title: Effect of fibroblasts on breast cancer cell mammosphere formation and regulation of stem cell-related gene expression.
abstract: The purpose of this study was to investigate the regulatory effects of breast cancer fibroblasts (BCFs) vs. normal mammary fibroblasts (NMFs) on mammosphere formation and stem cell-related gene expression in breast cancer cells. Breast cancer cells (MCF-7) were cultured in suspension to generate primary and secondary mammospheres. The proportion of CD44+/CD24low/- cells was assessed by flow cytometry (FCM), and Wnt1, Notch1, β-catenin, CXCR4, SOX2 and ALDH3A1 gene expression was detected by quantitative real-time PCR. The fibroblasts from either breast cancer tissue or normal mammary tissue were purified from tissue specimens and co-cultured with breast cancer cells. The mammosphere formation efficacy was approximately 180/10,000 MCF-7 cells. FCM analysis showed that, compared to the 2.1% positive expression in the MCF-7 monolayer culture cells, the expression of CD44+/CD24low/- in MCF-7 mammosphere cells was significantly elevated to 10.4% (P<0.01). The proportion of the CD44+/CD24low/- subpopulation of the cells in mammospheres was nearly 5-fold higher than that of general MCF-7 cells. Compared with MCF-7 monolayer culture cells, mammosphere cells showed significantly (P<0.01) enhanced expression of Wnt1 [fold-change (FC), 2.25], Notch1 (FC, 2.45), β-catenin (FC, 1.72), CXCR4 (FC, 4.68), SOX2 (FC, 4.25) and ALDH3A1 (FC, 5.38). When BCFs were co-cultured with MCF-7 cells under mammosphere culture conditions, the length of time of mammosphere formation decreased, the volume of the mammo-spheres increased and the mammosphere-forming efficiency (MFE) was higher than that of NMFs and the control group. Both the BCF and NMF groups showed enhanced gene expression for the following genes: Wnt1 (FC, 3.18 and 1.27, respectively), β-catenin (FC, 1.75 and 1.22, respectively), Notch1 (FC, 2.09 and 1.31, respectively), CXCR4 (FC, 2.77 and 1.33, respectively), SOX2 (FC, 2.77 and 1.80, respectively) and ALDH3A1 (FC, 5.23 and 1.85, respectively). Cancer fibroblast cells can promote the MFE and up-regulate stem cell-related gene expression in breast cancer cells.
authors.affiliation: Department of Oncology, Suzhou Kowloon Hospital, Shanghai Jiaotong University School of Medicine, Suzhou 215021, PR China.
authors.forename: Fengchun
authors.initials: F
authors.lastname: Zhang
authors.forename: Caili
authors.initials: C
authors.lastname: Song
authors.forename: Yue
authors.initials: Y
authors.lastname: Ma
authors.forename: Lei
authors.initials: L
authors.lastname: Tang
authors.forename: Yingchun
authors.initials: Y
authors.lastname: Xu
authors.forename: Hongxia
authors.initials: H
authors.lastname: Wang
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000032:analysis / Q000235:genetics; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000378:metabolism*; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D051881:Receptor, Notch1 / Q000032:analysis / Q000235:genetics; D019718:Receptors, CXCR4 / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000032:analysis / Q000235:genetics; D015398:Signal Transduction; D015854:Up-Regulation; D051155:Wnt1 Protein / Q000032:analysis / Q000235:genetics; D051176:beta Catenin / Q000032:analysis / Q000235:genetics
chemical_list: C495270:CTNNB1 protein, human; C527276:CXCR4 protein, human; C497761:NOTCH1 protein, human; D051881:Receptor, Notch1; D019718:Receptors, CXCR4; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; C495247:WNT1 protein, human; D051155:Wnt1 Protein; D051176:beta Catenin; C480021:ALDH3A1 protein, human; D000444:Aldehyde Dehydrogenase