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A Detailed Mammosphere Assay Protocol for the Quantification of Breast Stem Cell Activity

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Abstract

Since the discovery that neural tissue contains a population of stem cells that form neurospheres in vitro, sphere-forming assays have been adapted for use with a number of different tissue types for the quantification of stem cell activity and self-renewal. One tissue type widely used for stem cell investigations is mammary tissue, and the mammosphere assay has been used in both normal tissue and cancer. Although it is a relatively simple assay to learn, it can be difficult to master. There are methodological and analytical aspects to the assay which require careful consideration when interpreting the results. We describe here a detailed mammosphere assay protocol for the assessment of stem cell activity and self-renewal, and discuss how data generated by the assay can be analysed and interpreted.

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Abbreviations

CSC:

Cancer stem-like cells

DCIS:

Ductal carcinoma in situ

ER:

Oestrogen receptor

EGF:

Epidermal growth factor

IDC:

Invasive ductal carcinoma

MFE:

Mammosphere forming efficiency

PE:

Pleural effusion

PR:

Progesterone receptor

References

  1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. GLOBOCAN 2008 v1.2, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 10 [Internet]. Lyon, France: International Agency for Research on Cancer; 2010. Available from: http://globocan.iarc.fr, accessed on 20/2/2012.

  2. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997;3(7):730–7.

    Article  PubMed  CAS  Google Scholar 

  3. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA. 2003;100(7):3983–8.

    Article  PubMed  CAS  Google Scholar 

  4. Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D, et al. Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res. 2005;65(13):5506–11.

    Article  PubMed  CAS  Google Scholar 

  5. Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, Wu MF, et al. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst. 2008;100(9):672–9.

    Article  PubMed  CAS  Google Scholar 

  6. Creighton CJ, Li X, Landis M, Dixon JM, Neumeister VM, Sjolund A, et al. Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. Proc Natl Acad Sci USA. 2009;106(33):13820–5.

    Article  PubMed  CAS  Google Scholar 

  7. Diehn M, Cho RW, Lobo NA, Kalisky T, Dorie MJ, Kulp AN, et al. Association of reactive oxygen species levels and radioresistance in cancer stem cells. Nature. 2009;458(7239):780–3.

    Article  PubMed  CAS  Google Scholar 

  8. Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science. 1992;255(5052):1707–10.

    Article  PubMed  CAS  Google Scholar 

  9. Dontu G, Al-Hajj M, Abdallah WM, Clarke MF, Wicha MS. Stem cells in normal breast development and breast cancer. Cell Prolif. 2003;36 Suppl 1:59–72.

    Article  PubMed  CAS  Google Scholar 

  10. Farnie G, Clarke RB, Spence K, Pinnock N, Brennan K, Anderson NG, et al. Novel cell culture technique for primary ductal carcinoma in situ: role of Notch and epidermal growth factor receptor signaling pathways. J Natl Cancer Inst. 2007;99(8):616–27.

    Article  PubMed  CAS  Google Scholar 

  11. Harrison H, Farnie G, Howell SJ, Rock RE, Stylianou S, Brennan KR, et al. Regulation of breast cancer stem cell activity by signaling through the Notch4 receptor. Cancer Res. 2010;70(2):709–18.

    Article  PubMed  CAS  Google Scholar 

  12. DeFriend DJ, Anderson E, Bell J, Wilks DP, West CM, Mansel RE, et al. Effects of 4-hydroxytamoxifen and a novel pure antioestrogen (ICI 182780) on the clonogenic growth of human breast cancer cells in vitro. Br J Cancer. 1994;70:204–11.

    Article  PubMed  CAS  Google Scholar 

  13. Grimshaw MJ, Cooper L, Papazisis K, Coleman JA, Bohnenkamp HR, Chiapero-Stanke L, et al. Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells. Breast Cancer Res. 2008;10(3):R52.

    Article  PubMed  Google Scholar 

  14. Reynolds BA, Rietze RL. Neural stem cells and neurospheres–re-evaluating the relationship. Nat Methods. 2005;2(5):333–6.

    Article  PubMed  CAS  Google Scholar 

  15. Cariati M, Naderi A, Brown JP, Smalley MJ, Pinder SE, Caldas C, et al. Alpha-6 integrin is necessary for the tumourigenicity of a stem cell-like subpopulation within the MCF7 breast cancer cell line. Int J Cancer. 2008;122(2):298–304.

    Article  PubMed  CAS  Google Scholar 

  16. Singec I, Knoth R, Meyer RP, Maciaczyk J, Volk B, Nikkhah G, et al. Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology. Nat Methods. 2006;3(10):801–6.

    Article  PubMed  CAS  Google Scholar 

  17. Pastrana E, Silva-Vargas V, Doetsch F. Eyes wide open: a critical review of sphere-formation as an assay for stem cells. Cell Stem Cell. 2011;8(5):486–98.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We would like to acknowledge the following funding sources: Breast Cancer Campaign (RBC and GF), Cancer Research UK (FLS), European Union FP6 (HH), European Union FP7 (KS and BMS), Biotechnology and Biological Sciences Research Council and Vertex Pharmaceuticals (Europe) Ltd. (MPA). We would like to thank Kate Williams for supplying the SUM-225 and MCF10DCIS.com micrographs.

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Authors and Affiliations

  1. Breast Biology Group, School of Cancer and Enabling Sciences, Paterson Institute for Cancer Research, The University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK

    Frances L. Shaw, Katherine Spence, Matthew P. Ablett, Bruno M. Simões & Robert B. Clarke

  2. Molecular Pathology, Breakthrough Breast Cancer Research Unit, School of Cancer and Enabling Sciences, Paterson Institute for Cancer Research, The University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK

    Hannah Harrison

  3. Cancer Stem Cell Research, School of Cancer and Enabling Sciences, Paterson Institute for Cancer Research, The University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK

    Gillian Farnie

Authors
  1. Frances L. Shaw
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  2. Hannah Harrison
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  3. Katherine Spence
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  4. Matthew P. Ablett
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  5. Bruno M. Simões
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  6. Gillian Farnie
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  7. Robert B. Clarke
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Corresponding author

Correspondence to Robert B. Clarke.

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Frances L. Shaw and Hannah Harrison, are joint first authors

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Shaw, F.L., Harrison, H., Spence, K. et al. A Detailed Mammosphere Assay Protocol for the Quantification of Breast Stem Cell Activity. J Mammary Gland Biol Neoplasia 17, 111–117 (2012). https://doi.org/10.1007/s10911-012-9255-3

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  • DOI: https://doi.org/10.1007/s10911-012-9255-3

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