Spatially-resolved protein and transcriptome mapping of senescent cells

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PROJECT SUMMARY The goal of this project is to generate a novel technological pipeline to phenotype senescent cell identity (defined by biomarkers, function-related transcriptional profiles, morphology, and microenvironment) and composition (defined by quantity, diversity, and distribution) across any mouse or human tissue. Use of diverse profiling methods has revealed that senescent cells are highly heterogeneous and adversely influence tissue health and function. However, due to biological heterogeneity and reliance on diverse methods, we do not comprehensively understand the identities of senescent cells and extent to which they contribute to age-related decline. This limits the ability to devise effective therapeutics that could have important societal benefit. To fill these significant knowledge gaps, we require new technologies that accurately characterize heterogenous senescent cell states in aged tissues. We will pioneer iterative and integrated use of imaging mass cytometry (IMC) and transcriptomic digital spatial profiling (DSP) to molecularly phenotype senescent cells in aged tissues. Our preliminary data generated from high-dimensional spatially-resolved and suspension-based mapping technologies demonstrate that microglia, neurons, and additional cell types display distinct senescent profiles in the aged mouse brain. Unique properties of the brain include substantial cell and regional heterogeneity, limited regenerative capacity, age-vulnerability, and pleiotropic presentation of senescence-related biomarkers, all within well-defined micro- environments. These features support an initial experimental focus on the brain for feasible and comprehensive resolution of the anticipated panoply of senescent identities and contexts. Thus, we will develop this novel tech- nology to map senescent cells, first, in the hippocampus and cortex of aged mice (UG3 phase) and subsequently, across brain regions in female and male mice throughout the lifespan (UH3 phase). Critically, our innovative technological pipeline, by design, will be broadly applicable to any tissues through customization of cell-identity and senescence biomarkers. We will leverage experience studying cell senescence and the SASP in diverse tissues to collaboratively adapt and scale IMC and DSP to generate mouse and human senescent cell atlases. High-dimensional multimarker imaging and spatial transcriptomics are anticipated to revolutionize the ability to rigorously and comprehensively characterize and map senescent cells in distinct tissue contexts. Ultimately, use of this novel technology may fundamentally advance understanding of how cell senescence contributes to age- related tissue dysfunction and may reveal new strategies to disrupt senescence-mediated pathology.