Expression Of FSP-1 In Fibroblasts Co-cultured With Breast Cancer Cells And Its Effect On Breast Cancer Growth

ObjectiveIn order to investigate the molecular mechanisms of stromal fibroblast`s effect on breast cancer,FSP-1(fibroblast-specific protein-1)expression was researched in human fibroblasts co-cultured with human breast cancer cells and its impact on breast cancer growth.This study could provide new information for research on mechanism of the occurrence and development of breast cancer and experimental basis for molecular targeted therapy of breast cancer.Methods1.Cell culture and cell co-cultureCell subculture: Human fibroblast line ESF or human breast cancer cell line MDA-MB-231 was cultured,respectively,in DMEM-H media containing 10% fetal bovine serum,100U·m L-1penicillin,100?g·mL-1streptomycin at 37? in a humidified atmosphere with 5%CO2.Cells were subcultured when the cells confluence reached to 80% ~ 90%.Cell co-culture: After cell attachment in culture plates,the transwells inoculated with cells were plated into the culture plates to co-culture ESF cells with MDA-MB-231 cells.Two kinds of cells interacted with each other in the same medium.The cells implanted into culture plates were used in the experiments.2.RT-qPCRTrizol reagent kit was used to extract total RNA of the cultured cells.The extracted RNA was examinated by spectrophotometer.cDNA was synthesized by reverse transcription.Q-PCR amplification reaction was used with 39 cycles and melting curve of 64?~94?.The experimental results were automatically calculated by Bio-Rad CFX Manager,which is analytical software of fluorescent quantitative PCR.3.Western blotAfter cultured cells were washed and collected,cell lysis buffer was added at4? for 30 min,and 12000 rpm centrifuge at 4?for 5 min.According to theinstruction of BCA protein assay kit,protein standart was prepared,and standard curve was drawn.10% separated gel and 10% concentrated gel were prepared.20 ?g protein of each lane were put for protein polyacrylamide gel electrophoresis.The electrophoresed protein was transferred to the PVDF membrane.The PVDF membrane was incubated using the first antibody at 4? overnight,tthen the second antibody for 2h.Finally,the membrane was developed by ECL.The optical density of each electrophoresis band was analyzed by ImageJ software.4.Cell proliferation assayCCK-8 assay was used to detect the proliferation of MDA-MB-231 cells in vitro.Five wells in each group were taken in the assay.The proliferation of MDA-MB-231 cells was detected from day 1 to day 6.According to the instruction of CCK-8 kit,20?L CCK-8 solution were added to each well of culture plates,and the cells were incubated for 2h.100?L from each sample were moved to 96-well plates.Absorbance values were detected by enzyme micro-plate reader at 450 nm wavelength.5.Establishing human breast cancer xenograft model in nude miceSix weeks old female nude mice were randomly divided into 2 groups:ESF+MDA-MB-231 group and MDA-MB-231 group(n=16).ESF cells and MDA-MB-231 cells were co-cultured for 72 h in vitro and then washed by PBS.100?L cell mixture were(MDA-MB-231cells: 2×106 and ESF cells:1×106)implanted in the breast of right chest of mice.The tumor length(a)and width(b)were measured with caliper every 1 day and tumor formation were determined when the tumor diameter reached to 5mm.The tumor volume was calculated according to the formula.The nude mice were killed at day 30 by cervical dislocation.Tumor tissues were fixed,then stained by hematoxylin and eosin(HE)to observe the general status.The expression of FSP-1 in tumor tissues was tested by immunofluorescence.6.Immunofluorescence and laser confocal microscopyThe tumor tissues were fixed in 10% formaldehyde solution,dehydrated in ethanol,sectioned,dewaxed and then detected by immunofluorescence assay.Paraffin or benzene was wiped off by xylene and ethanol respectively.Antigen was repaired in0.01 M citrate buffer(pH6.0)by the microwave.Non-specific antigen of samples were closed in 1%BSA at 37 ? for 30 min.The samples were incubated with the firstantibody overnight,and the second antibody conjugated fluorescence for 60 min in dark.DAPI was used to stain nucleus for 10 min at room temperature.Laser confocal microscopy was used to observe the fluorescence staining.These pictures were taken and the fluorescence intensity was analyzed by Image-Pro Plus software.7.Immunohistochemistry experimentSections were dewaxed,and incubated in 3%H2O2 at 37 ? for 10 min.The antigen in sections were repaired,then incubated with the first antibody overnight,the second