Article

[Influence factors on the formation of mammospheres from breast cancer stem cells].

Department of Oncology, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, China.
Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences 10/2008; 40(5):500-4.
Source: PubMed

ABSTRACT

To investigate the influential factors on the formation of mammospheres from MCF-7, MDA-MB-231 and primary breast cancer epithelial cells, and to detect the proportion of CD44+/CD24(-/low) among them.
MCF- 7, MDA-MB-231 and primary breast cancer epithelial cells were cultured in suspension to generate primary mammospheres. After 7 days of cultivation, the proportion of cancer stem cells was measured in cells derived from mammosphere cells or monolayer culture cells by flow cytometry.
MCF-7 had the highest efficiency of mammosphere formation (2.1%+/-0.3%), while MDA-MB-231 cells hardly had any mammosphere formation, and no mammosphere formation was observed in breast cancer epithelial cells. MCF-7 tended to adhere under stem cell culture conditions without B27. Flow cytometry analysis indicated that, compared with 2.0%+/-0.1% for the MCF-7 monolayer culture cells, 11.8%+/-0.3% and 8.2%+/-0.8% of the MCF-7 mammosphere cells from MCF-7 under different conditions were CD44+/CD24(-/low) (P < 0.01). The proportion of CD44+/CD24(-/low) expression in MDA-MB-231 cells and primary breast cancer epithelial cells was 92.2%+/-3.1% and 93.8%+/-2.4%, respectively.
Compared with monolayer culture cells, MCF-7 mammosphere cells contained higher proportion of breast cancer stem cells, while MDA-MB-231 and primary breast cancer epithelial cells did not. The expression of CD44+/CD24(-/low) in MDA-MB-231 and primary breast cancer epithelial cells did not correlate with mammosphere formation positively.

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    • "On the contrary, the tumorspheres culture of MDA-MB-231 cells remained unsuccessful. This observation was consistent with what some authors reported [22], [23], although others showed this cell line was actually able to form tumorspheres [24], [25]. We hypothesized that the extinction of the tumorspheres formation ability of our MDA-MB-231 cells could result from the fact that these cells had been passaged many times before our experiments. "
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    ABSTRACT: Cancer stem cells (CSC) have raised great excitement during the last decade and are promising targets for an efficient treatment of tumors without relapses and metastases. Among the various methods that enable to enrich cancer cell lines in CSC, tumorspheres culture has been predominantly used. In this report, we attempted to generate tumorspheres from several murine and human cancer cell lines: B16-F10, HT-29, MCF-7 and MDA-MB-231 cells. Tumorspheres were obtained with variable efficiencies from all cell lines except from MDA-MB-231 cells. Then, we studied several CSC characteristics in both tumorspheres and adherent cultures of the B16-F10, HT-29 and MCF-7 cells. Unexpectedly, tumorspheres-forming cells were less clonogenic and, in the case of B16-F10, less proliferative than attached cells. In addition, we did not observe any enrichment in the population expressing CSC surface markers in tumorspheres from B16-F10 (CD133, CD44 and CD24 markers) or MCF-7 (CD44 and CD24 markers) cells. On the contrary, tumorspheres culture of HT-29 cells appeared to enrich in cells expressing colon CSC markers, i.e. CD133 and CD44 proteins. For the B16-F10 cell line, when 1 000 cells were injected in syngenic C57BL/6 mice, tumorspheres-forming cells displayed a significantly lower tumorigenic potential than adherent cells. Finally, tumorspheres culture of B16-F10 cells induced a down-regulation of vimentin which could explain, at least partially, the lower tumorigenicity of tumorspheres-forming cells. All these results, along with the literature, indicate that tumorspheres culture of cancer cell lines can induce an enrichment in CSC but in a cell line-dependent manner. In conclusion, extensive characterization of CSC properties in tumorspheres derived from any cancer cell line or cancer tissue must be performed in order to ensure that the generated tumorspheres are actually enriched in CSC.
    Full-text · Article · Feb 2014 · PLoS ONE
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    • "In our previous studies, we have showed that MCF7 cell line had the highest mammosphere-forming efficiency (MFE) among many breast cancer cells, so MCF7 cells were chosen to generate mammosphere cells in vitro [17]. Cells were then washed twice with PBS and cultured in suspension at a density of 2 × 105/bottle in DMEM/F12 (HyClone, Logan, Utah) with high glucose, supplemented with 1 × B27 (Invitrogen), 20 ng/ml insulin-like growth factor I (Invitrogen), 20 ng/ml EGF (Sigma, St. Louis, MO) and 20 ng/ml b-FGF (Invitrogen). "
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    ABSTRACT: Breast cancer stem cells (BCSCs) have been recently identified in breast carcinoma as CD44+CD24- cells, which exclusively retain tumorigenic activity and display stem cell-like properties. Using a mammosphere culture technique, MCF7 mammosphere cells are found to enrich breast cancer stem-like cells expressing CD44+CD24-. The stromal cells are mainly constituted by fibroblasts within a breast carcinoma, yet little is known of the contributions of the stromal cells to BCSCs. Carcinoma-associated fibroblasts (CAFs) and normal fibroblasts (NFs) were isolated and identified by immunohistochemistry. MCF7 mammosphere cells were co-cultured with different stromal fibroblasts by a transwell cocultured system. Flow cytometry was used to measure CD44 and CD24 expression status on MCF7. ELISA (enzyme-linked immunosorbent assay) was performed to investigate the production of stromal cell-derived factor 1 (SDF-1) in mammosphere cultures subject to various treatments. Mammosphere cells were injected with CAFs and NFs to examine the efficiency of tumorigenity in NOD/SCID mice. CAFs derived from breast cancer patients were found to be positive for alpha-smooth muscle actin (alpha-SMA), exhibiting the traits of myofibroblasts. In addition, CAFs played a central role in promoting the proliferation of CD44+CD24- cells through their ability to secrete SDF-1, which may be mediated to SDF-1/CXCR4 signaling. Moreover, the tumorigenicity of mammosphere cells with CAFs significantly increased as compared to that of mammosphere cells alone or with NFs. We for the first time investigated the effects of stromal fibroblasts on CD44+CD24- cells and our findings indicated that breast CAFs contribute to CD44+CD24- cell proliferation through the secretion of SDF-1, and which may be important target for therapeutic approaches.
    Full-text · Article · Jun 2010 · Journal of Experimental & Clinical Cancer Research
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    ABSTRACT: The cancer stem cell hypothesis asserts that malignancies arise in tissue stem and/or progenitor cells through the dysregulation or acquisition of self-renewal. In order to determine whether the dietary polyphenols, curcumin, and piperine are able to modulate the self-renewal of normal and malignant breast stem cells, we examined the effects of these compounds on mammosphere formation, expression of the breast stem cell marker aldehyde dehydrogenase (ALDH), and Wnt signaling. Mammosphere formation assays were performed after curcumin, piperine, and control treatment in unsorted normal breast epithelial cells and normal stem and early progenitor cells, selected by ALDH positivity. Wnt signaling was examined using a Topflash assay. Both curcumin and piperine inhibited mammosphere formation, serial passaging, and percent of ALDH+ cells by 50% at 5 microM and completely at 10 microM concentration in normal and malignant breast cells. There was no effect on cellular differentiation. Wnt signaling was inhibited by both curcumin and piperine by 50% at 5 microM and completely at 10 microM. Curcumin and piperine separately, and in combination, inhibit breast stem cell self-renewal but do not cause toxicity to differentiated cells. These compounds could be potential cancer preventive agents. Mammosphere formation assays may be a quantifiable biomarker to assess cancer preventive agent efficacy and Wnt signaling assessment can be a mechanistic biomarker for use in human clinical trials.
    Full-text · Article · Nov 2009 · Breast Cancer Research and Treatment
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Questions & Answers about this publication

