Quality Control for Single Cell Imaging Analytics Using Endocrine Disruptor-Induced Changes in Estrogen Receptor Expression.

BACKGROUND

Diverse toxicants and mixtures that affect hormone responsive cells [endocrine disrupting chemicals (EDCs)] are highly pervasive in the environment and are directly linked to human disease. They often target the nuclear receptor family of transcription factors modulating their levels and activity. Many high-throughput assays have been developed to query such toxicants; however, single-cell analysis of EDC effects on endogenous receptors has been missing, in part due to the lack of quality control metrics to reproducibly measure cell-to-cell variability in responses.

OBJECTIVE

We began by developing single-cell imaging and informatic workflows to query whether the single cell distribution of the estrogen (ER), used as a model system, can be used to measure effects of EDCs in a sensitive and reproducible manner.

METHODS

We used high-throughput microscopy, coupled with image analytics to measure changes in single cell ER nuclear levels on treatment with toxicants, over a large number of biological and technical replicates.

RESULTS

We developed a two-tiered quality control pipeline for single cell analysis and tested it against a large set of biological replicates, and toxicants from the EPA and Agency for Toxic Substances and Disease Registry lists. We also identified a subset of potentially novel EDCs that were active only on the endogenous ER level and activity as measured by single molecule RNA fluorescence hybridization (RNA FISH).

DISCUSSION

We demonstrated that the distribution of ER levels per cell, and the changes upon chemical challenges were remarkably stable features; and importantly, these features could be used for quality control and identification of endocrine disruptor toxicants with high sensitivity. When coupled with orthogonal assays, ER single cell distribution is a valuable resource for high-throughput screening of environmental toxicants. https://doi.org/10.1289/EHP9297.