Abstract B20: Single-cell profiling of EGFR-regulated protein changes involved in cell cycle, cell proliferation and apoptosis

Abstract

Abstract Single-cell molecular analysis has become increasingly important in biomedical research, especially in the field of cancer research, where cell heterogeneity is a characteristic feature. We developed a multiplexed single-cell protein detection assay for the C1™ system that can isolate and process 96 individual cells in parallel. The assay uses oligonucleotide-conjugated antibody binders and monitors the specific oligonucleotide levels instead of protein levels. The resulting DNA amplicon is detected by qPCR on the Biomark™ HD system. Data are analyzed using Singular™ Analysis Toolset. A total of 50 antibody binders were developed for this assay to target proteins related to apoptosis, cell cycle, cell proliferation, tumor suppressors, biomarkers, stem cells, growth factors and proteins associated with specific cancers, such as breast and prostate cancers. The assay can be performed in a 48-plex format which yields over 4,600 data points in two days. Assay sensitivity and specificity were assessed using recombinant proteins and various cell lysates. To demonstrate the usefulness of this assay, a panel of antibody binders was selected to profile the changes of proteins involved in cell cycle, cell proliferation and apoptosis in single cells. We treated cells that overexpress epidermal growth factor receptor (EGFR) with either the EGF ligand or the gefitinib receptor inhibitor and analyzed the changes in protein levels. Cells without EGFR expression were used as the control. EGFR is a member of ErbB family of Type I tyrosine kinases. EGFR overexpression and its mutations are involved in a variety of cancers. While much effort has been made to investigate the consequences of EGFR activation or inhibition, hardly any information is available regarding how EGFR regulation changes protein expression in single cells. Single-cell analysis—determining protein changes and analyzing the correlation between different proteins at the single-cell level—yields valuable information that is not achievable using bulk-cell analysis. Citation Format: Ilona Holcomb, Gajalakshmi Dakshinamoorthy, Benjamin Liu, Marc Unger, Ramesh Ramakrishnan, Haibiao Gong. Single-cell profiling of EGFR-regulated protein changes involved in cell cycle, cell proliferation and apoptosis. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B20.