Senescence-associated morphological profiles (SAMPs): an image-based phenotypic profiling method for evaluating the inter and intra model heterogeneity of senescence.

Senescence occurs in response to a number of damaging stimuli to limit oncogenic transformation and cancer development. As no single, universal senescence marker has been discovered, the confident classification of senescence induction requires the parallel assessment of a series of hallmarks. Therefore, there is a growing need for "first-pass" tools of senescence identification to streamline experimental workflows and complement conventional markers. Here, we utilise a high content, multidimensional phenotypic profiling-based approach, to assess the morphological profiles of senescent cells induced via a range of stimuli. In the context of senescence, we refer to these as senescence-associated morphological profiles (SAMPs), as they facilitate distinction between senescent and proliferating cells. The complexity of the profiles generated also allows exploration of the heterogeneity both between models of senescence and within an individual senescence model, providing a level of insight at the single cell level. Furthermore, we also demonstrate that these models are applicable to the assessment of senescence in vivo, which remains a key challenge for the field. Therefore, we believe SAMPs has the potential to serve as a useful addition in the repertoire of senescence researchers, either as a first-pass tool or as part of the established senescence hallmarks.

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

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.

The bright and dark side of extracellular vesicles in the senescence-associated secretory phenotype.

Senescence is a state of proliferative arrest which has been described as a protective mechanism against the malignant transformation of cells. However, senescent cells have also been demonstrated to accumulate with age and to contribute to a variety of age-related pathologies. These pathological effects have been attributed to the acquisition of an enhanced secretory profile geared towards inflammatory molecules and tissue remodelling agents - known as the senescence-associated secretory phenotype (SASP). Whilst the SASP has long been considered to be comprised predominantly of soluble mediators, growing evidence has recently emerged for the role of extracellular vesicles (EVs) as key players within the secretome of senescent cells. This review is intended to consolidate recent evidence for the roles of senescent cell-derived EVs to both the beneficial (Bright) and detrimental (Dark) effects of the SASP.