Developing a lung organoid signalome for real-time analysis of senescence-associated cellular cross talk

Cells in our body suffer damage on a daily basis, and this is particularly true in the lungs which are constantly exposed to dangerous agents in the air that we breathe. In isolation, the effect of pollution, diesel particles, tobacco smoke, allergens and viral insults may be mild, but repeated exposure over many years can lead to development of diseases which have long term effects on lung health including Idiopathic Pulmonary Fibrosis (IPF) and Chronic Obstructive Pulmonary Disease (COPD).
After exposure to an insult, the cells in the lung have to decide how to respond. In younger healthy individuals, lung cells will quickly repair themselves in response to a mild insult, restoring ormal lung function. As we age, the effect of years of repeated mild injuries leads the lung cells to respond in a different way, a process called cellular senescence. Senescent cells so not divide, so they are unable to aid in the repair of the lung following even mild injury. They also do not die. Instead they remain in the lungs and release signals which encourage neighbouring cells to also become senescent. Accumulation of these senescent, defective cells appears to contribute to the development of chronic lung disease although the exact process is unclear.
We will create a 3D model of the human lung which contains two of the most important lung cell types, epithelial cells and fibroblasts. These cells will be modified so that they can change colour in response to signals generated by senescent cells. We will use this model to try and understand how healthy cells in the lung respond to signals from senescent cells, and how neighbouring cells influence each other's behaviour.
Understanding how senescent cells increase in number in the aging lung could help us to identify new treatment to prevent these dysfunctional cells from collecting in the lung and causing disease.

Accumulation of senescent cells is linked to diseases of aging increasing interest in the use of senotherapeutic drugs for conditions affecting mutiple organ systems including the lung. However, a better understanding of the mechanisms regulating senescence, its biological consequences and role in disease is required before widespread use of these drugs can be considered.
We will develop a next generation 3D human lung organoid which incorporates specific 'Signalome' reporter cell lines to permit real-time live cell imaging of cell signalling and epithelial-mesenchymal cross talk to test the hypothesis that within the lung, dysfunctional senescent epithelial cells alter the behaviour of neighbouring cells, triggering senescence and inducing pathological changes in adjacent fibroblast populations.
Visually barcoded lung fibroblasts, alveolar and bronchial epithelial cells will be infected with 12 lentiviral fluorescent reporters corresponding to key signalling pathways or senescence markers. These 'Signalome' clone cells will initally be treated with mediators of senescence or lung injury and live imaged over time to compare the temporospatial signalling pathway dynamics in each cell type, assess whether pathways are activated sequentially or in parallel during senescence and if the responses are cell type and/or stimulus specific. We will study the role of paracrine SASP signalling in promoting bystander cellular senescence in neighbouring healthy cells, determine whether this occurs via direct cell-cell contact or the release of soluble (sSASP) or exosomes/extracellular vesicles (eSASP) SASP components and seek to understand the signalling pathways involved. Finally the Signalome clones will be combined in 3D organoids to investigate the impact of epithelial-mesenchymal cross talk on the acquisition of a senescence and understand the potential for a small population of senescent cells to modify the behaviour of adjacent cells and cause aging-related disease.