Multiple-Protein Detections of Single-Cells Reveal Cell–Cell Heterogeneity in Human Cells

Abstract

Cell population represents an intrinsically heterogeneous and stochastic system, in which individual cells often behave very differently in molecular contents, functions and even genotypes from the population average in response to uniform physiological stimuli. The traditional bulk cellular analysis often overlooks cellular heterogeneity and does not provide information on cell-cell variations. Single-cell measurements can reveal information obscured in population averages, and enable us to determine distributions rather than averaged properties within a cell population. The level of complexity, with numerous variables acting at the same time, requires multiparametric and dynamic investigation of a large number of single cells. Multiplexed study can provide quantitative correlations or inter-relationships among multiple cellular components and molecular markers within a protein network or family in biological processes. In this paper, we applied multiple fluorophore-conjugated primary antibodies to detect multiple proteins expressed on the same singe cells from a clonal population. To reveal cell-cell heterogeneity, we quantified the histograms of six proteins within a cell population as functions of TNF-α stimulation time. Then, we quantified noise and noise strength of these protein histograms as functions of TNF-α stimulation time. Thirdly, we quantified correlation coefficients of multiple proteins expressed on same single-cells as functions of TNF-α stimulation time. Above parameters demonstrated nonlinear relationships with TNF-α stimulation. Quantification of above parameters on independent cell subpopulations further reveals the cell-cell heterogeneity when exposed to identical environmental conditions. Such cellular heterogeneity will be useful to characterize the disease progression and disease diagnoses.