Senescent cells accumulate with age and are increasingly linked to a variety of age-related diseases. At present, senescent cells are operationally characterized based on a set of morphological, biochemical, and molecular properties. However, senescence is unlikely to represent a single uniform entity. Rather, analogous to what has been learned from studying tumors, the cell type, in conjunction with the specific endogenous driver of senescence, will likely determine the unique molecular fingerprint of a given senescent cell. Understanding this fingerprint, like our understanding of the specific genetic alterations in a given tumor, will, in turn, inform prognosis and response to therapy. Much of our understanding of the biology of senescence has come from experimental animal models, while our understanding of senescence in human cells and tissues remains, at best, incomplete. Similarly, while a number of exogenous stresses can trigger a senescent phenotype for cells in culture, the physiological relevant drivers of human senescence are not known. Finally, while for proliferating cells, senescence is characterized by a permanent withdrawal from the cell cycle, the relevant senescent cell markers and biology for critical post-mitotic cells (e.g., cardiac myocytes, neurons) is not well established. To begin to address these significant knowledge gaps, the TriState SenNet Tissue Mapping Center (TMC) will analyze human lung and heart tissue to provide a high-resolution map of the senescent cell population in these organs. With the combined expertise of the University of Pittsburgh and Carnegie Mellon University, Ohio State University, and the University of Rochester School of Medicine, this consortium will provide a comprehensive assessment of two organs linked to a variety of age-related diseases, one of which is largely post-mitotic. Our analysis will involve in situ mapping using established targeted assays, as well as high-content unbiased approaches involving single cell RNA/ATAC sequencing, proteomics, and spatial transcriptomics. In addition, as part of our mapping endeavor, the TriState SenNet TMC will analyze the relationship between the initiating trigger for senescence and the subsequent biology and phenotype of the senescent cell. This relationship will be probed using precision cut tissue sections from human lungs and hearts that will be perturbed ex vivo by specific senescent triggers. This procedure will allow us to increase the precision and sensitivity of detection and localization of senescent cells in the lung and heart. Finally, we will purify and isolate senescent and non- senescent cells from an individual donor lung and used these isogenic primary cells to deconvolute the physiologically relevant driver of human senescence and delineate the molecular basis for the observed selectivity of senolytic therapy. Together, this combined analysis of tissue and tissue slices/cells from the same individual will provide an unrivaled, unprecedented, in-depth, high-resolution map of the senescent cell population in the human lung and heart, define the physiological drivers of senescence, and provide a rational approach to understand the therapeutic potential of senolytic therapy.

NARRATIVE Senescence cells accumulate with age and are thought to contribute to age-related diseases. Here, we propose to comprehensively map senescent cells in high resolution in the human heart and lung. We will use multi-modal, high-content, and high-throughput approaches in whole tissues, organ slices, and isolated cells in order to characterize and map the senescent cell population, identify the relevant endogenous triggers for senescent cell formation, and delineate the specific senescent cell properties that modulate the response to senolytic therapy.