SASP-ER platform to identify spatiotemporal senescence-associated secretome

PROJECT SUMMARY The goal of this R21 project is to build a research platform to assess senescence-associated secretory phenotype (SASP) in vivo. A prominent feature of cellular senescence is the secretion of diverse biologic factors (cytokines, chemokines, proteases etc), collectively referred to as the SASP. Indeed, the SASP is hypothesized to be a key player in driving age-related diseases. However, SASP has been characterized almost exclusively in vitro due to the challenge of identifying the source of a secreted protein in vivo. Moreover, the field has been challenged by disentangling SASP from inflammation markers that may respond to other stressors, such as an infection. Cellular senescence is remarkably heterogeneous, and cellular responses can vary significantly both between cell types and in response to different senescence-inducing stressors. In addition to this heterogeneity, several studies show that although valuable, high resolution unbiased transcriptome analysis does not provide direct information about secreted proteins. To address these significant knowledge gaps, we will combine an innovative research infrastructure platform to assess SASP and multiple well-established aging paradigms to elucidate the molecular identity of the SASP-secretome in vivo. To accomplish this, we will take advantage of a novel SASP- ER platform that permits the conditional tagging of secreted proteins in specific cell types to identify the SASP signature. Tagged proteins can be purified from biological fluids and analyzed via mass spectrometry (MS). In addition, this data will allow integration with other -omic datasets to define robust composite biomarkers to disentangle senescence and SASP signature from distinct tissues. In Aim 1, we will asess secretory signature of multiple cell types from the lung and liver in response to two robust inducers of senescence—telomere dysfunction and persistent DNA damage. In Aim 2, we will perform cross-modal analysis to analyze the overlap between the transcriptome, proteome and secretome of senescent cells in vivo. We anticipate that our study will contribute to the development and validation of a diagnostic test to assess senescence burden and allow for translation in human disease in future studies. The research infrastructure assembled here is portable and highly adaptable, providing the research community with a tool to identify SASP in tissues of their interest. Together, the significance of this R21 proposal is that it will set-up an infrastructure to unambiguously identify secretome in vivo, capture the heterogeneity of senescence and SASP in vivo, provide a new tool to identify SASP in a cell type and stress-specific manner, as well as in the future test the efficacy of interventions such as senotherapeutics.