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

Authors

Fabio Stossi
Pankaj K Singh
Ragini M Mistry
Hannah L Johnson
Radhika D Dandekar
Maureen G Mancini
Adam T Szafran
Arvind U Rao
Michael A Mancini

Publication Date

2-1-2022

Journal

Environmental Health Perspectives

DOI

10.1289/EHP9297

PMID

35167326

PMCID

PMC8846386

PubMedCentral® Posted Date

2-15-2022

PubMedCentral® Full Text Version

Post-Print

Published Open-Access

yes

Keywords

Endocrine Disruptors, In Situ Hybridization, Fluorescence, Quality Control, Receptors, Estrogen, Single-Cell Analysis

Abstract

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

METHODS: We used high-throughput microscopy, coupled with image analytics to measure changes in single cell ER nuclear levels on treatment with

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

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.