Abstract
We have developed a 2D heterotypic co-culture technique between fibroblasts and cancer cells that enables the study of the stromal reaction. For such, stromal cells are seeded and cultured immediately around a tumour cell line, and the cells establish cell-cell contacts, as well as a gradient of soluble factors throughout the stromal cells, similar to that found in tissues. Thus, this system also enables the researcher to distinguish between events that are caused by direct cell-cell contact and secreted factors.
Keywords: Cancer, Stromal reaction, Fibroblasts, Extracellular matrix and proteoglycans
Background
The growth and survival of a tumour within a tissue depends upon interactions with surrounding stromal cells, such as fibroblasts, inflammatory cells, endothelial cells and lymphatic cells. Research has shown that as tumours grow there is extensive cross-talk between the cancer cells and the surrounding fibroblasts. Moreover, the tumour cells may activate these fibroblasts into tumour-associated fibroblasts (TAFs). In some instances, these fibroblasts may restrict tumour growth (Coulson-Thomas et al., 2011 and 2013); however, in many cases these TAFs aid tumour cell growth and survival (Coulson-Thomas et al., 2010 and 2015). Therefore, in vitro cancer studies should also take into account the protective effects TAFs can have on cancer cells. Taking this into account, we developed a 2D heterotypic co-culture technique between fibroblasts and cancer cells that enables the study of TAFs and cancer cells in the same system.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
This method has previously been used to analyse the prostate and colorectal stromal reaction (Coulson-Thomas et al., 2010 and 2011). In these studies the extracellular matrix (ECM) production was analysed in the fibroblast only region, cancer cell only region and interface which contained both the fibroblasts and cancer cells. The interface containing both fibroblasts and cancer cells can be viewed in Figure 2. The ECM was analysed by immunocytochemistry using a range of antibodies against different ECM components. We repeated the experiments at least three times in triplicates which yielded statistically significant results, with the Student’s t-test using the Prism-GraphPad software. Figure 2. Image of the O-ring area containing both the prostate cancer cells and fibroblasts. Fibroblasts are evidenced by vimentin staining in green and the prostate cancer cells are evidenced by tubulin staining in red. Scale bar = 20 μm.
Notes
Recipes
Acknowledgments
This protocol was from Coulson-Thomas et al. (2010). This work was funded by FAPESP, CAPES and CNPq.
References
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