AbstractBarrett’s metaplasia (BM) is a pathological condition in which the squamous epithelium of the distal oesophagus is replaced by intestinal-like columnar epithelium in response to acid-biliary reflux from the stomach. BM is important to study because it is the only known precursor to oesophageal adenocarcinoma (OA). The main risk factor for the development of BM is gastro-oesophageal reflux disease (GORD), which can cause chronic cell injury in the distal oesophagus due to periodic pulses of acid and bile salts refluxing from the stomach. At the molecular level, BM has linked to the induction of key (master switch) transcription factors, including HNF4α and CDX2, both of which are involved in the regulation of intestinal gene expression. Both HNF4α and CDX2 are also expressed in BM and it was previously shown that ectopic expression of HNF4α could induce a columnar-like phenotype in an ex vivo model of stratified squamous epithelium.
Whatever the cell of origin, BM cannot develop unless the columnar epithelium colonises the squamous area of the oesophagus. The mechanisms by which this happens are still unknown, however, an element of cell competition driven by environmental and possibly mutational factors may have an effect on the competitive ability of columnar cells with regard to cell migration and infiltration into the squamous domain. It is therefore possible that in BM, changes in the expression of key transcription factors may be involved in conferring a competitive advantage, turning clonal populations of cells from losers into winners. To examine whether HNF4α and CDX2 could trigger cell competition, I developed an in vitro co-culture model using the human oesophageal squamous cell line, Het-1A, as a cell model. To further investigate the effects of HNF4α and CDX2 expression in Het-1A cells, in vitro proliferation and migration assays were performed in monoculture. An additional model using Caco-2 (an intestinal columnar epithelial) and Het-1A (squamous epithelial) cell populations were also established to examine their fitness in homotypic and heterotypic environments. Moreover, given the function of the extracellular matrix (ECM) in promoting tumour progression, I determined the impact on the competitive behaviour of all cell lines cultured on different ECM substrata, including fibronectin, collagen I and Matrigel.
The data showed that stable expression of HNF4α, but not CDX2, conferred a competitive ability to Het-1A cells in regard to cell migration and infiltration into the wild-type domain. In addition, a role for fibronectin, collagen I and Matrigel was demonstrated in promoting cell migration of all cell lines examined (Het-1A, Het-1A-HNF4α, Het-1A-CDX2 and Caco-2 cells). However, there were differences in the degree of infiltration depending on the ECM component. Here, Matrigel enhanced the Het-1A-HNF4α c1 cell capacity to infiltrate into the wild-type domain, while collagen I did not increase the competitive behaviour of these cells. These findings suggest a function for the ECM in regulating the progression of BM in the distal oesophagus. Lastly, intestinal Caco-2 cells showed an increased cell fitness when compared to squamous Het-1A cells in monoculture and co-culture experiments. These data provide evidence that HNF4α may induce a columnar-like phenotype and confer a competitive advantage to invade the adjacent stratified squamous epithelium.
|Date of Award||22 Feb 2023|
|Supervisor||David Tosh (Supervisor) & Ute Jungwirth (Supervisor)|