A Facile 3D Cryogel Co-culture System Used To Study The Role Of CAFs In EMT Of MDA-MB-231 Cells

Background:Breast cancer is one of the most common epithelial tumors and seriously endangers women's health.Studies showed that carcinoma-associated fibroblasts(CAFs)possessed myofibroblast features and played an important role in the growth and metastasis of breast cancer.Due to the lack of applicable animal model,the exact role of CAFs in tumor metastasis is still unclear.Here we developed a facile 3D cryogel co-culture system to evaluate the role of CAFs in the breast cancer metastasis.We found that CAFs could promote EMT of MDA-MB-231 cells in 3D cryogel co-culture system in vitro.Then our co-culture human breast cancer system was used to construct the xenograft tumor mouse model in vivo.We found that CAFs could promote deterioration and metastasis of MDA-MB-231 cells in xenograft tumor mouse model in vivo.Objective1.Prepare cryogel by GelMA,then co-culture with MDA-MB-231,MCF-7 cells,characterization.Establish appropriate three-dimensional scaffold to the breast cancer cell growth to provide a better platform to further study EMT mechanism in tumor development under the condition of three-dimensional.2.Developed a facile 3D cryogel co-culture system through layer by layer displayed method to evaluate the role of CAFs in the breast cancer metastasis in vitro.3.Selectthe nude mice as experimental object,compare the state of tumor genesis and migration,preliminarily in nude mice of cells from among traditional two-dimensional culture conditions,three-dimensional model and the 3D cryogel co-culture system and explore the advantage of the facile 3D cryogel co-culture system,in order to provide a convenient approach to study the EMT mechanism and a useful platform for anti-cancer drug screening in vitro.Methods1.Use clinical breast cancer tumor samples to separate carcinoma-associated fibroblasts(CAFs).2.Cell culture.Buy human breast cancer cell line MDA-MB-231,MCF-7.One group culture in traditional 2D,the other groups co-culture with cryogel,building a 3D model of tumor tissue.3.Verify the effect of the facile 3D co-culture system on EMT in vitro.4.Use BALB/c-nu nude mice to verify further in vivo,.5.Statistical analysis.Complete statistical analysis using SPSS 13.0 statistical software,p<0.05 think the difference was statistically significant.Results1.Succeeded in good biocompatibility and elasticity cryogel preparation.2.Succeeded in isolating and culturing CAFs.3.Build the three dimensional cryogel model of breast cancer which used CAFs as extracellular matrix layer.4.The model can promote the EMT transformation of MDA-MB-231 cells.5.The model can promote deterioration and metastasis of MDA-MB-231 cells in xenograft tumor mouse model in vivo.ConclusionIn conclusion,the GelMA cryogels are desirable scaffolds for the study of cells interaction between the breast cancer cells and stromal cells under 3D co-culture conditions.According to our developed 3D cryogel co-culture system,we found that CAFs could promote EMT of MDA-MB-231 cells in vitro and promote deterioration and metastasis of MDA-MB-231 cells in vivo.This facile 3D cryogel co-culture system may be a convenient approach to study the EMT mechanism and a useful platform for anti-cancer drug screening in vitro.