Culture In Vitro And Isolation By Magnetic-activated Cell Separation Of Human Mammary Cancer Stem Cell And Research Of Breast Cancer Associated Gene Expression

Backgrouds and Objectives:Breast cancer is a common female malignant tumor. It is common disease and frequently-occurring disease with a high incidence in Western Europe and North America, and is a serious threat to women's health. Into the 21st century, about 120 million women worldwide each year suffer from breast cancer,and 500,000 people die from breast cancer. The United States as countries with high incidence of breast cancer, showed a clear upward trend of breast cancer incidence from 1973 to 1976, the U.S. An estimated 10 million people suffering from breast cancer each year and 3 million people die of the disease. In 1990, of 15 million new cases, there are 44000 people were killed, according to the estimated incidence rate trends, in the 21st century in the United States, every eight women will have a person's life with breast cancer. Low incidence of breast cancer is showed in China, but in recent years, clinical observation suggest that breast cancer incidence rates in China continue to rise, growing at about 3 percent per year, in Beijing, Tianjin, Shanghai and other major cities, breast cancer has leapt the first female malignancy, age, significantly ahead of the trend, and the clinical diagnosis of breast cancer in our side late stage of disease. For this reason, breast cancer research reaches growing concern.Cancer stem cell theory concern that tumor cells exist in a group of cells with stem cell properties(called cancer stem cells or tumor cells, or cancer initiating cells, CSCs or CICs), subject to external regulation and control of malignant proliferation of multi-differentiation to form tumors. Growing number of studies confirmed the brain glioma, breast cancer, prostate cancer and malignant melanoma and other solid tumors exist in cancer stem cells.Hedgehog family members are including SHH, PTCH, GIi-1, and SMOH. Specific mechanism of activation of Hedgehog signaling pathway is currently not entirely clear, most scholars believe mechanisms including ligand-dependent activation and ligand-independent activation. Ligand-dependent activation is that ligand, such as SHH, IHH (IndianHedgehog) combined with the receptor PTCH, Smo-induced activation of proto-oncogene, causing the activation of Hedgehog signaling pathway, which has been confirmed in small cell lung cancer, liver cancer, breast cancer, brain cancer and digestive tract cancer. Ligand-independent signaling pathway activated mainly refers to members such as PTCH or SMOH mutations in the Hedgehog signaling pathway initiated by the activation, which has already in the Gor-lin-associated tumors (such as basal cell carcinoma, into the neural tube cell carcinoma and rhabdomyosarcoma, etc.) been confirmed.GIi-1 gene's highly expression is relatively recognized a marker of activation of the Hedgehog signaling pathway. A number of studies confirmed the Hedgehog signaling pathway in stem cells of multiple myeloma, breast cancer and malignant glioma and other cancers that highly expressed Hedgehog signaling pathway in stem cells, and the Hedgehog signaling pathway is confirmerd play an important role in their self-renewal. SHH signaling knock-out mice can lead to significantly reduced neural stem progenitor cells, so that in vitro almost can not form neurospheres. On the contrary, activating SHH ways can make the final adult mouse brain's stem progenitor cells increased significantly. IHH mutation can reduce the intestinal stem cell proliferation and differentiationCytokeratin (cytokeratin, CK) is distributed in the epithelial cells of the intermediate filament silk, mainly expressed in the epithelial cells, were found in human epithelial tissue of at least 20 members, its expression is highly tissue-specific and differentiation-specific. Each type of epithelial tissue in different stages of growth and differentiation express different CK, its expression by regulating cell differentiation, such as the main expression of typeⅠepithelial CK (including CK7, CK8, CK18, CK19, CK20), while the main expression in squamous cellⅡ-type CK (including CK1, CK5/6, CK14). Therefore, the positioning of different CK research and observation CK expression under different conditions, is certainly significant to understand epithelial cell proliferation and differentiation status, disease occurrence and development.Research suggests that, CK5 is a sign of grass-roots keratin in proliferative cells in normal breast, CK5 always positive, but in the majority of invasive ductal carcinoma and ductal carcinoma in situ, CK5 often negative, so CK5 is often use to identify invasive and duct in situ cancer and common type of hyperplasia. CK8 is simply a sign of epithelial keratin, it can penetrate the outer surface of breast cancer cells, and in a soluble form of hybrid polymer is released from the cancer cells, CK8 present in many simple epithelial cells and carcinoma, but also for the differential diagnosis of breast cancer. Breast cancer resistance protein (breast cancer resistance protein, BCRP) is located in the membrane of the ABC half-transporter proteins, there are researches suggesting that it may be a sign of poorly differentiated breast epithelial tissue, in the abnormal proliferation of tissue high expression of BCRP is a cell undifferentiated maturity, BCRP may be related to the presence of drug-resistant tumor cells and cause tumor recurrence and treatment failure.In this report,breast cancer cell line MCF-7 and human breast cancer tissues were studied by real-time quantitative PCR technology and immunohistochemistry, in order to detect Hedghehog pathway in MCF-7 cells CD44+/CD24-/low stem cell subsets in the Hedghehog path expression, as well as some important