Single Cell Analysis of Nuclear Receptor Transcription

Michael Mancini Baylor College of Medicine, United States of America

Abstract
The main focus of our research has been to identify, characterize and quantify mechanistic steps of steroid nuclear receptor action at the single cell level by using state-of-the-art microscopy and bioinformatics-based approaches. To this end, we created stable cell lines and automated image analysis routines that facilitate multi-parametric small molecule, RNAi and endocrine disruptor screens to identify effectors of estrogen and androgen receptors, and also for use in primary hybridoma screening for imaging-quality monoclonal antibodies (mAbs) to receptors/coregulators. Increasingly, these studies are shifting to analysis of endogenous gene expression through multiplex mRNA FISH coupled with immunofluorescence using compatible custom-screened mAbs, and a novel, low-mag/high-resolution high throughput microscopy platform (e.g., StellarVision™). These efforts have led to the creation of biological response fingerprints that identify mechanistic and phenotypic changes in biosensor and native cell lines in response to various treatments. Screens have identified novel, disease relevant estrogen receptor coregulators (i.e., UBR5), receptor-specific EDCs (Bisphenol A analogs), and several repurposed drugs for use in advanced prostate cancer cell models; moreover, novel multiplex immunization and screening strategies led to generation of imaging-quality mAbs, and ‘shotgun’ mAb panels to tumor resistance. Collectively, these new imaging-based approaches are leading towards an improved understanding of steroid receptor and coregulator action in prostate and breast cancer models.