Identifying differences in the tumour microenvironment between ER-positive and ER-negative invasive ductal breast cancer

The tumour microenvironment plays an essential role in tumour growth and progression. The tumour microenvironment is composed of different cell types, including macrophages, fibroblasts, various inflammatory cells, fat cells and endothelial cells, collectively known as stroma. Most recent studies believe that the tumour microenvironment in many solid tumours including breast cancer defines outcome in oestrogen receptor positive (ER+) and triple negative breast cancer (TNBC). This PhD project was established in order to test the hypothesis that cells within the tumour microenvironment influence breast cancer outcome and this differs in ER+ versus TNBC disease. As part of the project, 7 biomarkers were used to identify stromal cells including fibroblasts and various immune cells. Furthermore, our results from positive pixel count (PPC) revealed CD3+ (n=4.01), CD4+ (n=1.28) and CD8+ (n=4.71) numbers were positively correlated with higher histologic grade in TNBC cases, while in ER+ samples, the cell number of CD3+ (n=2.1), CD4+ (n=0.5) and CD8+ (n=2.3) was negatively correlated with high grade tumour. High levels of CD68 were found to be associated with high tumour grade in both ER+ (n=5.5) and TNBC (n=6.41) breast cancer samples. Pirfenidone PFD drug displayed a sign of acute toxic at 100µm, while it showed inhibitory effect on cell proliferation of MCF-7, MDA-MB-231 and CAFs derived from ER+ positive breast cancer. Our data from qRT-PCR and immunofluorescence revealed that MCF-7 expressed ER-α gene but it was absent in MDA-MB-231 breast cancer cells. Human monocytic Thp-1 cells have been considered as an alternative model for human peripheral-blood macrophages. 25ng/ml PMA concentration was chosen as an optimal concentration among others to differentiate monocytic Thp-1 into macrophages. The co-culture of macrophages with MCF-7 or MDA-MB-231 has shown lower proliferative rate in both breast cancer subtypes, whereas breast cancer cells were more proliferative when co-cultured with fibroblasts and macrophages using triple culture model. Moreover, breast cancer cells co-cultured with macrophages for different time points were termed as following; control or untreated were labelled as (C), short-term co-culture (3-5 days) were labelled as (S) ,long-term co-culture (30 days) were labelled as (L) and those cells that grew individually after 1 month incubation with macrophages were labelled as programmed (P). However, long-term co-culture of macrophages with breast cancer subtypes has chronically promoted cell migration of programmed MDA-MB-231 but not MCF-7 clones compared to those co-cultured under short-term and control samples. Differential gene expression analysis revealed that programmed MDA-MB-231 clones share the highest number of up-regulated (5591) and down-regulated (6093) genes among other samples including short-term and long-term in contrast with control, indicating that the incubation of macrophages with MDA-MB-231 for 30 days has chronically regulated gene expression in programmed MDA-MB231 which has reflected in their migration and proliferation behaviour