Spatially-resolved proteome mapping of senescent cells and their tissue microenvironment at single-cell resolution

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PROJECT SUMMARY/ABSTRACT Cellular senescence is a permanent state of cell cycle arrest induced by many different stresses. Although senescent cells (SNCs) have been demonstrated with beneficial roles in normal physiological processes, they are increasingly recognized as the key determinants of many aging-related diseases, such as cancer, osteoarthritis, and type 2 diabetes. The SNCs and senescence-associated secretory phenotype (SASP) are found to be highly heterogeneous and vary in different types of cells and tissue regions. Currently, there are no “universal” biomarkers for identifying the SNCs in vivo. The first step towards advancing our understanding of cellular senescence and developing SNC-targeting therapy approaches is to comprehensively characterize cellular senescence in various human tissues. Mass spectrometry (MS)-based spatial proteomics can provide direct insights into cellular heterogeneity and reveal novel protein markers. However, current spatial proteomics technologies are limited by their poor spatial resolution and low analysis throughput. The overall objective of this project is to significantly advance our microfluidics-based spatial proteomics platform, termed laser capture microdissection coupled with nanodroplet processing in one-pot for trace samples (LCM-nanoPOTS), and apply this technology to map SNCs and their SASP in different mouse and human tissues. In the UG3 phase, we will establish a high-throughput and robust single-cell isolation system and couple it with nanoPOTS-MS. We will modify the Zeiss LCM system to enable reliable single-cell isolation and collection by designing and assembling a robot-addressable capillary sampling probe. Next, we will deploy the spatial single-cell proteomics platform for mapping of SNCs and their SASP in mouse skin tissue. We will develop a streamlined workflow to identify SNCs from FASST mouse skins. Immunohistochemistry or immunofluorescence will be used to validate novel protein marker candidates. In the UH3 phase, we will significantly enhance proteome coverage and analysis throughput of the nanoPOTS-LC-MS platform. We aim to achieve a throughput of >300 samples/day and a proteome coverage of >1500 proteins. The nanoPOTS sample preparation will be optimized for formalin-fixed paraffin- embedded (FFPE) tissues for broad applications of spatial proteomics technology. Next, we will demonstrate the improved platform in different tissue types, including mouse liver, skin draining lymph nodes, as well as human liver, breast, and lung tissues. We will establish collaborations with different TMCs to characterize SNCs and SASP using spatial proteomics in various human tissues and contribute to multiomics mapping of the tissues. Statement of Impact: The capability to generate unbiased and comprehensive proteome maps at single-cell resolution will enable the discovery of SNC protein markers across different cell types and organs, and advance our understanding of the impact of SNCs on tissue microenvironment.