The Use of 2D and 3D Models to Study Competition in the Context of Barrett’s Oesophagus and Oesophageal Adenocarcinoma

**Introduction**: Oesophageal adenocarcinoma (OAC) is a cancer with poor five-year survival which is mainly due to its late diagnosis. OAC develops through an identifiable pre-cursor stage, Barrett’s Oesophagus (BO). BO can progress via the accumulation of further genetic and morphological changes into low-grade and then high-grade dysplasia. This development process is believed to take place over many years or decades and offers multiple opportunities for medical intervention to prevent progression to cancer if diagnosed. **Purpose**: The change in the tissue landscape from squamous epithelia to BO and then further progression through dysplasia to OAC, in addition to the high heterogeneity in BO and OAC tissues, suggests a long history of cell competition which is yet to be understood. *Sawas et al*. in 2018 found that OAC patients with detectable BO tissue at the time of their cancer diagnosis were found to have increased survival compared to those without detectable BO. Although the driver-gene landscape of OAC is very heterogeneous no clear genetic differences have been found between the tumours according to their BO phenotype. This observation adds further weight to the hypothesis, and I hypothesise that cell competition may play a role in this differing survival. The aim of this project was to explore cell competition between BO and OAC using model systems. A secondary aim was to compare the different model systems to inform future studies of cell competition. **Methods**: BO and OAC patient derived epithelial organoids were utilised to study competition in 3D. Co-cultures were generated from fluorescently tagged BO and OAC organoids and imaged for analysis. Fusion events between BO and OAC organoids were captured by wide-field microscopy and quantified, providing an additional method to study competition. In addition, 2D cell-lines with loss of tumour suppressors *p53 and SMAD4* which are well defined events associated with progression from BO to dysplasia and to OAC respectively, were utilised. BO lines were fluorescently labelled and co-cultured. Evenly mixed and spatially separated confluent patches of cultures were utilised to study competition. Spatially separated co-cultures were generate through adaption of protocols for wound healing, with experiments with a single and double boundary generated to quantify changes in cell:cell boundaries. Removal of epidermal growth factor (EGF) from the media allowed competition dynamics to be revealed between cells. RNA-seq was performed for each knock-out (KO) in media with or without EGF to compare the effect of the specific KO effects on BO cells and each conditions effect on gene transcription. **Results**: Competition mixing experiments using BO cell lines revealed an EGF dependant competition effect. Upon EGF removal WT BO cells underwent growth arrest, however co-culture of WT BO cells with either P53 KO or P53 SMAD4 KO cells led to WT growth and reduction in KO cell proliferation. Since WT growth rescue was possible in response to conditioned media from KO cells, this suggests an effect from diffusible factors. RNA analysis from conditions with and without EGF suggest increased cell cycle arrest in WT cells. Increased expression of EGF receptor (EGFR) ligands from KO cells, with EGF removal, suggest a potential pathway for increased growth in KO cells and quiescent rescue of WT cells. Expression levels of EGFR ligands matched observed proliferation differences between all three lines in EGF removal conditions. I also observed that seeding of spatially separated confluent patches of WT BO cells on either side of KO lines under EGF removal conditions, caused a constriction of the KO growth area compared to controls. This suggests that in addition to biochemical factors, there is also some physical competition between WT and KO cells. The mixed, single and double boundary confluent patch experiments demonstrated different methods for studying biochemical and physical competition dynamics. Co-culture of epithelial organoid cultures has major limitations due to the differences in proliferation, shape, and density of BO and OAC cultures. I found that this can be overcome to some extent by investigating individual fusion events between BO and OAC organoids. I categorised these according to which organoid type resulted after the fusion event, namely: a “BO win”, “OAC win” or “neutral”. OAC wins were significantly higher than BO wins at three time points, at approximately 30% to 10% respectively. Repeat passaging of the same BO:OAC cultures showed an increase in OAC wins with increasing passage number with a decrease to 0% BO wins by the final passage. In keeping with this, time tracking of fusion events showed a dynamic takeover of BO organoids by OAC cells/organoids due to encircling and compression of BO organoids by OAC cells. **Conclusion**: The utilisation of 2D and 3D model systems has allowed multiple methods to be developed to study competition in the context of BO and OAC. The use of 2D spatial models, whether through mixing or via distinct boundaries, allows the potential for both physical and biochemical competition to be queried. The 2D boundary models in addition more effectively model how clones may interact in the oesophagus than in the evenly mixed co-culture model. All three 2D models showed WT BO cells demonstrating a plasticity to adapt to their environment in KO co-culture. In 3D cultures, fusion events allow a novel way to investigate competition in real time. The establishment of these different methods will allow researchers a tool to investigate competition in their specific context, such as specific KO effects and clonal competition.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Cassie, Charlotte

Advisor dc:contributor.advisor

• Fitzgerald, Rebecca

• Barrett's Oesophagus
• cell competition
• EGF
• Oesophageal Adenocarcinoma
• organoids

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/22e69200-c508-4a3a-9246-220d3d445f9e/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.113190

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/375490