Abstract
Oestrogen receptor-positive (ER+) breast cancer is the most diagnosed cancer in females annually and accounts for ~80% of all breast cancer cases. Metastatic breast cancer is responsible for 90% of all breast cancer related deaths and remains incurable. There is currently a need for a model system to be developed in which treatments of metastatic ER+ breast cancer can be tested however, all animal models are currently developed using immunocompromised animals, leading to non-representative results for therapies.
This thesis aimed to investigate Trp53, Arid1a and Cdh1 as genetic drivers of metastasis in the SSM3 cell line, an ER+ breast cancer murine cell line, and to utilise this knowledge to develop an ER+ metastatic breast cancer model in immunocompetent mice. Confirmation of CRISPR-mediated edits to SSM3 cells through western blots and qPCR revealed significant changes to expression of all genes in related cell lines. Attempts to generate three-dimensional spheroids in CRISPR-edited cell lines failed, indicating the loss of key epithelial cell markers compared to wildtype SSM3 which formed well compacted three-dimensional structures. Following spheroid attempts, epithelial-mesenchymal transition (EMT) markers expression was investigated using qPCR of SSM3 cells grown in a three-dimensional environment. qPCR analysis of Cdh1, Snai1 and Vimentin revealed significant changes in expression in all edited cell lines, indicating the induction of EMT under correct growth conditions of SSM3 cell carrying knockouts of Arid1a, Trp53 or Cdh1. Further analysis of metastatic processes, migration and invasion further confirmed the shift of CRISPR-edited cells to a more invasive phenotype showing a significant increase in migrated and invaded cells in three-dimensional assays. Successful in vitro characterisation of CRISPR-edited cell lines prompted in vivo investigations. To facilitate in vivo metastasis tracking of tumour burden, all cell lines underwent stable transfection with pSBbi-Luc2-RP plasmid containing the firefly luciferase gene. Following successful integration of firefly luciferase, the Trp53- KO cell line and SSM3 wildtype cell lines were implanted via tail vein injections to six 129S6/SvEv immunocompetent mice. Bi-weekly bioluminescent imaging revealed the development of significant metastasis in 1/3 mice injected with the Trp53-KO cells at 10- weeks post injection and no metastatic development in SSM3 wildtype injected mice.
The results obtained suggest that Arid1a, Trp53 and Cdh1 play significant roles in metastasis suppression in ER+ breast cancer. In vivo analysis of Trp53-KO cell lines metastatic potential suggests enhanced metastatic potential compared to wildtype SSM3 cells. These results provide promise for the development of an immunocompetent ER+ metastatic breast cancer model. Future research may investigate Arid1a- and Cdh1-KO cell lines for an experimental metastasis model to identify the optimal cell line to proceed for future immunocompetent model development. Following these studies, an increased biologically accurate implantation method such as the mouse intraductal injection method could be explored. This would provide a significant development in the research field of ER+ metastatic breast cancer and allow for the investigation of new therapies such as immunotherapies for ER+ metastatic breast cancer.