Primary Research On The Phenotype Of Breast Cancer Stem Cells Regulated By Tumor Associated Fibroblasts/Mir-100

BackgroundBreast carcinoma, one of the most common female malignant tumors, has a dramaticallyboost in its incidence and relapse rate, which is seriously threatening the female’s health.Many metastatic advanced-stage patients are suffering from severe complications and highmortality and even losing curative opportunities. Therefore, it is considerably vital to find outthe mechanism of the occurrence and progression of breast cancer for a new approach toanticarcinogen.Breast cancer stem cells（CSCs）are considered as the main reason for the occurrenceand metastasis, and CSCs may have a negative impact on the survival and prognosis of thepatients. In recent years, it is increasingly accepted that malignant tumor may be the result ofthe complex cross-talk between tumor cells and their microenvironment which is composed ofmesenchymal cells and extracellular matrix. The stromal cells are mainly constituted byfibroblasts. Tumor-associated fibroblasts may play a central role in promoting and regulatingthe carcinogenesis[1], proliferation[2], metastasis[3,4], angiogenesis[6]，epithelia-mesenchymaltransition（EMT）[5]，CSCs[7]and therapy resistance[9]of breast carcinoma cells via manydifferent kinds of cell factors and chemokines. That may be a novel important target fortherapeutic approaches for breast carcinoma. Yet little is known about the underlyingmechanism.Many studies have pointed out that miRNA may be associated with the differentiationproficiency. Tarantino C shows mir-100may regulate the differentiation ability of embryonicstem cell[10].To study and investigate the relation between miRNA and CSCs may provide usfor a novel exploration of potential targets for anti-cancer therapy.Objective1、Based on our previous research datas, this study aims to investigate whether tumorassociated fibroblasts can regulate the phenotype of breast cancer stem cells by observing theinteraction with primary cell line carcinoma associated fibroblas（tCAF）and breast cancer cell lines, and we also detect the expressions of their relevant markers of clinical samples, in orderto approach the relations between breast cancer stem cells and tumor associated fibroblastsand to seek a novel direction and evidence of the dignosis and treatment of breast cancer.2、we detect the expressions of mir-100and breast cancer stem cells of clinical samples, inorder to approach the relations between mir-100and breast cancer stem cells, and to seek anovel direction and evidence of the treatment of breast cancer.Materials and Methods1、Cell and paraffin section source and cell cultureHuman Breast Cancer cell lines MCF-7and BT474are from the lab of Prof. Song EW.And the primary cell lines CAF, tumor counterparts fibroblast（TCF）, and normal fibroblast（NF） are separated and cultured from operative specimens of breast cancer tissues, thecounterparts and normal breast tissues respectively. The paraffin sections of182breastcancers and36normal or benign breast diseases are also from Sun Yat-sen memorial hospital.2、Methods①Identification of the three primary fibroblasts by immunocytochemistry/immunofluorescence and western blot②Co-culture of the three primary fibroblasts with breast cancer cell linesrespectively③Enrichment of breast cancer mammosphere cells by serum free medium④Detection of the proportion of CD44^high/CD24^lowbreast cancer stem cells by flowcytometry⑤Evaluation of the mammosphere formation of each group of breast cancer cells bylimiting dilution assay⑥Protein quantitation of the expression of cancer stem cells’ markers ALDH1ofeach group of breast cancer cells⑦Detection of the proportion of ALDH1+breast cancer stem cells by flow cytometry⑧Detection of the expression of α-SMA and ALDH1by immunohistochemical ofclinical samples⑨Detection of the expression of mir-100by chromogenic in situ hybridization ofclinical samples3、Statistical methods①A llquantitative variables are presented as mean±standard deviation. The significanceof multiple groups is compared by one-way analysis of variance, and the comparison betweentwo groups is performed with LSD-test. ②All quanlitative variables are presented as frequency/percentage. The significance oftwo or multiple groups is compared by chi-square test or Fisher’s exact probability.③Spearman order correlation is applied to analyze the association between pairs of theexpressions of two markers.④The five-year survival is computed by life table, and survival curves are plotted by theKaplan-Meier method and compared by the log-rank test; multiple prognostic analysis iscompared by multifactorial Cox regression.⑤The statistical analyses are applied with SPSS13.0statistical software. P＜0.05in allcases is considered to be statistically significant.Results1、Identification of CAF: The primary CAF expresses α-SMA and vimentin, eliminatingCK positive cancer cells.2、CAF promotes the percentage of CD44high/CD24lowbreast cancer stem cells.3、CAF promotes the mammosphere formation of breast cancer cells by enlarging themammospheres and increasing the numbers of mammospheres.4、The protein level of ALDH1increases slightly in the mammospheres co-cultureedwith CAF.5、CAF promotes the percentage of ALDH1+breast cancer stem cells.6、The high expression of ALDH1is positively related to the high expression of α-SMAin clinical samples.7、The high expression of mir-100is negatively related to the high expression of ALDH1in clinical samples.Conclusions1、Tumor associated fibroblasts may be involved in the regulation of the formation ormaintenance of the phenotype of breast cancer stem cells.2、The high expression of ALDH1is positively related to the high expression of α-SMAin clinical samples, and tumor associated fibroblasts may support the growth of the grosstumor volume. The expressions of α-SMA and ALDH1are negatively associated with theprognosis of clinical breast cancer patients.3、The high expression of mir-100is negatively related to the high expression of ALDH1in clinical samples, and the down regulation of mir-100may be related with breast cancerstem cells. The expressions of mir-100are positively associated with the prognosis of clinicalbreast cancer patients.