Modelling the development of Barrett’s Oesophagus: Towards better treatment of Oesophageal Adenocarcinoma

Abstract

© 2020 Elhadi IichBarrett’s oesophagus (BO) is a metaplastic condition in which the normal squamous epithelium of the oesophagus is replaced by a columnar gastro-intestinal like epithelium due to repeated gastro-intestinal reflux. BO is generally accepted to be a precursor condition with the potential to develop into oesophageal adenocarcinoma. Consensus for the cell of origin for Barrett’s oesophagus is still lacking. Different sources for the cell of origin have been proposed, one of which is the submucosal gland (SMG) and duct cells of the oesophagus. This hypothesis, however, has not been properly studied due to the lack of proper model systems. In this thesis, pig and human SMGs and ducts were characterised and compared to each other to assess the suitability of pig SMGs as a substitute for human SMGs using immunohistology and multiplex immunofluorescence staining. Pig epithelial SMG cells were also further characterised using single cell RNA sequencing. Furthermore, organoid culture systems were developed to functionally assess the potential of the progenitor cells found using histologic and transcriptomic characterisation. Pig and human SMGs show different distributions at the distal end of their respective oesophagi but are largely similar in cell type and progenitor cell marker expression as demonstrated by histologic characterisation. Both pig and human SMGs showed progenitor cell marker expression in their respective basal duct cells (CD49f and p75) and the myoepithelial cells (CD49f), suggesting that both the ductal and glandular compartments to contain their own respective pool of progenitor cells. The transcriptomic analysis of pig SMGs single cell RNA sequencing largely supports their role in maintaining oesophageal homeostasis, as previously is known for human. Furthermore, the pseudotime trajectory inference data support the notion of the basal duct cells to be progenitor which give rise luminal duct cells. Interestingly, the data suggest the myoepithelial cells to be progenitor cells that could give rise to both basal duct cells and the gland compartment cells. Sorting and organoid culturing of basal duct cells demonstrated their capacity to grow into squamous spheroids, supporting their role in contributing to normal oesophageal repair. This process could be inhibited by treatment with retinoic acid. Similarly, culture of gland compartment cells gave rise to spheroids with two distinct morphologies. Dense type spheroids showed a squamous morphology but also mucin production found in BO. The second type of spheroid showed a cystic morphology and similarly produces BO type mucin. Finally, viral induction of intestinalisation in submucosal basal duct cells, in the current culture system, did not show metaplastic changes similar to BO. In summary, pig SMGs show great similarity to human SMGs. The pig SMGs contain progenitor cells in their ductal and gland compartments. These progenitors can be purified and cultured in vitro. The current developed protocols could be used to test the hypothesis that the SMGs contain the cell of origin of BO