Project Summary/Abstract Cellular senescence is a biological program whereby cells exit the cell cycle, typically as a consequence of DNA damage accumulation. After exerting their beneficial effects in young individuals by playing a role in tissue homeostasis, wound healing and tumor suppression, senescent cells are recognized and cleared by the immune system in response to their senescence-associated secretory phenotype (SASP). With age, immunosurveillance becomes increasingly dysregulated, senescent cells accumulate in tissues and their SASP leads to chronic inflammation which has been linked to many age-associated diseases including neurodegeneration, diabetes, osteoarthritis, fibrosis, heart disease and cancer. In addition, radiation and chemotherapy cause the accumulation of senescent cells in both normal and cancer tissues and create a microenvironment which can promote tumor relapse. Hence, methods to characterize the diversity of senescent cells in tissues are critical to improve our understanding of their roles in both normal physiology and disease and to develop more precise and effective senolytic and senostatic interventions. We propose to develop new technologies and integrative solutions to map the spatial epigenomic, transcriptome and proteomic states of senescent cells and their microenvironment in complex tissues. In the UG3 phase, we will develop spatial genomics, transcriptomics and proteomics methodologies to delineate senescent cells in a diverse set of murine tissues, including brain, liver and adipose tissue. We will also perform feasibility studies in a limited number of human samples available through tissue banks and collaborators, and in human cell culture. Our goal is to provide a proof of principle and evaluate the specificity and sensitivity of our proposed tools, technologies and methods (TTM) to identify and characterize the heterogeneity of senescent cells in multiple tissues. In the HG3 phase, we will optimize and expand our methodology to a variety of human tissues obtained from normal samples available from tissue banks, and via collaborations with other SenNet Tissue Mapping Centers (TMCs).