1. Mesenchymal Stem Cells and Breast Cancer
Maria Duignan, R.M. Dwyer, S. Khan and M.J. Kerin
Discipline of Surgery, School of Medicine, National University of Ireland Galway `
Breast cancer is the most common type of malignancy among women with over 2400 new cases diagnosed in Ireland each year. Despite improvements in detection methods and treatment options it remains the leading cause of female cancer related death world wide.
Mesenchymal stem cells (MSCs) have been shown to specifically home to sites of tumours. MSCs thus have huge potential for tumour-targeted gene delivery but equally they have also been found to be an element of the primary tumour microenvironment. It is crucial to understand the precise interactions between breast cancer cells and MSCs so that it can be established whether these interactions promote or inhibit tumour development.
Chemokines and growth factors play a central role in breast cancer progression. Two particular cytokines of interest for the purpose of this study were CCL5, also known as Regulated upon Activation, Normal T cell Expressed and presumably Secreted (RANTES), and Transforming Growth Factor Beta-1 (TGF-β1).
CCL5 is an 8kDa Chemotactic cytokine which has a principal role in the inflammatory response. It is known to induce angiogenesis and its effects are mediated through binding to the receptor CCR5.
TGF-β1 is a Pleiotrophic cytokine with a role in many regulatory processes which influence tissue Repair, immune defence, inflammation and angiogenesis. It initially binds to TGF-βII receptor.
There have been a number of studies reporting changes in levels of these two chemokines in mixtures of MSCs and breast cancer cells. Further understanding of their relevance and mechanism of action in breast cancer progression is required to support development of novel therapeutic strategies.
The main aim of this study was to investigate the interactions between these cells using 3D culture and analyse factors secreted by the cells and changes in cell morphology.
Cell Lines:
Three different breast cancer cell lines representing three different luminal subtypes were employed in this study, MDA-MB-231, SK-BR-3 and T47D. MDA-MB-231 is a hormone receptor negative invasive cell line, while SK-BR-3 is a less invasive hormone receptor negative cell line and T47D cells are HER 2 receptor positive and non-invasive. MSCs were consistently cultured in Dulbecco’s Modified Eagle’s Medium Alpha (DMEM). All cells were maintained at 37°C and 5% CO2 in a humidified atmosphere. The media was changed three times a week and the cells were routinely passaged at 75-90% confluence.
Introduction
3-dimensional culture:
This research employed Biotek 3D Insert™ PCL scaffolds for the culture of MSCs and breast cancer cell lines. Three dimensional in vitro models create a physiologically more relevant setting for the breast cancer cells, more accurately mimicking the structural architecture of the cells than a cellular monolayer set-up would.
Culture alone and co-culture of breast cancer cells and MSCs was performed. MSCs and breast cancer cell lines were co-cultured in a ratio of 1:3.
Results II
Table 1: Levels of TGF-β1 secreted in the conditioned media were quantified over a 21 day time period using ELISA. . All cell populations secreted relatively high levels of TGF-β1 when cultured individually. The levels of TGF-β1 secreted in co-culture varied for each cell line.
Collection of conditioned medium and ELISA:
Conditioned media, containing all factors secreted by the cells, was collected from the scaffolds at a number of time points over a 21 day time frame. The levels of CCL5 and TGF1 were then quantified using Enzyme-Linked Immunoassay (ELISA).
Objective
Fig. 2: Cpmditioned media was collected from the scaffold over a 21 day time period and then analysed using ELISA.
Results I
Figure 5: All cell populations secreted relatively high levels of TGF-β1 when cultured individually. There was a net decrease in the level of TGF-β1 secreted by cells in co-culture. Co-culture of MSCs with SK-BR-3s led to a net decrease in TGF-β1 secretion on all days.
Materials and Methods
Conclusion
In this study, it was reported that the quantity secreted of CCL5 (RANTES) and TGF-β1, by MSCs varied throughout differentiation over 21 days. CCL5 (RANTES) has recently been reported for having a central role in the interactions between MSCs and breast cancer cells, whileTGF-β1 plays a role in the epithelial to mesenchymal transition. In particular cell lines, CCL5 (RANTES secretion was observed to peak starkly in the initial period and then remain at a fairly constant high level at the culmination of the co-culture process.
The different levels of CCL5 secreted varied for each cell line. Such cell type specific effects highlight the importance of including a range of different cell lines in a study.
 This study clearly illustrates that MSCs have an impact on the genotype and phenotype of cells in their vicinity, potentially with cell type-specific outcomes. This may be exerted through changes in secretion of factors known to have a central role in breast cancer progression. Increased understanding of the prevalence of MSCs in the primary breast tumour microenvironment will support elucidation of their true relevance in breast cancer.
Fig. 3: Quantification of CCL5 levels was carried out on cell conditioned medium collected on days 3, 7, 14 and 21 using ELISA. This revealed that the quantity of CCL5 (pg/ml) secreted by breast cancer cell lines was relatively low for all. The graph above displays secretion of CCL5 in MDA-MB-231 cells +/- MSCs. There was a significant increase in the level of CCL5 detected following co-culture of MSCs with MDA-MB-231 cells. The levels of CCL5 secreted peaked on day 3 and remained at a high level on consequent days.
Fig. 1: Plate plan of cell populations cultured individually and in co-culture with MSCs