Small Extracellular Vesicles Are Key Regulators of Non-cell Autonomous Intercellular Communication in Senescence via the Interferon Protein IFITM3

Michela Borghesan,Juan Fafián-Labora,Olga Eleftheriadou,Paula Carpintero-Fernández,Marta Paez-Ribes,Gema Vizcay-Barrena,Avital Swisa,Dror Kolodkin-Gal,Pilar Ximénez-Embún,Robert Lowe,Belen Martín-Martín,Hector Peinado,Javier Muñoz,Roland A Fleck,Yuval Dor,Ittai Ben-Porath,Anna Vossenkamper,Daniel Muñoz-Espin,Ana O’Loghlen

doi:10.1016/j.celrep.2019.05.095

Abstract

SummarySenescence is a cellular phenotype present in health and disease, characterized by a stable cell-cycle arrest and an inflammatory response called senescence-associated secretory phenotype (SASP). The SASP is important in influencing the behavior of neighboring cells and altering the microenvironment; yet, this role has been mainly attributed to soluble factors. Here, we show that both the soluble factors and small extracellular vesicles (sEVs) are capable of transmitting paracrine senescence to nearby cells. Analysis of individual cells internalizing sEVs, using a Cre-reporter system, show a positive correlation between sEV uptake and senescence activation. We find an increase in the number of multivesicular bodies during senescence in vivo. sEV protein characterization by mass spectrometry (MS) followed by a functional siRNA screen identify interferon-induced transmembrane protein 3 (IFITM3) as being partially responsible for transmitting senescence to normal cells. We find that sEVs contribute to paracrine senescence.

Highlights

SEVs and Soluble Factors from Senescent Fibroblasts Mediate Paracrine Senescence To investigate whether EVs act as intercellular mediators during senescence, we took advantage of HFFF2 human foreskin primary fibroblasts expressing an empty vector or oncogenic H-RASG12V in a 4-hydroxytamoxifen (4OHT)-inducible form (ER:EV or ER:H-RASG12V)
MVs isolated from iC or iRAS induced a reduction in the incorporation of BrdU, no other markers of senescence were observed, suggesting that only the SN and small extracellular vesicles (sEVs) from iRAS cells transmit paracrine senescence
To confirm sEV-PS induction, we treated HFFF2s with increasing concentrations of sEVs derived from iRAS compared to the highest dose of sEVs from iC and observed a dose-dependent senescent response by quantifying the percentage of cells staining positive for SA-b-Gal and the levels of p53+ and BrdU+ cells by IF (Figures S1B and S1C)

Summary

Methods

MV and sEV isolation All cells were maintained in sEV-depleted FBS. The supernatant was filtered through a 0.22mm filter prior to the 100,000 g centrifugation step and after in some cases (for the CreLoxP experiments). The supernatant was collected after a 1h and 20min 100,000 g centrifugation step and concentrated using a 10K column (Amicon Ultra-0.5 Filter) at 14,000 g for 10min obtaining a concentration factor 10X. The final 100,000 g pellet was washed once in 15ml of PBS and resuspended in 100 mL of 10% FBS-depleted media for the functional cell culture experiments. For the MCF7 functional experiments the pellet was resuspended in 0.5% FBS-depleted media. A Sorvall 100SE Ultra Centrifuge, with a Beckmann Fixed Angle T865 rotor was used for all sEV isolations. We have submitted all relevant data of our experiments to the EV-TRACK knowledgebase (EV-TRACK ID: EV190024)

Results

Discussion

Conclusion