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Optimization and Application of an Image-based Phenotypic Profiling Assay to Estimate in vitro Points of Departure for Chemical Bioactivity
Citation:
Nyffeler, J., C. Willis, AND J. Harrill. Optimization and Application of an Image-based Phenotypic Profiling Assay to Estimate in vitro Points of Departure for Chemical Bioactivity. Presented at SETAC North America Focused Topic Meeting : High-Throughput Screening, Durham, NC, April 16 - 18, 2018. https://doi.org/10.23645/epacomptox.7033271
Impact/Purpose:
Image-based phenotypic profiling is a chemical screening method that measures a large variety of morphological features of individual cells in in vitro cultures. It has been successfully used for functional genomic studies and in the pharmaceutical industry for compound efficacy and toxicity screening. It may be used as an efficient and cost-effective method for evaluating chemical bioactivity.
Description:
Imaging-based phenotypic profiling is a high-throughput screening method that combines automated microscopy and image analysis to measure a large variety of features at the single cell level. Here, we describe workflows for concentration-response screening, image analysis and calculation of in vitro point-of-departure (POD) estimates using a phenotypic profiling assay that evaluates changes in organelle morphology. MCF7 or U-2 OS cells were plated in 384-well plates. After 24 h, cells were treated with 7 concentrations (0.1–100 µM, n = 3/plate, 3 independent plates) of 16 reference chemicals in a randomized pattern using an Echo 550 acoustic dispenser. After 48 h of treatment, cells were live labeled with MitoTracker (mitochondria), fixed, permeabilized and labeled with Hoechst-33342 (nuclei), SYTO14 (nucleoli) and fluorescent conjugates of concanavalin A (ER), phalloidin (actin cytoskeleton), and wheat germ agglutinin (Golgi/plasma membrane). A multiplexed cell viability and apoptosis assay was run in parallel. Confocal images were acquired using an Opera Phenix HCS system and analyzed using Harmony software, yielding > 300 features per cell. Cell-level data were median absolute deviation (MAD) normalized using DMSO controls. Benchmark dose (BMD) modeling was performed on well-level median values with BMDExpress 2.0. Most chemicals (n=13) significantly affected cell morphology. Distinct patterns of affected cellular features were observed across the chemical set and, in most cases, were consistent with observations from the literature. Changes in the actin cytoskeleton/Golgi/plasma membrane (AGP) domains were the most sensitive for Ca-074-Me, fluphenazine and tetrandrine, whereas the DNA/RNA, mitochondria, ER and DNA/morphology domains were the most sensitive for fenbendazole, berberine chloride, oxibendazole and taxol, respectively. For a subset of chemicals, phenotypic profiling BMDs were well below cytotoxicity BMDs. Most chemicals produced similar profiles in both cell lines, however, U-2 OS were more sensitive to a subset of chemicals. In summary, the described protocol was successfully applied to MCF7 and U-2 OS cells using automated liquid handling tools. Testing of diverse compounds yielded distinct patterns of affected parameters below the threshold for cytotoxicity, indicating that this test method could be used to derive conservative in vitro PODs for screening level risk assessments. This abstract does not necessarily reflect USEPA policy.
URLs/Downloads:
DOI: Optimization and Application of an Image-based Phenotypic Profiling Assay to Estimate in vitro Points of Departure for Chemical Bioactivity
SETAC2018 NYFFELER CYTOLOGICAL PROFILING_POSTER_FINAL_M.PDF (PDF, NA pp, 1225.275 KB, about PDF)