Skip to main content
SpringerLink
Log in

In Vitro Assays of Breast Cancer Stem Cells

  • Protocol
  • First Online:
Hypoxia

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1742))

Abstract

Aldehyde dehydrogenase and mammosphere assays enable the cost-effective quantification and characterization of cancer stem cells (CSCs) from cancer cell lines as well as cancer tissue. Here we describe the quantification of CSCs in breast cancer cell lines using aldehyde dehydrogenase and mammosphere assays under hypoxic (1% O2) and non-hypoxic (20% O2) culture conditions. Using this method, a significant enrichment of CSCs compared to bulk populations is observed when breast cancer cells are exposed to 1% O2 for 72 h.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 79.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 99.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Al-Hajj M, Wicha MS, Benito-Hernandez A et al (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 100(7):3983–3988

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Oskarsson T, Batlle E, Massague J (2014) Metastatic stem cells: sources, niches, and vital pathways. Cell Stem Cell 14(3):306–321

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Vaupel P, Hockel M, Mayer A (2007) Detection and characterization of tumor hypoxia using pO2 histography. Antioxid Redox Signal 9(8):1221–1235

    Article  CAS  PubMed  Google Scholar 

  4. Semenza GL (2015) The hypoxic tumor microenvironment: a driving force for breast cancer progression. Biochim Biophys Acta 1863(3):382–391

    Article  PubMed  PubMed Central  Google Scholar 

  5. Ginestier C, Hur MH, Charafe-Jauffret E et al (2007) ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1(5):555–567

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Dontu G, Abdallah WM, Foley JM et al (2003) In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 17(10):1253–1270

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Jones RJ, Barber JP, Vala MS et al (1995) Assessment of aldehyde dehydrogenase in viable cells. Blood 85(10):2742–2746

    CAS  PubMed  Google Scholar 

  8. Chute JP, Muramoto GG, Whitesides J et al (2006) Inhibition of aldehyde dehydrogenase and retinoid signaling induces the expansion of human hematopoietic stem cells. Proc Natl Acad Sci U S A 103:11707–11712

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ghiaur G, Yegnasubramanian S, Perkins B et al (2013) Regulation of human hematopoietic stem cell self-renewal by the microenvironment’s control of retinoic acid signaling. Proc Natl Acad Sci U S A 110(40):16121–16126

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Pastrana E, Silva-Vargas V, Doetsch F (2011) Eyes wide open: a critical review of sphere-formation as an assay for stem cells. Cell Stem Cell 8(5):486–498

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Dontu G, Al-Hajj M, Abdallah WM et al (2003) Stem cells in normal breast development and breast cancer. Cell Prolif 36(Suppl 1):59–72

    Article  CAS  PubMed  Google Scholar 

  12. Shaw FL, Harrison H, Spence K et al (2012) A detailed mammosphere assay protocol for the quantification of breast stem cell activity. J Mammary Gland Biol Neoplasia 17(2):111–117

    Article  PubMed  Google Scholar 

  13. Patrawala L, Calhoun T, Schneider-Broussard R et al (2005) Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2− cancer cells are similarly tumorigenic. Cancer Res 65:6207–6219

    Article  CAS  PubMed  Google Scholar 

  14. Christgen M, Ballmaier M, Bruchhardt H et al (2007) Identification of a distinct side population of cancer cells in the Cal-51 human breast carcinoma cell line. Mol Cell Biochem 306:201–212

    Article  CAS  PubMed  Google Scholar 

  15. Fillmore CM, Kuperwasser C (2008) Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy. Breast Cancer Res 10:R25

    Article  PubMed  PubMed Central  Google Scholar 

  16. Conley SJ, Gheordunescu E, Kakarala P et al (2012) Antiangiogenic agents increase breast cancer stem cells via the generation of tumor hypoxia. Proc Natl Acad Sci U S A 109980:2784–2789

    Article  Google Scholar 

  17. Xiang L, Gilkes DM, Hu H et al (2014) Hypoxia-inducible factor 1 mediates TAZ expression and nuclear localization to induce the breast cancer stem cell phenotype. Oncotarget 5(24):12509–12527

    Article  PubMed  PubMed Central  Google Scholar 

  18. Zhang H, Lu H, Xiang L et al (2015) HIF-1 regulates CD47 expression in breast cancer cells to promote evasion of phagocytosis and maintenance of cancer stem cells. Proc Natl Acad Sci U S A 112(45):E6215–E6223

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Brooks DL, Schwab LP, Krutilina R et al (2016) ITGA6 is directly regulated by hypoxia-inducible factors and enriches for cancer stem cell activity and invasion in metastatic breast cancer models. Mol Cancer 15:26

    Article  PubMed  PubMed Central  Google Scholar 

  20. Samanta D, Park Y, Andrabi SA et al (2016) PHGDH expression is required for mitochondrial redox homeostasis, breast cancer stem cell maintenance, and lung metastasis. Cancer Res 76(15):4430–4442

    Article  CAS  PubMed  Google Scholar 

  21. Zhang C, Samanta D, Lu H et al (2016) Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m6A-demethylation of NANOG mRNA. Proc Natl Acad Sci U S A 113(14):E2047–E2056

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Zhang C, Zhi WI, Lu H et al (2016) Hypoxia-inducible factors regulate pluripotency expression by ZNF217- and ALKBH5-mediated modulation of RNA methylation in breast cancer cells. Oncotarget 7(40):64527–64542. 10.18632/oncotarget.11743

    PubMed  PubMed Central  Google Scholar 

  23. Van den Beucken T, Koch E, Chu K et al (2014) Hypoxia promotes stem cell phenotypes and poor prognosis through epigenetic regulation of DICER. Nat Commun 5:5203

    Article  PubMed  PubMed Central  Google Scholar 

  24. Iriondo O, Rabano M, Domenici G et al (2015) Distinct breast cancer stem/progenitor cell populations require either HIF-1α or loss of PHD3 to expand under hypoxic conditions. Oncotarget 6(31):31721–31739

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

Cancer research in the authors’ laboratory is supported by grants from the American Cancer Society, Armstrong Family Foundation, Department of Defense Breast Cancer Research Program, and the Cindy Rosencrans Fund for Triple Negative Breast Cancer. G.L.S. is an American Cancer Society Research Professor and the C. Michael Armstrong Professor at the Johns Hopkins University School of Medicine.

Author information

Authors and Affiliations

  1. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Debangshu Samanta & Gregg L. Semenza

  2. Johns Hopkins Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Debangshu Samanta & Gregg L. Semenza

  3. Departments of Pediatrics, Medicine, Oncology, Radiation Oncology, and Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Gregg L. Semenza

Authors
  1. Debangshu Samanta
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gregg L. Semenza
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gregg L. Semenza .

Editor information

Editors and Affiliations

  1. Department of Neurosurgery, Clinical Neurosciences Center, University of Utah, Salt Lake City, Utah, USA

    L. Eric Huang

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Samanta, D., Semenza, G.L. (2018). In Vitro Assays of Breast Cancer Stem Cells. In: Huang, L. (eds) Hypoxia. Methods in Molecular Biology, vol 1742. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7665-2_21

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7665-2_21

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7664-5

  • Online ISBN: 978-1-4939-7665-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation