Abstract 898: Evaluation of cellular senescence through fluorescence characterization

Abstract

Abstract Aging is considered a risk factor for developing many chronic diseases which includes cardiovascular diseases, cancer, and neurodegenerative diseases. Researchers are now showing that many of these diseases are associated with cellular senescence. Cellular senescence is an important process for the removal of damaged cells, but if senescent cells are not removed a chronic pro-inflammatory environment ensues, increasing the risk of many age-related diseases. These cells accumulate as a result of cancer chemotherapy, are associated with age-related diseases, and are important for development. Recent work has shown that specific targeting of senescent cells results in increased life expectancy in a progeroid Ercc1-/Δ mouse model. As such there is great interest in identifying, characterizing and targeting senescent cells. Senescent cells are identified by the release of pro-inflammatory cytokines and chemokines, increased beta galactosidase activity (b-gal), senescence associated heterochromatin foci (SAHF), and changes in morphology which are increased size and a flattened appearance. There is no single good marker for determining senescence, so these cells are identified through the collective interpretation of multiple markers. Senescence associated b-gal (SA-b-gal) is considered the “gold standard” for identifying senescent cell, and it is detected with the blue-green precipitating colorimetric x-gal substrate. However, this substrate is limited use in that it cannot be used in flow cytometry or other fluorescence based platforms. Even though C12FDG, a fluorescein based b-gal substrate, has been used since the mid 90’s, it has limited utility restricted by its lack of fix-ability. Newer b-gal substrates are demonstrated here which do not leak out of cell; enabling researchers to more easily identify and evaluate cellular senescence. In this study we use a fluorescence approach to assess the senescence phenotype based on a combination of western blots, fluorescence based imaging and ICC, flow cytometry, cell sorting, and RT-qPCR. We examined several cell-based models include replicative senescence characterized by telomere shortening in primary fibroblasts, cellular senescence characterized through DNA damage pathway by doxorubicin, oxidative stress senescence induced by confluency in atmospheric oxygen environment (SIPS), and senescence induced with palbociclib treatment (a CDK 4/6 inhibitor). Overall, we found that phenotypic characteristics of senescence vary depending on the cellular model and that a multiplex fluorescence based approach to identifying senescent cells will contribute to the understanding of cellular senescence and the age-related diseases. Citation Format: Scott T. Clarke, Taryn Jackson, Amanda Kelley, Quentin Low, Timothy Huang, Yi-Zhen Hu, Veronica Calderon, Rong Wang, Michael O'Grady. Evaluation of cellular senescence through fluorescence characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 898.