Single-cell proteomic identification of novel markers of senescence

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PROJECT SUMMARY Cellular senescence is a stable form of cell cycle arrest associated with pro-inflammatory responses. On the one hand, senescent cells are a barrier for tumorigenesis and promote wound healing and embryogenesis. On the other hand, senescent cells accumulate in aged and diseased tissues, where they impair tissue renewal and contribute to inflammation and disease progression. Identification and characterization of senescent cells in human tissues will contribute to our understanding of human diseases. Thus, mapping senescent cells at the 3-dimensional level and single-cell resolution in human tissues is an important biomedical objective. Accurate mapping of senescent cells requires reliable markers to specifically identify senescent cells. Currently the senescence field has limited markers to unambiguously distinguish between senescent cells and cells in other pathological states. In addition, the available markers do not address the heterogeneity of senescent cells in tissues. To overcome these limitations, we propose to employ a novel single-cell proteomic technology to investigate the proteomes of senescent cells at the single-cell level, with the goal to reveal novel markers of senescence which can be used to identify and map senescent cells in human tissues. Our group has recently developed a novel technology termed Single-Cell ProtEomics by Mass Spectrometry (SCoPE2). This platform combines automated cell lysis, improved detection sensitivity, and optimized data acquisition and analyses, allowing detection and quantification of thousands of proteins within a single cell. We have applied this technology to study embryonic stem cell differentiation and macrophage polarization, revealing their heterogeneity and alterations of proteomes at the single-cell level. In this application, we will use the lung as a model system, which accumulate senescent cells that contribute to aging and lung diseases. In the UG3 phase, we will isolate senescent cells from the lungs of naturally aged mice. The isolated senescent cells together with control cells will be subjected to SCoPE2 procedure to quantify their proteomes at the single-cell level, allowing us to create unique signatures for potentially diverse senescent cell populations. This will help discover novel markers of senescent cells that are not possible with traditional technologies. In the UH3 phase, we will apply this technology to human lungs, to validate and to identify new markers of senescence. In aggregate, we will establish new ways of identifying senescent cells that should offer new tools to probe senescent cells in human tissues, facilitating tissue mapping of senescent cells of the SenNet initiative. In addition, this study has the potential to reveal new biology of senescence, addressing the heterogeneity and proteome alterations at the single-cell level.