Senescence is a cellular phenotype characterized by an irreversible cell cycle arrest and the secretion of inflammatory proteins, denominated senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, until now this role has been mainly attributed to soluble factors.
Here, researchers from Queen Mary University of London report that extracellular vesicles also alter the environment by transmitting the senescent phenotype to other cells via exosomes (extracellular vesicles of endocytic origin). A combination of functional assays, Cre-loxP reporter systems, proteomic analysis and RNAi screens confirm that exosomes form part of the senescent secretome and mediate paracrine senescence via the activation of a non-canonical interferon (IFN) pathway. Altogether, the researchers speculate that exosomes could be drivers of tissue degeneration both locally and systemically during aging and age- related disease.
Exosomes form part of the senescent secretome and mediate a cell cycle arrest in normal HFFF2s
(A) Schematic representation of the proof-of-concept experiments performed to show that exosomes form part of the senescent secretome. CM was taken from iC or iRAS HFFF2s and tested for the ability to induce senescence in HFFF2 as a whole(Figure S2A)or (B) processes by serial centrifugation to evaluate the effect of the different fractions, supernatant (SN), macrovesicles(MV) or exosomes(Ex), to induce senescence in HFFF2s.(B) HFFF2 fibroblasts were treated for 72h with the different fractions of the CM: SN, MV or ExfromiC or iRAS cells and the percentage of cells incorporating BrdU by IF was quantified. The graph represents the mean ±SD of 2-5 independent experiments.(C) Representative pictures show HFFFs treated for 72h with either the SN or the Ex fraction and stained for p53 or p-gH2AX. The quantification of the percentage of HFFF2s staining positive for each antibody is shown (mean ±SD of 2 independent experiments).(D) Immunoblotting for HFFF2s treated for 72h with Ex derived from iC and iRAS show an increase in p21CIP, p16INK4Aand integrin b3 when treated with iRAS Ex. b-Actin is used as loading control. (E) RNA from HFFF2 cells treated for 72hwith exosomes isolated from iRAS (mimicking OIS) or HFFF2 treated with Etop (mimicking DDIS) was isolated from 3 independent experiments and sent for RNA sequencing. (F) Gene ontology (GO) analysis for genes involved in Cellular Processes with >2 log2fold differential expression and p-value < 0.05 in both OIS-and DDIS-treated HFFF2s. The pie chart shows a high proportion of genes related to the “Cell cycle” and “Cell proliferation” pathways.(G,H) To confirm the RNA sequencing data, HFFF2 were incubated with an equal number of exosomes from HFFF2 treated with Etop with or without SpE and (G) the relative mRNA levels were determined for genes implicated in cell cycle regulation (CDKN1A, CDKN1A) by qPCR or (H) p53 protein levels were determined by IF. Data represent mean ±SD of (G) 2-3 and (H) 2 independent experiments