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- Title
Quality Control for Single Cell Imaging Analytics Using Endocrine DisruptorInduced Changes in Estrogen Receptor Expression.
- Authors
Stossi, Fabio; Singh, Pankaj K.; Mistry, Ragini M.; Johnson, Hannah L.; Dandekar, Radhika D.; Mancini, Maureen G.; Szafran, Adam T.; Rao, Arvind U.; Mancini, Michael A.
- 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 receptor-a (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 ~100 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 in situ 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, ERsingle cell distribution is a valuable resource for high-throughput screening of environmental toxicants.
- Subjects
RNA analysis; CELL analysis; PROTEIN analysis; ENDOCRINE disruptors analysis; POLLUTANTS; PREDICTIVE tests; RESEARCH evaluation; CELL culture; MICROSCOPY; CULTURE media (Biology); ESTROGEN receptors; QUALITY control; FLUORESCENCE in situ hybridization; RESEARCH funding; TRANSCRIPTION factors; SENSITIVITY &; specificity (Statistics); ENDOCRINE disruptors; ANALYTICAL chemistry
- Publication
Environmental Health Perspectives, 2022, Vol 130, Issue 2, p027008-1
- ISSN
0091-6765
- Publication type
Article
- DOI
10.1289/EHP9297