PROJECT SUMMARY – Core C (Biological Analysis Core) Cellular senescence in human primary lymphoid organs such as bone marrow (BM) and thymus, and secondary lymphoid organs such as tonsil and lymph nodes (LNs) have been reported to show complex roles in normal hematopoiesis, immune function, aging, and disease development. These organs are among the most active, dynamic, and plastic physiologic systems. How cellular senescence is associated with stress- induced or age-related functional impairment, what types or subtypes of senescent cells are present and their spatial heterogeneity in the lymphoid tissues and how these cells impact the tissue environments remain poorly understood, precluding the development of strategies to target these cells to improve healthspan/lifespan or harnessing these cells or secreted factors to promote tissue remodeling and repair. Yale TMC Core C: Biological Analysis Core (BAC) will deploy a suite of high-resolution, high-content and high-throughput single- cell & spatial omics technologies to construct comprehensive maps of cellular senescence and the associated environments in primary human cells and human lymphoid tissues. Four major biological analyses will be performed: single-cell high-plex protein secretome profiling, single-cell proteo-transcriptomic sequencing (scCITE-seq), spatial proteo-transcriptomic sequencing at cellular level (DBiT-seq), and spatial molecular imaging (SMI) at single molecule resolution. Specifically, we will conduct (1) single-cell protein secretome profiling of SAPS heterogeneity using induced senescent cell models and body fluids, (2) single-cell and spatial proteo-transcriptomic sequencing to map senescent cells and environments in human lymphoid tissues. (3) high-plex proteo-transcriptomic spatial molecular imaging of lymphoid tissues. We envision that the proposed project will generate extensive multiscale, multimodality, and multifaceted data to characterize phenotypic, functional and spatial heterogeneity of senescent cells in response to multiple inducers or modifiers, identify biomarkers of senescent cell subtypes and neighborhoods, and construct maps and atlases of cellular senescence and environments in primary and secondary lymphoid organs of humans over longitudinal time frames, representing a highly valuable resource for a wide range of biomedical research.