Cellular Phenotype sequencing (CePh-seq): Decoding the morphological and molecular age-related phenotypes of single cells

All the cells in our body have the same genetic information, yet different cells which have the same genome display very different properties and do very different things. A large part of these differences comes down to which genes are actually turned on within each cell. It has also been seen that cells which have the same combination of genes being transcribed often look very similar and behave in a similar way, yet understanding the connection between active genes and appearance is very difficult, as there is no automated way to collect cells which all look similar.
In this BBSRC project, we will develop a new biotechnology instrument which can take a mixed sample of cells, accurately measure their morphological properties, then select those cells with a particular morphology of interest, barcode them with a unique molecular identifier, and finally measure the activity of all of their genes. The use of the barcode is essential because the process of analysing the genes destroys the cells and only looks at the molecules within the cell. By linking this molecular barcode to the image of the cell before it was destroyed, it will be possible to investigate the relationship between particular genes and how cells look.
We will use this new technology to investigate a so far unsolvable problem in ageing which may shed light on how old cells pack their DNA and how this can change the active genes. In particular, we will investigate the changes in the nuclei of cells when they become senescent - a process by which cells stop dividing which is linked to ageing and cancer. It has been observed that the DNA in some senescent cells can rearrange to form a distinctive pattern of bright spots, however the causes and function of such rearrangements are still unknown and difficult to investigate as it is only some of the cells which display the change. By isolating cells based on the arrangement of their DNA, we will be able to investigate what makes these cells different from their companions, which may provide critical insight into understanding the cellular changes of growing old.

Fluorescence microscopy has provided detailed insights into the structures and functions of living cells, tissues and organisms. Its ability to provide spatial information on the molecular scale has revolutionised our understanding of the cellular world. Advances in image analysis have led to sophisticated cellular phenotyping, scoring hundreds of morphological parameters. At the same time, breakthroughs in single-cell 'omics techniques, provide vast quantities of molecular data about an individual cell. Single-cell transcriptomics, for example looks at the mRNA molecules within a given cell, reporting the activity of activated genes across the entire genome. So far, insights in one field are generally isolated from the other, as there is no technology capable of linking the morphological features of a cell to its transcriptome. Here we propose to develop Cellular Phenotype sequencing (CePh-seq) which will synergise these two rapidly evolving fields in biology and medicine, enabling scientists and clinicians to isolate distinct and rare cells from a heterogeneous population based on any optical phenotype by linking morphological fluorescence imaging with single-cell sequencing via unique DNA barcodes. We propose to test our method on a so far intractable question in the field of nuclear morphology: What is the transcriptional consequence of the heterogeneous emergence of heterochromatic foci in oncogene induced senescent cells?

This interdisciplinary project will have potential impact across a several fields. In the short-term, the PDRA employed on the project will benefit from interdisciplinary training and exposure to a variety of cutting-edge science and techniques. The collaboration which this project will establish between the labs of GW and TC will extend interdisciplinary and inter-institutional research across Edinburgh. The frequent project meeting and exchange of ideas will benefit the wider research communities in both institutions. Beyond the project team itself, the technology development inherent in the project will allow other researching to build upon the techniques developed, the integrated morphology-based sorting and single-cell barcoding will enable new experiments to answer biological questions relating to the cause and effects of spatial patterning at the single cell level. By enabling morphology-based sorting to be performed, and building a strong user-base, interest in the capabilities may spur biotechnology companies to invest in similar technologies which are currently missing from the marketplace. The biological question addressed in this study is the role of the nuclear morphology and heterochromatin in cellular senescence. As senescence is a stress response common to cancer and ageing, our results will impact the cancer and ageing scientific communities. Since senescence is the first response to a genotoxic insult, senescence biology has the potential to particularly impact on early events in oncology, such as cancer detection and prevention. If our results do highlight an important role of heterochromatin in senescence and ageing, this could trigger pharmaceutical companies to fund research into the control and manipulation of heterochromatin through small molecules. We may also identify more specific gene targets and pathways that get differentially regulated as a consequence of heterochromatin changes, which could be of interest to pharmaceutical companies too. In summary, our proposed research could have a significant long-term benefit for the society. Even influencing the course of ageing at a small scale could provide an enormous benefit to society, because of the amount of people affected from age-related disabilities. Short-term benefits would most likely be through pushing the boundaries of genomic technology and the creation of disease related information, which can be implemented and further explored by the wider academic community and biotechnology companies.