The Expression Of FAP In Breast Cancer Stroma And The Relationship Between FAP And MVD

INTRODUCTIONWhether tumor epithelium or stroma can not singlely determine the tumor-genesis or growth of neoplasms. While the balance of tumor micro - environment can result in that. Tumor - associated fibroblast (TAF) , one of the most important of host cells, can secret a sort of protease and cytokines and play a role of controlling.The human fibroblast - activation protein, one of marker products of TAFs, a member of the serine protease family, was discovered as an inducible type — H cell - surface glycoprotein selectively expressed by reactive stromal fibroblast of epithelial cancer and healing wounds. It's structure anchor the membrane and make it accept other cell signal, then regulate the adhesion and migration of epithelial cells. The latest article suggest that overexpressioning FAP increase the expression of matrix metalloproteinase - 2 and CD44 and reduce intergrin - β₁ expression in 293T cells, while in LX -2 FAP overexpression cause increased cell adhesion and migration on ECM proteins.In our study , we detect FAP expression in breast fibroadenoma, breast ade-nosis , breast intraductal carcinoma, breast invasive ductal carcinomas and MCF -7 - CCC - HPF - 1,MDA - MB -231 - CCC - HPF - 1 co - culture model by methods of immunohistochemistry and Western Blotting analysis and we investigate the relationship between FAP and MVD or lymphatic metastasis.MATERIAL AND METHODS1. Tissue SampleA total of thirty - eight cases of fresh breast cancer tissue samples were ob-tained from surgical resection at the first Affiliated Hospital of China Medical U-niversity in 2005. According to the World Health Organization breast carcinoma histological classificational criteria (2003), histological type, including 18 breast intraductal carcinomas, 20 breast invasive ductal carcinomas.2. Cell lineCCC - HPF - 1 cell line was provided by Peking Union medical college;MCF -7, MDA - MB -231 was provided by cell bank in Shanghai.3. ReagentThe primary anti - human FAP mouse monoclonal antibody was provided by Multi - Science Company in Peking. The anti - human CD34 mouse monoclonal antibody was provided by Duko Company. The Ultro - sensitive S - P kit and DAB agent kit were bought from Fujian Maxin Biological Company.4. Methods1) Cell cultureMCF-7-CCC-HPF-1 or MDA-MB-231 -CCC-HPF-1 co-culture model was supplemented with medium of MCF -7 or MDA - MB -231 and DMEM containing 20% FBS (1:1).2) ImmunohistochemistryIHC was performed according to indirect streptavidin - hyperoxidase method , as manufacture protocol. Controls: It was positive control that the membrane of human tumor stroma fibroblast was with intense staining for FAP. Primary antibody was omitted as negative controls. Evaluation: the cells with browned cell membrane or cytoplastic staining were considered FAP positive and cell membrane staining were considered CD34 positive. Results are indicated as follows: + + , + : abundant (>? 50% ) , intermediate (10 -50% ) number of FAP+ stro-mal fibroblast respectively;- , antigen negative;- / + , focal presence FAP+ fibroblast ( < 10% ). Microvessels were determined by immunohistochemisty for CD34. After the area of highest vascularization had been chosen under low power, vessel count in tumors was carried in high power.3) Western BlottingWestern Blotting analysis was made as previously described with some modification. The transferred sample was incubated with the primary antibody. (di-lution: FAP: 1;400) overnight at 4X.. (3 - actin levels were shown as protein loading control. Changes of protein content in invasive ductal carcinoma compared with breast intraductal carcinoma were calculated by densitometry.4) Statistical analysisStatistical Product and Service Solutions (SPSS) was applied for data analysis. The result of immunohistochemistry was compared using chi -squared test and Spearman rank correlation. The results of Western Blotting were compared using independent sample t - test. P < 0. 05 was considered statistically significant.RESULTS1. Expression of FAP in breast cancer stroma.The result of the immunohistochemistry FAP was observed on the fihroblast cell membrane or in trie cytoplastic. No staining was founding in the breast fi-broadenomas, breast adenosis. The expression of FAP in breast invasive ductal carcinomas is more abundant than that of breast intraductal carcinoma. But after statistical analysis, there was no statistically difference of FAP expression between breast intraductal carcinomas and invasive ductal carcinomas. (P >0. 05)2. Western Blotting analysis of FAP protein content in breast fibroadeno-ma, breast intraductal carcinoma, breast invasive ductal carcinomas and MCF -7 - CCC - HPF - 1 x MDA - MB - 231 - CCC - HPF - 1 co - culture model.(1) The results showed that no detectable expression of FAP in breast fibro-adenoma was observed. The protein content of FAP of breast invasive ductal carcinomas is more abundant than that of breast intraductal carcinoma. But, no statistically difference of FAP protein content was found between breast intraductal carcinomas and invasive ductal carcinomas (P > 0. 05).(2) The results showed the protein content of FAP of MDA - MB -231 -CCC - HPF - 1 co - culture is more abundant than that of MCF - 7 - CCC -HPF - 1 co - culture. No statistically significant difference of FAP protein content was found between MCF - 7 - CCC - HPF - 1 and MDA - MB - 231 - CCC- HPF - 1 co - culture model ( P > 0.05).3. The relationship between the expression of FAP and CD34.There was a significantly positive correlation was found between FAP and CD34. ( P<0.05).4. The relationship between the expression of FAP and lymphatic metastasis.A significantly positive correlation was found between FAP and lymphatic metastasis. (P<0.05)DISCUSSIONThe human fibroblast - activation protein, one of marker products of TAFs, a member of the serine protease family, was discovered as an inducible type -H cell - surface glycoprotein selectively expressed by reactive stromal fibroblast of epithelial cancer and healing wounds. Its structure anchor the membrane and make it accept other cell signal, then regulate the adhesion and migration of epithelial cells.This article found that the expression of FAP was negative in the breast fi-broadenoma, breast adenosis. And it only was abundant in the breast cancer stroma. Western blotting analysis of FAP expression in tissues and co - culture model agreed with the result of IHC. In this article, there was significant relation between expression of FAP and CD34 or lymphatic metastasis. It suggests that the expression of FAP is related to migration and invasion of neoplasm.CONCLUSION1. There was overexpression of FAP in breast intraductal carcinoma, breast invasive ductal carcinomas. And no expression of FAP in the breast fibroadeno-ma and breast adenosis was found.2. The expression of FAP in breast invasive ductal carcinomas is more a-bundant than that of breast intraductal carcinoma. But, after statistical analysis, no detectable difference was found between breast invasive ductal carcinomas and breast intraductal carcinoma.3. The overexpre:ssion of FAP was significantly related to the increase of micro vessel density.4. The overexpression of FAP was significantly related to the increase of lymphatic metastasis.