SUMMARY Yale-murine TMC (mTMC), through application of high-content and high throughput single-cell and spatial omics technologies, we aim to accelerate the discovery of senescent biomarkers, apply them to mouse models, and generate hypotheses to test mechanisms of organismal aging. Our proposed unbiased analyses will also answer the question whether senescent cells (stromal or hematopoietic lineage) exist in sufficient quantities to alter the inflammatory landscape and biology. Yale-mTMC will use lineage-marked mouse to classify types or subtypes of senescent cells, their spatial heterogeneity and how these cells impact the tissue environments. Yale-mTMC will assemble 1) a multidisciplinary team to generate the molecular and cellular maps of cellular senescence in Thymus, Bone marrow, Spleen, PBMCs, Mesenteric and Inguinal adipose tissue. 2) develop and deploy a suite of high-resolution, high-content and high throughput single-cell and spatial omics technologies to characterize these specimens and 3) perform integrated informatics to identify biomarkers of senescent cell heterogeneity and to construct comprehensive molecular and cellular maps of cellular senescence and including an i.v CD45 antibody labeling/sorting approach to study tissue resident immune cells in non-lymphoid tissues. We will utilize the analysis platforms established through the Yale human TMC to enable cross-species verification of biomarkers. Three major biological analysis pipelines are: (A) Multiplex Imaging (MI) including CODEX, IMC, and SMI, complemented by 3D light sheet microscopy of cleared tissues, (B) Single Cell Analysis (SCA) including scCITE-seq for protein and mRNA profiling, CyTOF for high-plex immunophenotyping, and single-cell protein secretome profiling to measure SASP heterogeneity, and (C) Spatial Multi-Omics Sequencing (SMOS) using DBiT-based spatial-CITE-seq for spatially resolved proteo-transcriptomic mapping at genome scale. The combination of these pipelines allows for highly sensitive and single-cell resolution mapping of senescent cells and associated tissue environments. Yale-mTMC aims to assemble a multidisciplinary team led by PI: Dixit (Director, Yale Center for Research on Aging), and MPI: Montgomery (Immunologist, Associate Dean for scientific affairs) with Core Leads Dr. Fan (Bioengineer), Dr. Kluger (informatics and data analytics) and Key personnel Dr. Booth (mouse pathologist), Dr. Lucas, Haberman (Immunologists, expert in pet store mouse model, imaging) with IAB composed of scientific leaders Drs. Medzhitov, Iwasaki and Ruddle. We have experience in management of large, multi-component programs and prior experience with generating significant high-quality imaging and omics data as part of a consortium. A dedicated program manager will manage and coordinate all activities across the center and with the SenNet consortium. Complementary and multidisciplinary scientific expertise of the team will translate into the collective capability of the TMC to foster integration of multi- dimensional, multiparameter data generation and coordination with SenNet and other TMCs for greater impact

PROJECT NARRATIVE The Yale-murine Tissue Mapping Center project aims to use high-throughput, high-resolution single-cell and spatial omics technologies to characterize senescent cell heterogeneity with special emphasis on the immune cells in various tissues. This data resource will advance the understanding of senescent cells, their biomarkers and mechanism of aging.