proteins, cytokeratin, chromogranin proteins in human breast cancer tissue expression. We analyze the correlation of their different breast lesions, so that provide a new way of thinking for breast cancer prevention and improving the diagnosis and treatment of breast cancer. Proved for these academic problems will provide valuable experimental evidence for early diagnosis of breast cancer, prognosis evaluation and screening of gene therapy targets.Materials and Methods:Breast cancer cell line MCE-7 and human breast lesions were selected from the Dalian Medical University and China-Japan Center for Clinical Pathology. Of total 89 human breast tissue samples from 2004 to 2008, there were 13 cases of normal breast tissues,16 cases of breast hyperplasia,12 cases of atypical ductal hyperplasia,6 cases of ductal carcinoma in situ and 42 cases of invasive ductal carcinoma. Of total 75 human breast tissue samples from 2008 to 2009, there were 10 cases of normal breast tissues,26 cases of breast fibroadenoma tissues,39 cases of invasive ductal carcinoma. Patients aged 28 to 75 years with an average age of 45 years. Samples were collected from specimens resected, had not received preoperative radiotherapy and chemotherapy, all patients were clearly pathological diagnosed and got informed consent of patients and their families, access to the Dalian Medical University Ethics Committee. All sections were confirmed by two pathologists referral of high qualification, of which 13 cases of normal breast tissue adjacent to breast tissue from the tumor 5cm. In this study, immunohistochemistry (IHC), immunocytochemistry (ICC), real-time quantitative polymerase chain reaction (Realtime-PCR) and cell culture methods and magnetic cell separation system(MACS) were used to detect the expression of Hedghehog pathway in MCF-7 cells CD44+/CD24-/low stem cell subsets, and to detect expression and distribution of Hedghehog pathway important protein, cytokeratin, chromogranin proteins in human breast cancer tissue. SPSS 11.5 package was use for statistics analysis with the experimental data, distribution of all factors in different pathological were analysed significantly by Mamm Whitney and Kruskal-Wallis test methods, and Spearmen correlation test was applied to judge the statistical relationship between relevant factors.Results:1 CD44+/CD24-/low subpopulation ratio (8.90±1.73)% in the breast cancer cell line MCF-7. Hedgehog signaling pathway of the CD44 +/CD24-/low subgroups in breast cancer cell line MCF-7 is significantly activated, including Hedgehog signaling pathway important downstream signaling molecules GIi-1 in MCF-7 cells CD44+/CD24-/low cells subgroup expression is significantly higher than non-CD44+/CD24-/low cell subsets (P= 0.007).2 Positive expression rate of PATH 1 and Gli-1 in normal breast tissue, breast tumor tissue, and breast cancer showed gradually increased, PATH1 expression rates were 10.0%,46.1% and 96.2%, Gli-1 expression rates were 20.0%,69.2% and 98.1%. With the differentiation of breast lesions decreased, PATH1 and Gli-1 positive expression rate was increased, showed strong positive expression. Their expresss in invasive ductal carcinoma tissues was significantly higher than normal breast tissue, was significantly higher than that of fibroadenoma tissue (P<0.05).3 Positive rate of CK5 and CK8 in the normal breast tissue, breast hyperplasia, atypical hyperplasia and breast cancer showed descending. Positive expression rates of CK5 in normal breast tissue, breast hyperplasia, atypical ductal hyperplasia, ductal carcinoma in situ and invasive ductal carcinoma were 84.6%,62.5%,41.7%,33.3%,19.0%. Positive expression rates of CK8 in the normal breast tissue, breast hyperplasia, atypical ductal hyperplasia, ductal carcinoma in situ and invasive ductal carcinoma positive expression rates were 100.0%,100.0%,75%,66.7%,66.7%. Most of the 28 cases of invasive ductal carcinoma (66.7%) were CK5-/CK8+, only 8 cases (16.7%) were CK5+/CK8-,8 cases presented CK5+/CK8+,14 cases (29.2%) showed CK5-/CK8-. With the decline of differentiation of breast lesions, BCRP positive expression was significantly increased in normal breast tissue, breast hyperplasia, ductal atypical hyperplasia, ductal carcinoma in situ and invasive ductal carcinoma, the positive expression rates were 15.4%,250%,25%,33.3%,42.9%. Positive expression in invasive ductal carcinoma tissues was significantly higher than in normal breast tissue, was significantly higher than breast hyperplasia (P<0.05).Conclusions:1 Hedgehog signaling pathway of CD44+/CD24-/low signs stem cell subsets in breast cancer cell line MCF-7 is significantly activated.2 Distribution and expression degree of PATH1 and Gli-1 in different breast diseases were significantly different, the lower differentiation, the stronger the expression,which was contribute to the pathological diagnosis of breast lesions.3 The expression of CK5 and CK8 were different in different degrees of breast ductal carcinoma. In different degree of differentiation, the higher differentiation, the weaker the expression of CK8, but CK5 expression was stronger, on the contrary, the lower degree of differentiation, the stronger the expression of CK8, expression of CK5 relatively weak or absent. Distribution and expression of BCRP in different breast diseases tissues were significantly different, suggesting that BCRP may have a role in breast cancer cells differentiation. Different expressions of CK5, CK8 and BCRP in different breast tissue may be contribute to the pathological diagnosis of breast lesions.In conclusion, immunomagnetic beads sorting of breast cancer stem cells is a simple and reliable method of sorting the CD44+/CD24-/low stem cell mark cell subsets, Hedgehog signaling pathway is significantly activated. Several cancer stem cell markers can be used for the pathological diagnosis of breast lesions...