Spatial omics technologies to map the senescent cell microenvironment

Project Summary Extracellular vesicles (EVs) are critical mediators of intercellular communication, released by cells to transfer bioactive molecules like RNA, DNA, proteins, and lipids. In the context of cellular senescence, EVs play a crucial role in the senescence-associated secretory phenotype (SASP), helping senescent cells communicate with their environment. These vesicles can induce paracrine senescence, promoting inflammation and tissue remodeling in neighboring cells. EVs have been associated with various age-related diseases such as cancer, fibrosis, and neurodegeneration, where their cargo, including specific proteins and RNA, can serve as biomarkers for senescence, offering non-invasive diagnostic potential. The project aims to profile the RNA, DNA, and protein content of EVs from human blood, focusing on markers linked to senescence. We will isolate and sequence these molecules, particularly focusing on senescence- related markers like LINE-1 and pro-inflammatory cytokines. By using super-resolution microscopy (STORM), we will image individual EVs and analyze their cargo. The integration of this data with the broader benchmarking project, which also includes lung and skin tissue analyses, will provide insights into the role of EVs in senescence-related diseases. In Aim 1, RNA, DNA, and protein profiling will be conducted using sequencing and immunoassay techniques, while Aim 2 will involve imaging the content of individual EVs at super-resolution. This will help identify senescence markers, which can be used for further clustering and analysis of EVs. The expected outcomes include identifying enriched senescence markers in EVs and integrating this data with existing datasets to better understand the role of EVs in cellular aging and disease progression.