| Breast cancer is a common malignant tumor with high occurrence and mortality rate.The high mortality rate of breast cancer is related to its predilection of metastasis andrecurrence as well as the drug resistance of tumor cells. Recently, the cancer stem cell theorybrings us new views of oncogenesis and progression of cancers. Based on this theory, theoccurrence and drug resistance of tumors are related to cancer stem cells. This theory explainswell the reason of failures in cancer treatments, brings us new cytological targets for cancerresearch and therapies, and provides us with theoretical basis for complete cure of cancer.Therefore, the aims of this study are first to establish clones of tumor stem cells fromprimary cell cultures of fresh breast tumor, and to identify the surface marks and stemcell-specific genes, and to explore the responses of the breast tumor stem cells to hypoxicmicroenvironment. We hope that our study can provide experimental clues to get insight ofbreast tumors, and provide reference for development of new clinic diagnosis and therapies ofbreast cancers.Part1Isolation and characterization of human breast tumor stem cellsOBJECTIVE: To isolate and cultivate human breast cancer stem cells, and characterize theirbiological properties in vitro. METHODS: Fresh tumor tissues of breast cancer patients werecollected from surgical rooms with written contest. The tissue samples were mechanicallysheared and enzyme-digested to get single cell suspensions. Cancer cell surface antigens wereanalyzed by Flow Cytometry analysis, and cell growth curve was established by MTT； RT-qPCR was employed to detect the expression of stem cell-specific genes Sox2and Nanogin the cancer cells; Immunofluorescence staining was performed to exam the expression ofbreast cancer-specific proteins BCL-2and progesterone receptor (PR). RESULTS: Humanbreast cancer stem cell cultures were successfully established as sphere-growth cells. Thesestem cells had a CD44~+/CD24~-phenotype as characterized by Flow cytometry analysis,expressed high levels of Sox2and Nanog genes, and positive for Bcl-2and PR.CONCLUSION：Human breast tumors contain CD44~+/CD24~-cancer stem cells which arecapable of self-renewal and proliferation, and can be long term cultured and expanded invitro.Part2Comparison of cancer stem cell characteristics between breast cancer stem cellscultured in normal and hypoxia conditionsOBJECTIVE: To study the different responses of the breast cancer stem cells obtained frompart1to normal and low oxygen conditions. METHODS: breast cancer stem cells obtainedfrom part1were cultured under either normal or hypoxia conditions. The cells were thenanalyzed by flow cytometry to identify the expression of breast cancer stem cell surfaceantigens, by MTT colorimetry to detect cell growth curve under different condition, and byreal-time quantitative PCR (qPCR) to detect the expression of stem cell-specific genes Sox2,Nanog, and HIF. RESULTS: comparing with normal oxygen conditions, breast cancer stemcells cultured under hypoxia conditions have a higher proliferation rate, and expressed higherlevels of stem cell-specific genes. CONCLUSION: the low oxygen environment has apromoting effect on breast cancer stem cell growth and maintenance and development of theirstemness. |
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