PROJECT SUMMARY Single cell profiling has recently exploded into mainstream biology. Single cell profiling has been employed in myriad applications in biology, including multiple diseases, embryonic development, comparative evolutionary studies and aging. Most recently, single cell profiling has informed the phenomena of cellular senescence. This proposal has two phases, UG3 (model systems), and UH3 (human validation). Cellular senescence is a multi-faceted cell fate that arrests cell proliferation, essentially irreversibly, and activates the production and secretion of pro-inflammatory cytokines, chemokines, growth factors, proteases and lipids, termed the Senescence Associated Secretory Phenotype (SASP). The SASP can influence tissue microenvironments, and thus senescent cells can strongly affect tissue function and likely the systemic milieu. Senescent cells increase with age and can drive a growing list of age-related pathologies, ranging from neurodegeneration to cancer, in part through the SASP. There is increasing evidence that there are no universal markers for senescent cells. Instead, senescent cells, while sharing certain characteristics and biomarkers, are remarkably heterogeneous, varying in characteristics with genotype, cell and tissue type, senescence inducer, tissue (and cell culture) microenvironment, and chronology (time after initial senescence induction). While some of the more commonly employed senescence markers have utility in superficially identifying senescent cells de novo, the onus remains on the investigator to demonstrate why a cell should be considered senescent, rather than relying on historical markers such as p16INK4a or p21Cip1. Thus, new technologies designed to identify novel senescent cells and phenotypes are necessary that will require validation both in culture and in tissue. The ultimate goal of this proposal is to develop new technologies to map senescent signatures back to intact human tissue. This goal will enable us to identify and spatially characterize senescent cells in each tissue, uncovering unique markers depending on tissue and cell type. Pilot 1: Identify senescent cells in the mouse vasculature, and determine if they can be detected by ultrasound, and verified in human tissue; Pilot 2: Develop a microphysiologic ex vivo tissue-on-a-chip to model ovarian senescence, and human tissue-tissue interactions via the SASP. These two pilots will use a combination of cell surface markers identified in our initial profiling, and Digital Spatial Profiling (DSP, Nanostring) or Visium (10x) to localize senescent signatures to morphological structures and cells in tissue sections. Our proposal will develop validated markers of senescence in multiple tissues and cell types not previously characterized to deploy these technologies to the broader community.

PROJECT NARRATIVE We will determine the feasibility of detecting senescent cells in two distinct tissues – the ovary and the vasculature using novel technologies. We will evaluate molecular markers of cellular senescence in single cells and biofluids using a combination of gene and protein expression, and secreted proteins. If successful in our initial pilots, we will validate our approaches in human biopsies, demonstrating broad clinical utility of these emerging technologies.