Lympnoids tissue senescence and multimodal imaging using REDCAT - SUMMARY Cellular senescence, a hallmark of aging, is an irreversible state of cell cycle arrest in otherwise proliferative cells. Senescence in immune organs is particularly significant in aging and disease, as immune cells circulate throughout the body and regulate the physiological functions of all major organs. Metabolic alterations play a crucial role in immunosenescence and are recognized as key mechanisms driving abnormal immune homeostasis. To better understand the distinct role of senescent cell metabolism – for example, in thymic aging and immune function – there is a pressing need for novel methods to map senescent cells and cell-type-specific metabolism within complex tissues to assess their impact on the local environment. Given the critical role of immune senescence in systemic, organism-level aging, we hypothesize that: (i) thymic epithelial cell (TEC) senescence plays a key role in driving thymic aging. (ii) abnormal metabolic dynamics, including lipid metabolism and accumulated adipocytes in aged thymi, contribute to reduced T-cell diversity, (iii) senescence and aging in the thymus lead to systemic immune function decline. To address these hypotheses, we propose to develop and deploy an integrated platform, Raman Enhanced Determination of Cell Atlas and Typing (REDCAT), for mapping single-cell metabolic activity in complex tissues and decoding the underlying transcriptional and epigenomic mechanisms. It will be applied to the study of thymic aging in wild-type, lineage-tracked, and FGF21 mouse models. Specifically, we will (1) develop REDCAT for single-cell resolution profiling of cellular senescence in complex lymphoid tissues, (2) examine senescence-associated transcriptomic mechanisms via integrating DBiT- Based spatial transcriptome sequencing, and (3) investigating dynamic phenotypic and metabolomic heterogeneity of senescent cells in thymic aging and the impact on tissue microenvironments and systemic immune function. The proposed techniques can be widely adopted by the cellular senescence and aging research community. This work will also generate a valuable data resource to unveil insights in thymic aging to advance fundamental understanding of immuno-senescence and propel translational developments in anti- senescence interventions aimed at improving immune function and overall healthspan.