antibody at 37 ? for 10 min.These sections were coloured by DAB.Cell nuclei were stained by hematoxylin.These sections were dehydrated,been transparent,sealed,finally analyzed by Image J sofeware.8.Statistical methodThe data were processed by SPSS statistical software 17.The results of the experiments were expressed by mean±SD and comparison between 2 groups was used by T test.P<0.05 meant the difference was statistically significant.P>0.05 meant the differefce was not statistically significant.Results1.FSP-1 expression was up-regulated in ESF cells co-cultured with MDA-MB-231 cellsFSP-1 mRNA levels in ESF+MDA-MB-231 groups were significantly higher than ESF groups at day 2,day 3 and day 4(P<0.05).The level of FSP-1 mRNA in the co-cultured groups reached to the peak at day 3,which had statistically different(P<0.05)compared with level of FSP-1 mRNA in the co-cultured groups at day 2.FSP-1 mRNA levels had not statistically different between day 3 and day 4(P>0.05).FSP-1 protein expression was significantly higher in ESF+MDA-MB-231co-culture groups than ESF mono-culture groups at day 2,day 3 and day 4(P<0.05).FSP-1 protein expression of co-culture groups was higher at day 3 and day 4 than day2(P<0.05).FSP-1 protein expression of co-culture groups was not statistically difference between day 3 and day 4(P>0.05).The above results indicated that the microenvironment of the co-culture of breast cancer cells with fibroblasts could up-regulate FSP-1 expression in fibroblasts.FSP-1expression tended to be stable at day 4 of the co-culture.2.MDA-MB-231 cells co-cultured with ESF cells could express FSP-1Compared with MDA-MB-231 mono-culture groups,FSP-1 mRNA levels in ESF+MDA-MB-231 co-culture groups were statistically different at day2,3 and day4(P<0.05).FSP-1 m RNA levels in ESF+MDA-MB-231 co-culture groups were significantly higher at day 3 and day 4 than day 2(P<0.05),and not statistically different between day 3 and day 4(P>0.05)FSP-1 protein expression was not detected in MDA-MB-231 mono-culture groups.FSP-1 protein expression of ESF+MDA-MB-231 co-culture groups was higher at day3 and day 4 than day 2(P<0.05).FSP-1 protein expression of the co-culture groups had not statistically different between day 3 and day 4(P>0.05)The above results indicated that the microenvironment of the co-culture of breast cancer cells with fibroblasts could induce FSP-1 expression in breast cancer cells,which suggested breast cancer cells of epithelial tumor type occurred epithelian-mesenchymal transition(EMT).3.Human breast cancer cell line MDA-MB-231 proliferation increased in co-culture in vitroCompared with MDA-MB-231 groups,MDA-MB-231 cell proliferation rate of ESF+MDA-MB-231 groups significantly increased from day 2 to day 6(P<0.05).The proliferation presented speediness from day 2 to day 5,especially at day 3.MDA-MB-231 cell proliferation activity was higher in ESF+MDA-MB-231 groups at day 3 than other time period(P<0.05).4.FSP-1 expression was high in transplantation tumor of breast cancer cells co-grown with fibroblasts in nude mice and promoted tumor growthImmunofluorescence laser confocal microscopy results showed FSP-1expression was high in tumor tissues of tumor-burdened nude mice in ESF+MDA-MB-231 groups,and FSP-1 expression was low in MDA-MB-231 groups.The results indicated that FSP-1 expression of the tumor tissues,which were formed by implantation of human breast cancer cells with human fibroblasts into breasts of nude mice,was higher than the tumor tissues formed by only implantation of human breast cancer cells(P<0.05).The tumor volumes of ESF+MDA-MB-231 groups were significntly larger than that of MDA-MB-231 groups(P<0.05),which indicated that tumor stromal fibroblasts and high expression of FSP-1 could promote tumor growth.5.High expression of FSP – 1 in clinic human breast cancer tissuesImmunohistochemical results showed that FSP-1 expressed in human breast cancer tissues.But FSP-1 expression was low or not in human breast non-cancer tissues.Conclusions1.FSP-1 expression of fibroblasts could be up-regulated in the co-culture microenvironment in vitro,and promotes breast cancer cell proliferation.2.FSP-1 expression o f breast cancer cells could be induced in the co-culture microenvironment in vitro.3.High expression of FSP-1 is shown in tumor tissues of the co-growth of human breast cancer cells with human fibroblasts in human breast cancer-burdened nude mice,and promotes the growth of transplantation tumor.4.FSP-1 expresses in clinic human breast cancer tissues.But FSP-1 expression is low or not in human breast non-cancer tissues.