Targeting, Quantifying, and Isolating Heterogeneous Populations of Senescent Cells from Tissues via Cell Surface and Secreted Proteomes

National Institute On Aging

Investigators

Linked publications, trials & patents

Abstract

In our first objective, substantial progress has been made in refining the list of SASP biomarker candidates based on human cohorts studies. In early collaborative studies with Drs. Luigi Ferrucci and Toshiko Tanaka, we identified a subset of the senescence-associated secretory phenotype (SASP) that are associated with aging in human plasma in the Baltimore Longitudinal Study of Aging. To refine this list into a panel of SASP biomarkers that is stable across geographically and genetically diverse human cohorts our collaborators were able to identify age-associated SASP proteins in the InCHIANTI study, a representative population-based study of older persons living in the Chianti geographic area (Tuscany, Italy). These studies are now published in the journals PLOS Biology and Elife. Toward our second objective, we have collected early data on the cell-surface proteome (surfaceome) of senescent cells, from which we will identify and prioritize the most specific surfaceome candidates for targeting senescent cells. To identify the most specific surface proteins, we have compared the cell surface proteins we identified on senescent fibroblasts with the cell-surface protein atlas database (Baush-Fluck et al. 2015. PLOS One) and rank our candidates based on the number of cell-types they are known to be expressed in. To expand and validate our list of senescent cell surface markers, we have also collected senescent and non-senescent cell surface proteins using an additional method termed glyco-cell surface capture, which isolates cell surface N-linked glycoproteins. In collaboration with Dr. Bernd Wollscheid (ETH Zurich), we have worked to implement these methods and new innovations in the protocol that will reduce the sample amounts required for cell surface capture. The initial materials for a pilot study of the new protocol have been collected and are awaiting mass spectrometry analysis. To validate cell surface markers in human specimens going forward, we are making arrangements with our colleagues at the BLSA to receive formalin-fixed paraffin embedded (FFPE) skin, fat, muscle, and plasma tissues from aged individuals. These specimens will be probed for candidate surfaceome proteins by immunofluorescence. To validate senescent markers in vivo in a tissue type that matches the cell culture experiments used for the initial discovery of the surfaceome, we have requested FFPE lung tissues from young healthy, old healthy, and idiopathic pulmonary fibrosis patients from Dr. Steven Huang from the Division of Pulmonary and Critical Care Medicine at the University of Michigan. We will use lung tissue as well to minimize variability due to cell-type differences from IMR90s, which are primary lung fibroblasts.