No abstract

Pseudomonas aeruginosa (PA) is known to chronically infect airways of people with cystic fibrosis (CF) by early adulthood. PA infections can lead to increased airway inflammation and lung tissue damage, ultimately contributing to decreased lung function and quality of life. Existing models of PA infection in vitro commonly utilize 1–6-hour time courses. However, these relatively early time points may not encompass downstream airway cell signaling in response to the chronic PA infections observed in people with cystic fibrosis. To fill this gap in knowledge, the aim of this study was to establish an in vitro model that allows for PA infection of CF bronchial epithelial cells, cultured at the air liquid interface, for 24 hours. Our model shows with an inoculum of 2 x 102 CFUs of PA for 24 hours pro-inflammatory markers such as interleukin 6 and interleukin 8 are upregulated with little decrease in CF bronchial epithelial cell survival or monolayer confluency. Additionally, immunoblotting for phosphorylated phospholipase C gamma, a well-known downstream protein of fibroblast growth factor receptor signaling, showed significantly elevated levels after 24 hours with PA infection that were not seen at earlier timepoints. Finally, inhibition of phospholipase C shows significant downregulation of interleukin 8. Our data suggest that this newly developed in vitro “prolonged PA infection model” recapitulates the elevated inflammatory markers observed in CF, without compromising cell survival. This extended period of PA growth on CF bronchial epithelial cells will have impact on further studies of cell signaling and microbiological studies that were not possible in previous models using shorter PA exposures.

The aggressive nature of certain cancers and their adverse effects on patient outcomes have been linked to cancer innervation, where neurons infiltrate and differentiate within the cancer stroma. Recently we demonstrated how cancer plasticity and TGFβ signaling could promote breast cancer innervation that is associated with increased cancer aggressivity. Despite the promising potential of cancer innervation as a target for anti-cancer therapies, there is currently a significant lack of effective methods to study cancer-induced neuronal differentiation, hindering the development of high-throughput approaches for identifying new targets or pharmacological inhibitors against cancer innervation. To overcome this challenge, we used CRISPR-based endogenous labeling of the neuronal marker β3-tubulin in neuronal precursors to investigate cancer-induced neuronal differentiation in nerve-cancer cocultures and provide a tool that allows for better standardization and reproducibility of studies about cancer-induced innervation. Our approach demonstrated that β3-tubulin gene editing did not affect neuronal behavior and enabled accurate reporting of cancer-induced neuronal differentiation dynamics in high-throughput settings, which makes this approach suitable for screening large cohorts of cells or testing various biological contexts. In a more context-based approach, by combining this method with a cell model of breast cancer epithelial-mesenchymal transition, we revealed the role of cancer cell plasticity in promoting neuronal differentiation, suggesting that cancer innervation represents an underexplored path for epithelial-mesenchymal transition-mediated cancer aggressivity.