The Effect Of Fibroblasts On The Growth And Differentiation Of Breast Epithelial Cells

Objective: By co-cultivating human fibroblasts with human breast cancer cells and human breast normal epithelial cells to simulate the tumor microenvironment of "seed" and "soil",observe the growth and differentiation of tumor and epithelial cells by fibroblasts,the main component of the stroma.Analyze the relationship between the physiological and biochemical changes of fibroblasts and the occurrence,development and prognosis of breast cancer,and explore the feasibility of the related physiological and biochemical changes of fibroblasts as biomarkers for tumor screening,diagnosis and prognosis to achieve the goals of moving forward the sentinel monitoring of tumor prevention.Methods:(1)Human fibroblasts(CCC-ESF-1),breast cancer cell lines(MDA-MB-231,MDA-MB-468)and human breast normal epithelial cells(MCF10A)were co-cultured with conditioned medium method : a.When the cells grow to about 80% confluence,discard the original medium,replace with serum-free medium and culture for 24 h,after centrifugation,collect the supernatant and aliquot into 5% FBS conditioned medium.b.According to this method,CCC-ESF-1-CM,MDA-MB-231-CM,MDA-MB-468-CM,MCF10A-C M were prepared,namely take fibroblasts,normal breast cells,and cancer cells as each other’s target cells and conditioned cells.c.Observe and compare the growth status of cells before and after co-cultivation in conditioned medium,and use real-time fluorescent quantitative PCR to detect the changes in fibroblasts,normal breast cells and cancer cells of the EGFR m RNA level;Western Blot detects the expression changes of EGFR,Vimentin and E-cadherin in protein level,and immunocytochemistry detects the changes in EGFR expression in normal breast cells and cancer cells.(2)Human breast cancer tissue microarray of the EGFR immunohistochemistry,comparing the expression of EGFR in normal breast cells and cancer cells in tissues with different fibroblast interstitial content,and analyzing the effect of fibroblasts on the growth and differentiation of breast epithelial cells.Results:1.In CCC-ESF-1 cells co-cultured with MDA-MB-231,MDA-MB-468 and MCF10A conditioned medium,the changes in the levels of EGFR m RNA and EGFR protein were up-regulated compared with the expression of EGFR alone,increasing by 30.75% respectively(P<0.05),7.20 times(P<0.05)and35.24 times(P<0.05).The expression of vimentin was all down-regulated,and decreased by 8.88%(P<0.05),17.94%(P<0.05)and 11.19%(P<0.05);the up-regulation of e-cadherin expression increased by 2.61 times(P<0.05),14.72times(P<0.05)and 1.56 times(P<0.05),respectively.2.After co-cultured with CCC-ESF1 conditioned medium,MDA-MB-231,MCF10 A cell EGFR m RNA and protein expression levels were down-regulated,compared with the independent culture,they were reduced by 98.83%(P<0.05)and 59.98%(P<0.05).In the MDA-MB-468 cells,EGFR m RNA and protein expression levels were up-regulated,protein expression increased by 4.79 times(P<0.05).In MDA-MB-231,MCF10 A cells,vimentin expression was up-regulated,increasing by 32.54%(P<0.05))and 30.51%(P<0.05).Vimentin expression in MDA-MB-468 cells was down-regulated by 62.43%(P<0.05).MDA-MB-231 cells hardly express e-cadherin,and the difference is not significant compared with single culture(decrease by 13.42%,P>0.05).In MDA-MB-468 and MCF10 A cells,E-cadherin expression is up-regulated,increased 2.40 times(P<0.05)and 15.33%(P<0.05),respectively.3.Human breast cancer tissue microarray analysis showed that the higher the interstitial content of distant cancer(normal)tissues,the stronger the expression of EGFR in breast epithelial cells.In cancer tissues,no matter how the higher or the lower of the interstitial content,the EGFR of breast tumor cells is not express in the two types.Conclusion:1.Breast epithelial cells can up-regulate the expression levels of EGFR and e-cadherin in fibroblasts,and reduce the expression levels of vimentin,indicating that normal fibroblasts may gradually undergo mesenchymal-epithelial transformation in the tumor microenvironment.2.Fibroblasts can reduce the expression of EGFR in MDA-MB-231 and MCF10 A,and increase the expression of EGFR in MDA-MB-468,while the expression trend of vimentin is the opposite.Fibroblasts may have different regulatory mechanisms for different breast cancer cells and normal epithelial cells.The expression of e-cadherin in MDA-MB-468 and MCF10 A cells was up-regulated,indicating that fibroblasts may promote adhesion between breast cells and reduce the degree of differentiation.3.Human breast cancer tissue microarrays show that fibroblasts in the mesenchyme can down-regulate the expression of EGFR in breast cancer cells.Consistent with the results of cell experiments,fibroblasts can regulate the growth and differentiation of breast epithelial cells.4.As an important intercellular substance,fibroblasts have a "soil" and "seed" relationship with breast epithelial cells,and play an important role in regulating the growth and differentiation of breast epithelial cells,and are closely related to the development and prognosis of breast cancer.Fibroblast-related physiological and biochemical changes are generally earlier than epithelial cells,and can be used as biomarkers for tumor screening,diagnosis and prognosis.It is feasible to move forward the "gateway" of tumor prevention sentinel monitoring.