Applying Cell Painting in Non-Tumorigenic Breast Cells to Understand Impacts of Common Chemical Exposures

Abstract

There are a substantial number of chemicals to which individuals in the general population are exposed which have putative, but still poorly understood, links to breast cancer. Cell Painting is a high-content imaging-based in vitro assay that allows for rapid and unbiased measurements of the concentration-dependent effects of chemical exposures on cellular morphology. We optimized the Cell Painting assay and measured the effect of exposure to 16 human exposure relevant chemicals, along with 21 small molecules with known mechanisms of action, for 48 hours in non-tumorigenic mammary epithelial cells, the MCF10A cell line. Through unbiased imaging analyses using CellProfiler, we quantified 3042 morphological features across approximately 1.2 million cells. We used benchmark concentration modeling to quantify significance and dose-dependent directionality to identify morphological features conserved across chemicals and find features that differentiate the effects of toxicants from one another. Benchmark concentrations were compared to chemical exposure biomarker concentration measurements from the National Health and Nutrition Examination Survey to assess which chemicals induce morphological alterations at human-relevant concentrations. Morphometric fingerprint analysis revealed similar phenotypes between small molecules and prioritized NHANES-toxicants guiding further investigation. A comparison of feature fingerprints via hypergeometric analysis revealed significant feature overlaps between chemicals when stratified by compartment and stain. One such example was the similarities between a metabolite of the organochlorine pesticide DDT (p,p-DDE) and an activator of canonical Wnt signaling CHIR99201. As CHIR99201 is a known Wnt pathway activator and its role in beta-catenin translocation is well studied, we studied the translocation of ꞵ-catenin following p,p-DDE exposure in an orthogonal high-content imaging assay. Consistent with activation of Wnt signaling, low dose p,p-DDE (25nM) significantly enhances the nuclear translocation of beta-catenin. Overall, these findings highlight the ability of Cell Painting to enhance mode-of-action studies for toxicants which are common exposures in our environment but have previously been incompletely characterized with respect to breast cancer risk.

Investigating phenotypic plasticity due to toxicants with exposure disparities in primary human breast cells in vitro

“…These proliferative effects have been attributed to genetic and epigenetic modifications following exposures in non-tumorigenic MCF-10A breast epithelial cells, with specific upregulation of human epidermal growth factor following BPS exposure, and upregulation of the PCNA gene following DDE exposure ( 22 , 30 , 42 ). We also recently found that low-concentration DDE (25 nM) activates Wnt signaling in MCF-10A cells, as reflected by increased translocation of beta catenin to the nucleus, which may, in part, explain increased cellular proliferation ( 47 ). The mechanisms behind the proliferative effects associated with these chemicals in these normal primary lines from diverse donors is worthy of future exploration.…”

DataSheet_1.docx

“…These proliferative effects have been attributed to genetic and epigenetic modifications following exposures in nontumorigenic MCF-10A breast epithelial cells, with specific upregulation of human epidermal growth factor following BPS exposure, and upregulation of the PCNA gene following DDE exposure (22,30,42). We also recently found that lowconcentration DDE (25 nM) activates Wnt signaling in MCF-10A cells, as reflected by increased translocation of beta catenin to the nucleus, which may, in part, explain increased cellular proliferation (47). The mechanisms behind the proliferative effects associated with these chemicals in these normal primary lines from diverse donors is worthy of future exploration.…”