Location, location, location: an imaging approach to identify homeostatic cell elimination mechanisms in the adult pancreas.

Epithelial tissues line our inner organs and act as protective barriers to the external environment. Cells are the building blocks of our tissues and in an epithelium, cells tightly connect to each other to form the impenetrable barrier. Epithelial cells also use cell-cell connections to communicate and monitor each other. This ensures that dying cells are removed and replaced with new cells at the appropriate time, and in a controlled manner. Any deviation from this process would result in a leaky barrier and would increase the risk of disease. As we age, our cells often acquire genetic mutations, some of which are harmful because they reduce the ability of a cell to work properly and respond to cues from the tissue. How do tissues respond to the presence of genetically mutant cells? To address this question, we use innovative experimental systems that allow us to randomly switch on a genetic mutation in a minority of epithelial cells in an otherwise healthy tissue. We label genetically mutant cells with a fluorescent tag so we can follow where mutant cells are in the tissue over time using microscopy. We focus on adult pancreas tissues because the genetic mutation expressed in cells is directly linked to pancreatic cancer and we currently know very little about how pancreas tissues stay healthy in adulthood. We discovered that adult pancreas tissues expel the genetically mutant cells and proper cell-cell connections with normal cells are essential for this to occur. Our research also revealed that some genetically mutant cells are never eliminated and instead remain in the tissue, where they slowly develop into the early stages of cancer. We recently recorded what genes are expressed in genetically mutant cells that are never eliminated and found that these cells switch on genes that control cell survival, tissue injury responses and stem cells. These data suggest that 'never eliminated' cells take on the characteristics of an injured or stem-like cell to avoid being eliminated. However, research shows that every epithelial cell in the pancreas has the potential to become a stem cell to repair a damaged or injured tissue. This raises the question as to what controls why some of the mutant cells are retained while others are eliminated. In this project, we will test the possibility that cell elimination outcomes (that is whether a genetically mutant cell is eliminated or not) are predictable and depend on where a mutant cell sits in the tissue. We will use new technologies to image genetically mutant cells in pancreas tissues before and after cell elimination has occurred and simultaneously measure changes in genes expressed in both mutant and normal cells. This will allow us to identify the communication signals between normal and mutant cells before and after cell elimination and determine whether the same signals operate across different parts of the tissue. By taking this approach, we will also ask whether a mutant cell is using signals from the local tissue architecture to avoid being eliminated. We will also experimentally test whether switching to a become more like a stem cell allows mutant cells to avoid cell elimination signals and remain in tissues. An improved understanding of the mechanisms controlling tissue health will lay the foundation for future studies to assess how these mechanisms decline with age and in disease.

As we age, our tissues frequently acquire genetic mutations, many of which are disease-causing. Our research shows that epithelial tissues protect against disease by actively eliminating genetically mutant cells. Cell elimination requires normal-mutant cell-cell interactions and dynamic remodelling of normal-mutant cell shape, cell volume and cell-cell adhesions. How genetically mutant cells are detected and subsequently eliminated by healthy neighbours, particularly in adult tissues, remains poorly understood.

We model sporadic mutagenesis in adult pancreas tissues of the mouse by expressing a genetic mutation linked to pancreatic cancer in low numbers of epithelial cells in an otherwise healthy tissue. Using a fluorescence reporter to label mutant cells and quantitative imaging approaches, we trace the fate of mutant cells in tissues over time. We discovered that while most genetically mutant cells are removed from the pancreas in vivo; remarkably, some mutant cells are never eliminated. This raises the question as to whether cell elimination is stochastic or is determined by predictive cell intrinsic or tissue-dependent factors.

In this project, we will combine quantitative imaging in 3D tissues with spatial transcriptomics and computational biology to identify the molecular mechanisms underpinning cell elimination in the adult pancreas. We provide supporting data to show that 'never eliminated' mutant cells activate cell dormancy and stemness gene signatures and require Wnt signalling to avoid cell elimination. Here, we will uncouple cell intrinsic factors (e.g., stemness, Wnt) and extrinsic cues from tissue neighbourhoods with spatial and temporal resolution. We will also functionally test whether stemness per se determines cell elimination outcomes. This interdisciplinary project will improve our understanding of the mechanistic basis of tissue health and will directly impact future research in lifelong health and detection/prevention of cancer.