| Emerging evidence have been carried out that the breast cancer cells originate from a rare population of tumor cells, which maintain the property of stem cells. These cells have enhanced self-renewal capacity, tumorigenic, and construct the whole population of tumor cells. These cells represent some main character of breast cancer such as unlimited growth pattern, invasion and metastasis, immunal released, irresponsable to radiation, chemotherapy resistance. Cancer stem cells were successfully isolated and identified in some solid tumors such as breast cancer, brain cancer, rectal cancer and prostate cancer. The population of breast cancer cells with the phenotype of CD44+/CD24- was considered to be enriched for breast cancer stem cells, and this population of tumor cells reflects some biology characteristics of breast cancer. Breast cancer cells with the phenotype of CD44+/CD24- isolated from metastatic pleural effussion show enhanced tumorigenesis and less to 100 cells with this type can form tumor in SCID/NOD mouse, further more other subpopulation of tumor cells 100 fold more fail to form tumor in vivo. Resent studies show that the breast cancer stem cells are the resource of the breast cancers. It is the key procedure to eliminate the breast cancer stem cells in treating breast cancers. The objective of this project is to isolate the population of CD44+/CD24- phenotype of breast cancer cells and identify its tumorigenic capacity and the correlation between the ratio of these cells in breast cancer and the pathological character of breast cancers.Octamer-4 (Oct-4) is a transcription factor in ESCs, it is correlated with the pluripotency, proliferative potential and self-renew capacity in embryonic stem cells and germ cells. Up to the present the research on Oct-4 is limited in cells with stem property, such as stem cells of skin basal layers, breast stem cells and gastric cells. With the profound investigation in cancer stem cells, role of stem related genes are more concerned. There is rare research about the mechanism of Oct-4 in breast cancer cells properties. We focus on the expression of Oct-4 in breast cancers with gene chip arrays and immunochemical analysis and illuminate its relationship with the clinical pathological character of breast cancer, in order to provide more evidence for breast cancer therapy.Materials and Methods一.Isolation of CD44+/CD24- phenotype breast cancer cells and identify its tumorigenic capacityThe flow cytometry(FCM) technique is performed to isolate the cell population with CD44+/CD24- phenotype from breast cancer specimens, and analysis the ratio of these cells in breast cancer and investigate its significant comparing with the breast cancer clinical parameters. Also we test the tumorigenic capacity of this population of cancer cells in vivo.二.Test of stem cell associate gene Oct-4Gene chip array and immunochemical analysis are used to prove the relationship between the expression of Oct-4 and breast cancer clinical parameters, immunochemical methods are also used to test the expression of ER, PR and Her-2 gene expression in breast cancer research.三.Statistic methodsDatas are analysis by SPSS13.0 statistic software. T-test and X2 test were used to analysis the relationship of these parameters. Kaplan-Meier method and Log rank method are used to analysis the survival rate and COX model is used to test prognosis factors.Results一.Ratio of CD44+/CD24- phenotype of breast cancer cells indicate clinicopathological characters and its tumorigenic capacityThe cell population of CD44+/CD24- phenotype take up the ratio of cancer cells between 3.75%-33.11%, the mean ratio is 13.67%. Compared with the NO stage of breast cancer, cases with positive lymph node metastasis, N1N2, exhibit an accelerated CD44+/CD24-cancer cell ratio (10.37% VS 16.16% P=0.037). In respect with the hormone receptor expression, CD44+/CD24-cancer cells have more enrichment in ER- cases than that in ER+ breast cancers (11.05% VS 16.27% P=0.043). Her-2+ breast cancers have higher CD44+/CD24- cancer cell ratio than Her-2- ones (9.47% VS 17.52% P=0.013). Breast cancer cells with phenotype of CD44+/CD24- isolated by FCM were injected in SCID/NOD mouse together with other subpopulation of cancer cells as negative a control, as little as 5000 CD44+/CD24- tumor cells can develop new tumor in mouse, nevertheless 105 other phenotype cancer cells fail to develop tumor, which indicate the enhanced tumorigenic facility in CD44+/CD24- subpopulation of breast cancer cells.二.Expression of Oct-4 in CD44+/CD24- breast cancer cells and its clinical implicationsThe difference of gene expression profile between CD44+/CD24- tumor cells and CD44+/CD24- negative tumor cells:the list of genes which are highly expressed:stem cells associated factors CD44, Oct4, nestin,(145.82,64.28,49.17);cell cycle regulators:APC,CDC2(4.79,33.0); growth factors:HGF, TGF (12.82,37.38). Oct-4 is highly expressed in breast cancer tissues compared with the adjacent normal tissues.Oct-4 positive breast cancer patients own shorter survival rate (P=0.001). COX model indicate that tumor size, histological type, stage of cancer, lymph node metastasis, Her-2 and Oct-4 expression are the independent prognosis factors of breast cancer (P=0.031;0.012; 0.001; 0.002; 0.030; 0.003).Conclusions1. Breast cancer cells contain a subpopulation of CD44+/CD24- tumor cells. CD44+/CD24- tumor cell ratio is correlated with N stage of breast cancer, and is with relationship to ER, Her-2 expression.2. The subpopulation of CD44+/CD24- phenotype of tumor cells are enriched for breast cancer stem cells and exhibit enhanced tumorigenicity which reflect its stem cell properties. 3. Oct-4 gene is highly expressed in CD44+/CD24- breast cancer cells. Its expression is correlated with clinicopathological character of breast cancer, and is an independent factor for the survival rate.
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