human epidermal growth factor receptor 2.eukaryotic initiation factor 4E-binding protein.Our study demonstrates that IMC data expand the number of measured parameters in single cells and brings higher-dimension analysis to the field of cell-based screening in early lead compound discovery. We show strong pairwise correlation between nuclear markers pHistone3 S28, Ki-67, and p4E-BP1 T37/T46 in classified mitotic cells and anticorrelation with cell surface markers. Predicted pharmacodynamic effects were measured in MCF-7 cells dosed with three target-specific compounds: growth stimulatory EGF, microtubule depolymerization agent nocodazole, and genotoxic chemotherapeutic drug etoposide. Human breast cancer-derived cell lines SKBR3, HCC1143, and MCF-7 were screened for expression of cellular markers to generate digital images with a resolution comparable to conventional fluorescence microscopy. CellProfiler was used to identify single cells through establishing cellular boundaries, followed by histoCAT™ (histology topography cytometry analysis toolbox) for extracting single-cell quantitative information visualized as t-SNE plots and heatmaps. As a proof of concept, we present here a workflow for multidimensional Imaging Mass Cytometry™ (IMC™) and data processing with open source computational tools. Measuring drug response in vitro by examining over 40 parameters, including biomarkers, signaling molecules, cell morphological changes, proliferation indices, and toxicity in a single sample, could significantly enhance discovery of new therapeutics. In pharmaceutical research, high-content screening is an integral part of lead candidate development.
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