  • Berrin Saracoglu asked a question in Cancer Stem Cells:
    How do B27 and insulin affect mammospheres?
    I reached your paper but it is in Chinese so I could not understand it. Could you help me to understand how B27 and insulin affect MCF-7 derived mammospheres please?

    Thank you
    • [Show abstract] [Hide abstract]
      ABSTRACT: To investigate the influential factors on the formation of mammospheres from MCF-7, MDA-MB-231 and primary breast cancer epithelial cells, and to detect the proportion of CD44+/CD24(-/low) among them. MCF- 7, MDA-MB-231 and primary breast cancer epithelial cells were cultured in suspension to generate primary mammospheres. After 7 days of cultivation, the proportion of cancer stem cells was measured in cells derived from mammosphere cells or monolayer culture cells by flow cytometry. MCF-7 had the highest efficiency of mammosphere formation (2.1%+/-0.3%), while MDA-MB-231 cells hardly had any mammosphere formation, and no mammosphere formation was observed in breast cancer epithelial cells. MCF-7 tended to adhere under stem cell culture conditions without B27. Flow cytometry analysis indicated that, compared with 2.0%+/-0.1% for the MCF-7 monolayer culture cells, 11.8%+/-0.3% and 8.2%+/-0.8% of the MCF-7 mammosphere cells from MCF-7 under different conditions were CD44+/CD24(-/low) (P < 0.01). The proportion of CD44+/CD24(-/low) expression in MDA-MB-231 cells and primary breast cancer epithelial cells was 92.2%+/-3.1% and 93.8%+/-2.4%, respectively. Compared with monolayer culture cells, MCF-7 mammosphere cells contained higher proportion of breast cancer stem cells, while MDA-MB-231 and primary breast cancer epithelial cells did not. The expression of CD44+/CD24(-/low) in MDA-MB-231 and primary breast cancer epithelial cells did not correlate with mammosphere formation positively.
      No preview · Article · Oct 2008 · Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences