Abstract
Heterogeneous prostatic carcinoma-associated fibroblasts (CAF) contribute to tumor progression. This was established using transgenic mouse models. Paracrine interactions between fibroblasts and epithelial cells were further interrogated using isolated 2D cell culture systems, but 3D culture systems currently being developed can better mimic reciprocal interactions potentially found in the native tissue. To understand paracrine and juxtacrine signaling among fibroblasts and epithelia, 3D co-cultures with species differences allows for further subsequent analysis of the cultures. The use of mouse and human cells, for example, in one system allows for species-specific FACS or quantitative PCR analysis. This protocol describes the use of a 3D Co-culture System of Mouse Prostatic Wild-type Fibroblasts with Human Prostate Cancer Epithelial Cells.
Keywords: 3D Co-culture, Prostate cancer, Fibroblasts, Paracrine signaling, Epithelia, Species
Background
Prostate cancer (PCa) is a heterogeneous disease that results in the second highest cancer mortality in men. From the early steps of PCa initiation, the associated stromal fibroblastic cells begin to co-evolve with cancer progression and are predictive of recurrent disease and survival (Ayala et al., 2003). Studying paracrine signaling from the epithelial cancer cells to the associated fibroblasts and vice versa is imperative to understand the complex interactions during all stages of cancer progression (Bhowmick et al., 2004; Placencio et al., 2008). 2D culture systems where conditioned media from one cell type is incubated with the target cell type as well as those utilizing Boyden chamber cultures enable the exchange of paracrine factors. However, the characteristics of the cells in collagen matrix have documented differences in their responses to hormones and growth factors (Ziaee and Chung, 2014; Naba et al., 2014). Utilizing a 3D co-culture system allows a more representative readout of paracrine interactions between the stroma and epithelia. This system can be adapted for multiple combinations of stromal fibroblasts and cancer epithelial cells. Combinations of mouse and human cells can be used to better differentiate the cell type from which the signals were derived/received. Furthermore, incubation in a hypoxic chamber can more closely model physiologic environments. We have used localized prostate cancer as an example for our studies.
Materials and Reagents
Equipment
Software
Procedure
Day 1 Note: Step one may be performed the day before, keep sterile.
Data analysis
Notes
Recipes
Acknowledgments
This work was supported by grants from the National Cancer Institute (CA108646 to NAB and CA098912 to VRP) and Veterans Affairs (BX001040 to NAB).
Competing interests
The authors declare that they have no conflict of interest.
Ethics
In accordance with institutional animal care and use committee approval, primary mouse fibroblasts were harvested and grown using approved procedures.
References
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