Tapaswi Anagha, Cemalovic Nicholas, Polemi Katelyn M, Sexton Jonathan Z, Colacino Justin A
Department of Environmental Health Sciences, University of Michigan, Ann Arbor, MI, USA.
Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.
bioRxiv. 2024 May 3:2024.04.30.591893. doi: 10.1101/2024.04.30.591893.
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 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 β-catenin translocation is well studied, we studied the translocation of β-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 β-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.
普通人群会接触到大量化学物质，这些物质被认为与乳腺癌存在联系，但目前仍了解甚少。细胞绘画是一种基于高内涵成像的检测方法，可快速、无偏地测量化学物质暴露对细胞形态的浓度依赖性影响。我们优化了细胞绘画检测方法，并在非致瘤性乳腺上皮细胞系MCF10A中测量了16种与人体暴露相关的化学物质以及21种具有已知作用机制的小分子暴露48小时后的影响。通过使用CellProfiler进行无偏成像分析，我们在约120万个细胞中量化了3042个形态特征。我们使用基准浓度模型来量化显著性和剂量依赖性方向性，以识别不同化学物质间保守的形态特征，并找出能够区分不同毒物作用效果的特征。将基准浓度与来自美国国家健康与营养检查调查的化学物质暴露生物标志物浓度测量值进行比较，以评估哪些化学物质在与人体相关的浓度下会引起形态改变。形态计量指纹分析揭示了小分子与优先考虑的美国国家健康与营养检查调查毒物之间相似的表型，为进一步研究提供了指导。通过超几何分析比较特征指纹发现，按区室和染色分层时，不同化学物质之间存在显著的特征重叠。一个例子是有机氯农药滴滴涕（p,p'-二氯二苯二氯乙烯）的代谢物与经典Wnt信号通路激活剂CHIR99201之间的相似性。由于CHIR99201是已知的Wnt信号通路激活剂，且其在β-连环蛋白易位中的作用已得到充分研究，我们在正交高内涵成像检测中研究了p'-p'二氯二苯二氯乙烯暴露后β-连环蛋白的易位情况。与Wnt信号通路激活一致，低剂量p',p'-二氯二苯二氯乙烯（25nM）显著增强了β-连环蛋白的核易位。总体而言，这些发现突出了细胞绘画在增强对环境中常见毒物作用机制研究方面的能力，这些毒物此前在乳腺癌风险方面的特征尚不完整。
