Isolation methodology is essential to the evaluation of the extracellular vesicle component of the senescence-associated secretory phenotype.

Ryan Wallis,Arturo Robles-Tenorio,Natasa Josipovic,Hannah Mizen,Cleo L Bishop,Eleanor J Tyler,Argyris Papantonis

doi:10.1002/jev2.12041

Abstract

A hallmark of senescence is the acquisition of an enhanced secretome comprising inflammatory mediators and tissue remodelling agents – the senescence‐associated secretory phenotype (SASP). Through the SASP, senescent cells are hypothesised to contribute to both ageing and pathologies associated with age. Whilst soluble factors have been the most widely investigated components of the SASP, there is growing evidence that small extracellular vesicles (EVs) comprise functionally important constituents. Thus, dissecting the contribution of the soluble SASP from the vesicular component is crucial to elucidating the functional significance of senescent cell derived EVs. Here, we take advantage of a systematic proteomics based approach to determine that soluble SASP factors co‐isolate with EVs following differential ultracentrifugation (dUC). We present size‐exclusion chromatography (SEC) as a method for separation of the soluble and vesicular components of the senescent secretome and thus EV purification. Furthermore, we demonstrate that SEC EVs isolated from senescent cells contribute to non‐cell autonomous paracrine senescence. Therefore, this work emphasises the requirement for methodological rigor due to the propensity of SASP components to co‐isolate during dUC and provides a framework for future investigations of the vesicular component of the SASP.

Highlights

1.1 Senescence 1.1.1 The Cell CycleThe division of eukaryotic cells is orchestrated in a highly regulated process known as the cell cycle (Figure 1.1)
Senescence induction was confirmed through the characterisation of multiple orthogonal markers including tumour suppressor expression (p16 and p21), loss of cellular proliferation and the appearance of senescence-associated heterochromatin foci (SAHF)
This panel demonstrated that Oncogene-induced senescence (OIS) was associated with an overall increased cellular area, whilst this enlarged shape was more irregular than that of the proliferating vector control

Summary

Introduction

1.1 Senescence 1.1.1 The Cell CycleThe division of eukaryotic cells is orchestrated in a highly regulated process known as the cell cycle (Figure 1.1). Progression through the cell cycle is driven by the interaction and complex formation between cyclin dependent kinases (CDKs) and cyclin sub-units (Satyanarayana and Kaldis, 2009; Lim and Kaldis, 2013) These allow advancement through regulatory checkpoints at which stage a cell is committed to the phase of the cell cycle (Malumbres and Barbacid, 2001; Vermeulen, Van Bockstaele and Berneman, 2003). These stimuli have been demonstrated to drive a context dependent response, with the emergent phenotype dependent on the particular senescence trigger, and on the cell type under insult This leads to varied secretory profiles emerging between different models of senescence, with the SASP driven by both cell type and senescence inducer. The contribution of EVs to the phenomenon of paracrine senescence is assessed in both IMR90 fibroblasts and MDA-MB-468 basal-like breast cancer cells (468s)

Objectives

Methods

Findings

Discussion

Conclusion