A High-Content Imaging Pipeline to Investigate Subcytotoxic Effects in RTgill-W1 Cells

Area of Science:

• Environmental toxicology
• Cellular imaging
• In vitro toxicology

Background:

• High-content screening (HCS) has potential in environmental toxicology but lacks standardized workflows and interpretable analysis.
• Existing methods struggle with reproducible application in environmental test systems.

Purpose of the Study:

• To establish and optimize a reproducible HCS pipeline for phenotypic profiling of rainbow trout gill cells (RTgill-W1).
• To identify robust, biologically meaningful endpoints for environmental chemical exposure.
• To demonstrate the pipeline's utility using Tebuthiuron as a case study.

Main Methods:

• Developed a workflow integrating organelle dyes for image-based phenotypic profiling.
• Systematically evaluated data processing steps: quality control, standardization, outlier removal, feature selection, and dimensionality reduction.
• Utilized Maximum Mean Discrepancy and Uniform Manifold Approximation and Projection (UMAP) for data visualization.

Main Results:

• The pipeline demonstrated robustness, detecting concentration-response relationships even with minimal processing.
• Optimized standardization and feature selection enhanced signal clarity.
• Identified 94 morphological features capturing Tebuthiuron's concentration- and time-dependent effects.
• Lysosomal redistribution and mitochondrial fragmentation were identified as early stress indicators.

Conclusions:

• The quality-controlled, transparent HCS pipeline can resolve concentration- and time-dependent subcellular responses in fish cells.
• This work establishes a methodological basis for future multichemical phenotypic profiling in environmental toxicology.
• The developed framework enables biologically interpretable analysis of cellular responses to environmental contaminants.