Single Cell Detection of the p53 Protein by Mass Cytometry

Simple Summary Investigation of protein expression in cancer cells is an important part of the diagnostic process. Increasing knowledge about expression of different proteins has been exploited for prognostic assessments and in some cases also for selection of treatment. The p53 protein has proven important in development of various cancers, and the expression of this protein and its signaling pathway is therefore of interest when examining cancer patient samples. Here, we present mass cytometry as a tool for detection of p53 expression. Mass cytometry allows for measurement of up to 50 parameters per sample with single cell resolution, and we aim to demonstrate its potential for p53-focused research. Purpose: The p53 protein and its post-translational modifications are distinctly expressed in various normal cell types and malignant cells and are usually detected by immunohistochemistry and flow cytometry in contemporary diagnostics. Here, we describe an approach for simultaneous multiparameter detection of p53, its post-translational modifications and p53 pathway-related signaling proteins in single cells using mass cytometry. Method: We conjugated p53-specific antibodies to metal tags for detection by mass cytometry, allowing the detection of proteins and their post-translational modifications in single cells. We provide an overview of the antibody validation process using relevant biological controls, including cell lines treated in vitro with a stimulus (irradiation) known to induce changes in the expression level of p53. Finally, we present the potential of the method through investigation of primary samples from leukemia patients with distinct TP53 mutational status. Results: The p53 protein can be detected in cell lines and in primary samples by mass cytometry. By combining antibodies for p53-related signaling proteins with a surface marker panel, we show that mass cytometry can be used to decipher the single cell p53 signaling pathway in heterogeneous patient samples. Conclusion: Single cell profiling by mass cytometry allows the investigation of the p53 functionality through examination of relevant downstream signaling proteins in normal and malignant cells. Our work illustrates a novel approach for single cell profiling of p53.