysical parameter when screening drugs for muscle-related diseases. In this study, we demonstrated the use of a micropillar-based force assay chip in combination with a fluorescence assay to evaluate the efficacy of various drugs currently used in treatment of neurodegenerative and neuromuscular diseases. Using this two-dimensional approach, we showed that the force assay was generally more sensitive in measuring efficacy of drug treatment in Duchenne Muscular Dystrophy and Parkinson’s Disease mutant worms as well as partly in Amyotrophic Lateral Sclerosis model. These results underline the potential of our force assay chip in screening of potential drug candidates for the treatment of neurodegenerative and neuromuscular diseases when combined with a fluorescence assay in a two-dimensional analysis approach.

A high-throughput screening assay based on automated microscopy for monitoring antibiotic susceptibility of Mycobacterium tuberculosis phenotypes

BackgroundEfficient high-throughput drug screening assays are necessary to enable the discovery of new anti-mycobacterial drugs. The purpose of our work was to develop and validate an assay based on live-cell imaging which can monitor the growth of two distinct phenotypes of Mycobacterium tuberculosis and to test their susceptibility to commonly used TB drugs.ResultsBoth planktonic and cording phenotypes were successfully monitored as fluorescent objects using the live-cell imaging system IncuCyte S3, allowing collection of data describing distinct characteristics of aggregate size and growth. The quantification of changes in total area of aggregates was used to define IC50 and MIC values of selected TB drugs which revealed that the cording phenotype grew more rapidly and displayed a higher susceptibility to rifampicin. In checkerboard approach, testing pair-wise combinations of sub-inhibitory concentrations of drugs, rifampicin, linezolid and pretomanid demonstrated superior growth inhibition of cording phenotype.ConclusionsOur results emphasize the efficiency of using automated live-cell imaging and its potential in high-throughput whole-cell screening to evaluate existing and search for novel antimycobacterial drugs.

Therapeutic Efficacy of GC1118, a Novel Anti-EGFR Antibody, against Glioblastoma with High EGFR Amplification in Patient-Derived Xenografts.

Simple SummaryGC1118 is a novel anti-EGFR monoclonal antibody with a distinct mode of epitope binding. Its therapeutic efficacy has been validated in preclinical studies of several cancers. We evaluated the anti-tumor efficacy of GC1118 against glioblastoma (GBM) using patient-derived xenografts (PDXs). GC1118 exhibited anti-tumor efficacy comparable to that of cetuximab in a subset of PDXs, and EGFR amplification was a potential biomarker for predicting its therapeutic efficacy. Growth inhibitory and direct apoptotic effects on GBM tumor cells were confirmed in in vitro analyses. In intracranial PDXs, GC1118 significantly improved survival outcome, indicating its potential to cross the blood–brain barrier. These results support the clinical potential of GC1118 in treating GBM, further prompting the requirement of a clinical trial.We aimed to evaluate the preclinical efficacy of GC1118, a novel anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb), against glioblastoma (GBM) tumors using patient-derived xenograft (PDX) models. A total of 15 distinct GBM PDX models were used to evaluate the therapeutic efficacy of GC1118. Genomic data derived from PDX models were analyzed to identify potential biomarkers associated with the anti-tumor efficacy of GC1118. A patient-derived cell-based high-throughput drug screening assay was performed to further validate the efficacy of GC1118. Compared to cetuximab, GC1118 exerted comparable growth inhibitory effects on the GBM tumors in the PDX models. We confirmed that GC1118 accumulated within the tumor by crossing the blood–brain barrier in in vivo specimens and observed the survival benefit in GC1118-treated intracranial models. Genomic analysis revealed high EGFR amplification as a potent biomarker for predicting the therapeutic efficacy of GC1118 in GBM tumors. In summary, GC1118 exerted a potent anti-tumor effect on GBM tumors in PDX models, and its therapeutic efficacy was especially pronounced in the tumors with high EGFR amplification. Our study supports the importance of patient stratification based on EGFR copy number variation in clinical trials for GBM. The superiority of GC1118 over other EGFR mAbs in GBM tumors should be assessed in future studies.

Thioridazine requires calcium influx to induce MLL-AF6–rearranged AML cell death

AbstractIn pediatric acute myeloid leukemia (AML), intensive chemotherapy and allogeneic hematopoietic stem cell transplantation are the cornerstones of treatment in high-risk cases, with severe late effects and a still high risk of disease recurrence as the main drawbacks. The identification of targeted, more effective, safer drugs is thus desirable. We performed a high-throughput drug-screening assay of 1280 compounds and identified thioridazine (TDZ), a drug that was highly selective for the t(6;11)(q27;q23) MLL-AF6 (6;11)AML rearrangement, which mediates a dramatically poor (below 20%) survival rate. TDZ induced cell death and irreversible progress toward the loss of leukemia cell clonogenic capacity in vitro. Thus, we explored its mechanism of action and found a profound cytoskeletal remodeling of blast cells that led to Ca2+ influx, triggering apoptosis through mitochondrial depolarization, confirming that this latter phenomenon occurs selectively in t(6;11)AML, for which AF6 does not work as a cytoskeletal regulator, because it is sequestered into the nucleus by the fusion gene. We confirmed TDZ-mediated t(6;11)AML toxicity in vivo and enhanced the drug's safety by developing novel TDZ analogues that exerted the same effect on leukemia reduction, but with lowered neuroleptic effects in vivo. Overall, these results refine the MLL-AF6 AML leukemogenic mechanism and suggest that the benefits of targeting it be corroborated in further clinical trials.

Patient-derived malignant pleural mesothelioma cell cultures: a tool to advance biomarker-driven treatments

Malignant pleural mesothelioma (MPM) is an aggressive cancer, associated with poor prognosis. We assessed the feasibility of patient-derived cell cultures to serve as an ex vivo model of MPM. Patient-derived...