Generation of an In Vivo Senescent Cell Atlas: Across the life-course and in pathology

Lead Research Organisation: University of Cambridge
Department Name: CRUK Cambridge Research Institute

Abstract

Senescence is a state of stable cell cycle arrest, triggered by physiological and pathological stressors. Whilst senescent cells cannot proliferate, they are biologically active, and have been shown to be functionally active in a variety of settings including (but not limited to) embryogenesis, ageing, wound healing, cardiovascular disease, and tumourigenesis. In these diverse contexts, senescence can be either a positive or detrimental component to organismal health. For example, senescent cells promote the prompt closure of wounds, and their absence delays this process. Whilst in ageing the accumulation of senescent cells across multiple tissues results in tissue dysfunction, in this context their ablation (pharmacological or genetic) has been shown to extend healthy life-span in mice. Many of these effects are believed to be mediated by the acquired secretory phenotype (Senescence-Associated Secretory Phenotype; SASP).

So how do senescent impinge on these diverse biological processes, across a variety tissues? Surely a senescent liver cell during fibrosis cannot engage the exact same programme as precancerous skin cell, as the functional consequences are widely divergent. To answer this question, our working hypothesis is that senescence cannot be described as one programme, and that different tissue and senescence-inducing contexts are associated with both unique and common functional states. Furthermore, we also posit that senescence in one pathophysiological setting (i.e. fibrosis) is not composed of one senescent population, but a number of sub-populations whose composite together orchestrates the phenotypic outcome. Thus, alterations to the proportions of these sub-populations may result in a pathological disease.

However, currently the majority of senescence data has been developed in vitro, and in a small number of lines (often primary fibroblasts). As such we lack a basic understanding of senescence in vivo, across these diverse states and tissue contexts, and importantly at a resolution that enables us to determine what sub-populations exist.

The aim of this proposal is to generate a high-quality in vivo atlas of these diverse senescent states to act as a catalyst for the research and biotech industry. Our proposal centres around the use of novel tools to isolate senescent cells (a p16-lineage tracing mouse, and a fluorescence dye for Senescence-Associated Beta Galactosidase that requires no fixation and pH modification), innovative sequencing approaches (enabling the transcriptome, chromatin accessibility, and methylation data to be isolated from the same single cell), and pioneering computational analyses (interrogation of multi-omic datasets). Combined these data will enable us to characterise the different senescent sub-populations, as well as understand how these diverse functional states are achieved. Integral to the proposal is our focus on maximal distribution of this resource, as such we intend to generate a user-friendly web-based platform for the rapid dissemination of processed data, in addition to sharing all technical and primary data with the community.

As such this resource has the potential to be used widely by the academic research community to quickly test hypotheses based on their own primary data. Furthermore, as targeting senescence pharmacologically has been shown to improve health and life-span in mice, understanding the functional senescence states is essential to develop future targeted agents, with reduced off-target effects. In this regard, this dataset will also be essential for the burgeoning healthy-ageing focused biotech industry.

Technical Summary

Senescence is functionally implicated in a myriad of pathophysiological settings, including ageing, wound healing and tumour development. Whilst there is a general consensus that this is in part mediated through the acquired senescence-associated secretory phenotype (SASP), much of this work has been performed in vitro, across a limited number of cell lines. We currently have a limited understanding of what constitutes senescence in vivo, in these diverse cellular and pathological settings, and whether the functional programmes underlying senescence contain tissue and context specific features. As such we intend to isolate senescent populations from a number of distinct pathophysiological states using two fluorescent reporters and acquire the transcriptional, DNA methylation status, and nucleosome accessibility information from the same single cell. Fundamentally, our study will test the hypothesis that senescence is not one functional state, but instead is a composite of multiple functional units. Furthermore, we aim to prove that different tissue and pathophysiological contexts are associated with the emergence of unique senescent sub-populations, with functionalities that reflect the biological context. In addition to the cutting-edge single cell multi-omic approaches and novel tools developed by collaborators, we have also ensured this is a community-driven project by forming an advisory board composed of prominent members of the senescence and ageing communities.

Planned Impact

We live in an ageing society, however while average lifespans have increased the average health-span for an individual has not kept apace. As such age-associated disorders and pathologies are becoming an increasing fiscal and social burden on our society. Senescence represents a cellular state that is a key component of organismal ageing and the development of age-associated disorders. Recent evidence in mice suggests that targeting senescence may lead to improvements in age-related disorders and promote healthy ageing. Yet the bulk of our knowledge regarding this state is based on in vitro data, and is relatively poorly understood as an in vivo phenomenon.

As such we have proposed to develop a 'Senescence Atlas' to generate data and methodologies that will have instant impact across a wide-range of research areas and diseases.

Academic Impact: Firstly, basic and translational researchers will benefit from these data, this is exemplified by the wide-range of scientific backgrounds, and respective research questions, that comprise the members of our scientific advisory board. Additionally, we aim to be a highly transparent working group, providing in-depth technical descriptions of senescent cell isolation and data analysis tools for the wider community. Finally, this project will benefit tremendously the post-docs, involving personal development, research-specific training, and ensuring efficient and complete dissemination of information to all stakeholders.

Economic impact: We live in an ageing society and as such the incidence of age-related diseases (for which senescent cells are functionally implicated) are increasing. This has produced a fertile environment wherein biotechnology and pharmaceutical companies are attempting to develop intervention strategies to treat these disorders, however our current knowledge of what to target, with regards to in vivo senescence populations, and how amenable they are to current targeting agents is relatively low. Our data can not only be used to address the former, but will specifically address the latter. As such this has the potential to act as a catalyst for these sectors.

Societal: As mentioned above, our society has an increasing burden of age-related disorders which result in a high cost to society to treat and manage. The combined impact academically and economically has the potential to provide a profound shift in society, by promoting an increased health-span, or healthy aged population.

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