Breast Cancer Stem Cell-derived Endothelial Cells Involved In Tumor Angiogenesis

Breast cancer stem cells (BCSCs) constitute a subpopulation of tumor cells that express stem cell-associated markers and have a high capacity for tumor generation in vivo. Identification of BCSCs from fresh tumor section samples or breast cancer cell lines has been based mainly on CD44+/CD24-/low or ALDH+phenotypes. BCSCs isolation has been established to the analyze the molecular mechanisms involved in their origin,self-renewal, differentiation into tumor cells, resistance to radiation therapy and chemotherapy, and invasiveness and metastatic ability. Molecular genetic analysis using knockout animals and inducible transgenics has identified a lot cytokines and enzymes involved in tumor angiogenesis of BCSC. Recent research indicated that there are glioblastoma cancer stem cell-derived endothelial progenitor cells involved in tumor angiogenesis. Whether breast cancer stem cell-derived endothelial progenitor cells may be involved in tumor angiogenesis of breast cancer?Objective:To investigate whether breast cancer stem cells have the potential to differentiate into endothelial cells involved in tumor angiogenesis.Methods:8fresh specimens of breast cancers were collected from Hunan Province People’s Hospital after the patients gave informed consents.The expression of CD31, VEGF, P53, HER-2were detected by immunohistochemistry (IHC), and co-expression of CD31and HER-2by immunofluorescence, and the HER-2gene amplification by fluorescence in situhybridization(FISH). Separated breast cancer cells were gained from fresh specimens by0.5%collagenase digestion and CD44+/CD24-/low cell subsets were isolated by magnetic activated cell sorting (MACS), and the expression of CD44and CD24of the isolated cells were detcted by flow cytometry techniques (FCM). Then the sorted CD44+/CD24-/low cell were cultured in stem cell medium DMEM/F12supplied with20μg/L EGF、20μg/L bFGF and2%B27. After1-2w, the colony forming was observed everyday. Then the colonies were analyzed CD105and CD31protein expression by FCM and digested into single cell suspension to culture in endothelial cell differentiation culture medium EGM-2then to passage. Passage1cells were identified by uptake of acetylated low-density-lipoprotein(Ac-LDL) and observed the formation of dimensional tube-like structure in vitro.Results:CD31, VEGF, mutant p53and HER-2was found in blood vessels and lined the lumens of capillaries and/or vascular glomeruli. CD31and HER-2protein co-expressed in blood vessels by confocal laser scanning microscopy. Meanwhile, HER-2gene amplification was confirmed by FISH. The separated cells after isolation by MACS, the percent of CD44+increased from7.1%±2.9%to94.5%±5.1%, and the percent of CD24+cells decreased from43.6%±9.7%to3.7%±0.9%. The CD44+/CD24-/low cells could form clones. After culture differentiation to endothelial cell, the CD105+cells increased from4.8%±0.6%to42.1%±7.8%, and the CD31+cells increased from0.5%±0.2%to92.1%±4.7%. And Passage1cells after culture differentiation to endothelial cell could uptake DiI-Ac-LDL and form dimensional tube-like structure in vitro.Conclusion:1) There are homologous between a part of vascular endothelial cells and tumor cells in breast cancer.2)CD44+/CD24-/low cells isolated from breast cancer cells could form colonies.3) CD44+/CD24-/low breast cancer stem cells could differentiate into endothelial cells in vitro.