CAFs are one of the most important components of the tumour microenvironment in pancreatic ductal adenocarcinoma (PDAC). CAFs have traditionally been associated to a tumour-promoting function by affecting tumour growth, immunosuppression, and metastatic spread. In contrast, genetic and pharmacological approaches have revealed the existence of both tumour-promoting and tumour-suppressing CAF activities. We have identified two stable fibroblast subpopulations that play unique roles in tumour formation. One of the fibroblast populations has a strong tumour suppressive impact, which is dependent on functioning innate and adaptive immunity. However, the precise process by which individual fibroblast populations coordinate restrictive immunological microenvironments remains unknown. In this thesis, I sought to get a deeper knowledge of pancreatic fibroblast transcriptional flexibility. I use in vitro model systems to study fibroblast inflammatory signalling states. Then, using CRISPR-Cas9-based screening and causal network analysis, I want to identify master regulators of inflammatory signalling in fibroblasts. After validating potential hits in publicly available scRNAseq datasets, fibroblast knockout clones for the individual master regulators are created. Finally, in vitro systems as well as mouse models are employed to evaluate the significance of these master regulators in fibroblast-specific inflammatory signalling and tumour growth. According to ingenuity pathway analysis (IPA), CCAAT-enhancer-binding proteins (CEBP) are significant upstream regulators of inflammatory gene expression in pancreatic fibroblasts. The CEBP transcription factors exhibit a bimodal expression pattern in fibroblasts from several scRNAseq datasets, and CEBPBPOS fibroblasts express more inflammatory mediators. CRISPR/Cas9-mediated knockouts and targeted qPCR analyses confirm that CEBP-transcription factors are mediating inflammatory gene expression in fibroblasts in vitro. Subcutaneous co-injection of pancreatic cancer cells with tumour restrictive wildtype fibroblasts effectively controls tumour development. Co-injections of tumour cells and CEBP-KO fibroblasts, on the other hand, result in altered immune cell infiltration and the formation of immune cold tumours, resulting in increased tumour growth. The findings in this thesis show that inflammatory signalling networks of tumour restricting fibroblasts are important for immune cell recruitment to the local microenvironment. Fibroblast-specific CEBP-signalling is a primary driver of this signalling and an important signalling network to drive tumour control.
|Date of Award
|1 Aug 2023
- The University of Manchester
|Tim Somervaille (Supervisor) & Claus Jorgensen (Supervisor)