Abstract
Approximately 80% of breast cancer diagnoses present as oestrogen receptor-positive (ER+), with ~10% of these patients developing metastatic ER+ breast cancer (mBC). The estimated five-year survival rate of mBC is between 20 – 30% and is effectively incurable with current treatments. However, the almost impossibly large number of potential therapy regimens means that relevant preclinical models are required to assess which combinations are mostly likely to be effective in mBC. While the models currently available are still important for therapeutic discovery, they largely utilise immunocompromised animal backgrounds, or focus on more highly aggressive forms of breast cancer. The distinct lack of models that fully recapitulate the biological processes involved in ER+ mBC has, therefore, significantly slowed progress towards effective treatment options. The work described in this thesis aimed to develop the first biologically relevant preclinical model of ER+ mBC in mice with fully intact immune systems.
In this research, the 129S6/SvEvTac immunocompetent mouse strain was utilised, along with the syngeneic SSM3 cell line. This cell line was derived from spontaneous mammary carcinomas from Stat1-/- 129S6/SvEv mice. SSM3 cells proliferate in response to oestrogen, and therefore, can be utilised as an ER+ model of breast cancer. This PhD investigated the advantages and limitations of this model.
The first aim of this research was to establish trackable SSM3 cell lines that could be used to monitor and visualise cancer progression in the 129S6/SvEvTac background. Fluorescently labelled plasmid vectors encoding Antares2 or Firefly luciferase were introduced to SSM3 cells. In vitro, comparisons of the two cell lines were performed to determine imaging potential and sensitivity. Luciferase expressing SSM3 cells were then introduced to 129S6/SvEvTac mice for development of the spontaneous and experimental metastasis models by mammary fat pad or tail vein injections, respectively. Bioluminescent imaging captured luciferase activity at multiple time points over six months following primary tumour removal (spontaneous model) or cell line injection (experimental model). SSM3 metastasis was determined through ex vivo imaging and histological analysis.
It was found that mammary fat pad injections of SSM3 cells produced primary tumours with luciferase activity. Tumour establishment rates and morphology indicated that the tagged SSM3 cells may result in a stronger immune response than the parental SSM3 cell line. The experimental model of mBC showed that luciferase signal can be detected in the chest of mice following tail vein delivery. It was also found that metastatic tumours formed in the lungs and reproductive tracts of mice receiving high concentrations of cells and oestrogen supplementation.
To continue the development of an ER+ mBC model, the mammary intraductal (MIND) injection model was established using the SSM3 cell line and 129S6/SvEvTac mice. This is the most biologically relevant model of luminal-derived cancer as cancer cells are delivered directly to the site of origin. It was found that SSM3 cells established in the ducts of mice and progressed to the stage of invasive breast cancer. With continued optimisation, this model will be highly advantageous in ER+ mBC research and therapy discovery.
Overall, this research has helped define the boundaries of the syngeneic 129S6/SvEvTac model as a model for ER+ metastasis. It was discovered that under the right experimental conditions, the SSM3 cells can form metastatic tumours. It also showed that there are both advantages and limitations to using trackable cell lines, and that further investigation surrounding this will be important for future studies involving luciferase imaging of live animals. Most importantly, it has contributed to the progress of ER+ mBC model development, as well as, establishing a new model of ER+ breast cancer, through the MIND model. This PhD has created a foundation for continued development of much-needed animal models that are essential for bridging the gap in available therapeutic options for patients with ER+ metastatic breast cancer.