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High Content Analysis of phenotypic assays in cancer drug discovery
4 INSTRUMENTS/DATA ANALYSIS2 ALGORITHMS DEVELOPED IN ONCOLOGY1 INTRODUCTIONCharles River Laboratories (CRL) has developed many disease-relevant cellular models to screen small molecules and geneticmodifiers for target and phenotypic based drug discovery. Our expertise and portfolio in bespoke cellular model developmentcovers several therapeutic areas including oncology and a wide variety of target classes.Complex cellular models derived from primary cells, patient derived embryonic stem cells and induced pluripotent stem cellshave been adapted for phenotypic screening. Models have been further developed to produce physiologically relevantmulticellular structures, such as 3D spheroids and co-cultures. Combining complex biological systems with multiparametric highcontent analysis provides detailed single cell quantification of cellular and subcellular biology.Such validated High Content (HC) assays can be applied to both target and phenotypic based discovery platforms to supportpreclinical drug discovery and improve translation of targets and compounds to the clinic.
3 EXAMPLES OF HIGH-CONTENT BASED ONCOLOGY ASSAYS
IN Cell 2200 IN Cell 6000 IncuCyte S3
Formats Assays developed
Cell viability and synergistic cytotoxic studies 2D: Clonogenic assay, cell viability assay (Hoeschst/PI), immunotherapy cytotoxic co-culture assays
Anchorage independence and colony forming assays (in 3D) 3D: Multiple cell lines and co-culture
Apoptosis, cell death, DNA damage response, cellular stress and degeneration
Early and late stage apoptosis markers, mitochondrial function, nuclear condensation, DNA damage assay and downstream signaling assays, foci, stress granules, protein inclusions
Cell cycle Cell cycle progression and S phaseCell motility and migration Scratch wound assay and Boyden chamber motility assay
Acquisition of invasive properties Epithelial to mesenchymal transition (EMT) assay
Post translational modification Multiple assays for signalling events (phosphorylation and acetylation)
Marker expression Multiple assays to assess transduction efficiency, epigenetic target and biomarker expression
Receptor internalization and degradation Nuclear receptor degradation, receptor internalisation, ligand binding
Translocation of transcription factors Multiple cytosolic/nuclear translocation assays
IMMUNO-ONCOLOGY 3D SPHEROID T CELL CYTOTOXIC ASSAY
Figure 2. 3D Spheroid T cell cytotoxicity Assay. (A) A549 cells were transfected with IncuCyte® Nuclight Green lentivirus reagent to generate a stable cell line. Cells wereseeded in ultra-low attachment plates to enable spheroid formation, and treated after 2 days of culture with activated T cells in the absence and presence of test compounds ortherapeutic antibodies. This method uses the IncuCyte® live-cell analysis system to generate data in the form of presentation-ready and time-lapse graphs and movies. (B)Example of data generated with IncuCyte® over a period of 5 days after treatment. (C) Examples of live cell imaging acquired with IncuCyte®.
No stimulation control
Staurosporine 10 uM
Stimulation + indoximod 100 uM
Stimulation + IgG4 control 10 ug/mL
10ng/mL Anti-CD3 + 10 ng/mL IL-2Stimulation + pembrolizumab 10 ug/mLA B
C
Rare diseasesRespiratory
Inflammation
Oncology
Neurodegeneration Our extensive experience in the development of complex cellular assays:Execution of high-throughput phenotypic screenings involving multi-parametric readouts, design of quantitative image segmentationalgorithms, and experience in a wide variety of disease models (incl. co-cultures, 3D and organoid systems) allows us to approach withconfidence a diversity of preclinical drug discovery programs.Multi-parametric feature extraction is performed by using analytical software tools such as IN Cell Developer Toolbox, IN Cell AnalyzerWorstation and Cell ProfilerTM.
Charles River’sHigh Content Team
Figure 3. Nuclear translocation assay. NF-κB signaling in inflammation and cancer. In the control situation NF-kB isdetected in the cytoplasm and not in the nucleus. Once cells are treated with IL-1α, nuclear translocation of NF-kB isevident. Using HC analysis the concentration- and time-dependent effects of IL-1α in the nuclear translocation ofNFκB could be studied in HeLa cervical cancer cells by ratiometric quantification of NF- κB expression in the nucleusversus the cytoplasm
No Stimulation Stimulation
Transport into the nucleus
NFκB nuclear translocation induced by IL-1α in HeLa
control 2 ng/ml IL-1α, 30 min 37°C-100
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time (min) Il-1α stimulation
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NUCLEAR TRANSLOCATION ASSAY
Hajo Schiewe1, João Carvalho2, Fleur Stevenhagen2, Servane Lachize2, Rhea van de Bospoort2, Melek Atalar2, Kimberly Lo2, Lianne van Beek2, Marta da Silva2, Niki van der Steenstraten2, Viola Tabel2, Jeroen Esselink2, Jeroen Overman2, Ian Gowers3, Roger Clark3, Shilina Roman4 and Shushant Jain2
1Charles River, Wilmington, Massachusetts, USA, 2Charles River Leiden NL, 3Saffron Walden UK and 4Harlow UKContact: [email protected]
Figure 1. Scratch migration assay. (A) Fibroblasts were transduced with shRNA encoding adenovirus 1 day post seeding,after 4 days of treatment, the cells were triggered with PDGF-BB. A scratch was created 1 day post trigger addition. (B)Graphical representation of the migration of fibroblasts into the open area created by the scratch. (C) Trigger induces fullwound closure after 18h. (D) Example of images depicting migration into open area and inhibition of cell migration. This assayuses a high content algorithm developed to segment and quantify the open area to identify migration inhibition by target geneknockdown.
SCRATCH MIGRATION ASSAYA
scratch
wound healing
D0 D1
Transducewith shRNA
Seed cells
Screen:Image at
T>8hr
D6
Scratch& trigger
Assay development:Image at t=0hr
D2
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D5
Trigger
PositiveControl shRNA
Negative Control (non targeting shRNA)
T = 0hr
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