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High-Throughput Phenotypic Profiling of 1470 ToxCast Chemicals in Five Biologically Diverse Human-Derived Cell Lines Using the Cell Painting Assay
Citation:
Willis, C., D. Haggard, F. Harris, G. Byrd, AND J. Harrill. High-Throughput Phenotypic Profiling of 1470 ToxCast Chemicals in Five Biologically Diverse Human-Derived Cell Lines Using the Cell Painting Assay. SOT Conference 2024: A New Approach Method (NAM) to Screen for the Impact of Endogenous Stress on Chemical Toxicity, Salt Lake City, UT, March 10 - 14, 2024. https://doi.org/10.23645/epacomptox.25499239
Impact/Purpose:
Presentation to SOT Conference 2024: A New Approach Method (NAM) to Screen for the Impact of Endogenous Stress on Chemical Toxicity
Description:
Background and Purpose: High-throughput phenotypic profiling (HTPP) is a modified Cell Painting assay proposed as a first-tier bioactivity screening tool within a tiered in vitro hazard evaluation strategy using New Approach Methodologies (NAMs). HTPP combines high-content imaging and image analysis to measure phenotypic features at the single cell level and detect changes to organelle and cell morphology. Organelles are labeled with a set of fluoroprobes that allow for a broad range of biological effects to be observed and quantified in a non-targeted manner. In this study, 1470 ToxCast chemicals were screened in four human-derived immortalized primary cell lines – HBEC3-KT (bronchial epithelia), TeloHAEC (vascular endothelia), RPTEC/TERT1 (kidney epithelia) and Ker-CT (skin epithelia) – and one human-derived immortalized cancer cell line, MCF7 (breast adenocarcinoma), using HTPP. These cell lines were selected using a data-driven cell line selection approach based on differences in baseline gene expression and represent a variety of tissue types. The objective of this study was to compare the relative sensitivity of the HTPP assay when applied to a biologically diverse set of human-derived cell models. Methods: HTPP was conducted in 384-well format. Seeding densities for each cell line were chosen from previous experimental data. Each cell line was allowed a 24h to recovery period prior to dosing except for RPTEC/TERT1 which required a 48h recovery period to ensure adequate attachment to the plating surface. Cells were dosed in a randomized pattern with 8 concentrations of test chemicals (1/2-log10 spacing). The highest dose tested for most chemicals was 100 µM. After a 24h exposure period, live cells were labeled with MitoTracker (mitochondria), fixed, permeabilized and labeled with Hoechst-33342 (nuclei), SYTO14 (nucleoli) and fluorescent conjugates of concanavalin A (ER), phalloidin (cytoskeleton), and wheat germ agglutinin (Golgi/plasma membrane). After staining, images were acquired using an Opera Phenix HCS system and analyzed using Harmony software, resulting in ~1300 morphological features per cell. For each feature, data were aggregated to the well-level and normalized to in-plate vehicle control wells using median and median absolute deviation (MAD). Normalized cell counts (nCC) were calculated and used to evaluate cell viability and cytostatic effects. Concentration response modeling of the nCC data was performed using the R package tcplfit2, and any treatments that produced a >50% decrease in nCC were considered cytotoxic and excluded from further analysis. Mahalanobis distance calculations were used for concentration response modeling of the well-level feature data. ‘Global Mahalanobis’ distance values were generated for each treatment from the combined ~1300 features and modeled using the R package tcplfit2. The benchmark concentration (BMC) for a chemical was defined as the concentration where the response exceeded the benchmark response (BMR) of 1*nMAD. A chemical was considered active if the continuous hitcall from tcplfit2 was greater than 0.9 and the BMC was below the highest tested concentration. Results: Of the cell types tested, MCF-7 were found to be the least sensitive with 576 total active chemicals (40.7%). Ker-CT were the most sensitive with 771 total active chemicals (54.4%). The remaining three cell lines listed from least to most sensitive were TeloHAEC with 653 active chemicals (46.1%), RPTEC/TERT1 with 657 active chemicals (46.4%) and HBEC3-KT with 680 active chemicals (48.0%). In total, there were 472 chemicals active across all cell types and 553 chemicals inactive across all cell types. There was a set of 65 chemicals that were found to be active across all cell types except for MCF7. A total of 58 chemicals . . .
URLs/Downloads:
DOI: High-Throughput Phenotypic Profiling of 1470 ToxCast Chemicals in Five Biologically Diverse Human-Derived Cell Lines Using the Cell Painting Assay
SOT 2024 POSTER CW_FINAL.PDF (PDF, NA pp, 2506.935 KB, about PDF)