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Assessment of Breast Cancer Stem Cell Activity Using a Spheroid Formation Assay

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Stem Cell Assays

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2429))

Abstract

Evidence is emerging that cancer cells are arranged as a hierarchy that spans from stem cells to lineage-restricted progenitor cells. The recent development of spheroid cultures with several tissue type has provided new opportunities to assess cancer stem cell (CSC) activity by allowing them to propagate under conditions that resemble the microenvironment for growth of tumors. One tissue type widely used for stem cell investigations is mammary tissue, and the sphere formation assay has been used in both normal mammary tissue and in breast cancer. Here, we describe detailed experimental methodology for generating and propagating spheres from normal mammary tissue and primary breast tumors of mice, patient derived xenografts (PDXs) and breast cancer cell lines. We further describe how these sphere cultures can be employed for coculture assays to assess the effect of tumor microenvironment (TME) on self-renewal ability of CSCs in breast cancer.

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References

  1. Ishiguro T, Ohata H, Sato A et al (2017) Tumor-derived spheroids: relevance to cancer stem cells and clinical applications. Cancer Sci 108:283–289

    Article  CAS  Google Scholar 

  2. Anderson K, Lutz C, van Delft FW et al (2011) Genetic variegation of clonal architecture and propagating cells in leukaemia. Nature 469:356–361

    Article  CAS  Google Scholar 

  3. Kitamura H, Okudela K, Yazawa T et al (2009) Cancer stem cell: implications in cancer biology and therapy with special reference to lung cancer. Lung Cancer 66:275–281

    Article  Google Scholar 

  4. Schulenburg A, Ulrich-Pur H, Thurnher D et al (2006) Neoplastic stem cells: a novel therapeutic target in clinical oncology. Cancer 107:2512–2520

    Article  CAS  Google Scholar 

  5. Jordan CT, Guzman ML, Noble M (2006) Cancer stem cells. N Engl J Med 355:1253–1261

    Article  CAS  Google Scholar 

  6. Bonnet D, Dick JE (1997) Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3:730–737

    Article  CAS  Google Scholar 

  7. 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 100:3983–3988

    Article  CAS  Google Scholar 

  8. Ricci-Vitiani L, Lombardi DG, Pilozzi E et al (2007) Identification and expansion of human colon-cancer-initiating cells. Nature 445:111–115

    Article  CAS  Google Scholar 

  9. Reynolds BA, Weiss S (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:1707–1710

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Ponti D, Costa A, Zaffaroni N et al (2005) Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 65:5506–5511

    Article  CAS  Google Scholar 

  12. Rosenbluth JM, Schackmann RCJ, Gray GK et al (2020) Organoid cultures from normal and cancer-prone human breast tissues preserve complex epithelial lineages. Nat Commun 11:1711

    Article  CAS  Google Scholar 

  13. Grimshaw MJ, Cooper L, Papazisis K et al (2008) Mammosphere culture of metastatic breast cancer cells enriches for tumorigenic breast cancer cells. Breast Cancer Res 10:R52

    Article  Google Scholar 

  14. Liu JC, Deng T, Lehal RS et al (2007) Identification of tumorsphere- and tumor-initiating cells in HER2/Neu-induced mammary tumors. Cancer Res 67:8671–8681

    Article  CAS  Google Scholar 

  15. Farnie G, Clarke RB, Spence K et al (2007) Novel cell culture technique for primary ductal carcinoma in situ: role of Notch and epidermal growth factor receptor signaling pathways. J Natl Cancer Inst 99:616–627

    Article  CAS  Google Scholar 

  16. Hughes CS, Postovit LM, Lajoie GA (2010) Matrigel: a complex protein mixture required for optimal growth of cell culture. Proteomics 10:1886–1890

    Article  CAS  Google Scholar 

  17. Shaw LM, Chao C, Wewer UM, Mercurio AM (1996) Function of the integrin alpha 6 beta 1 in metastatic breast carcinoma cells assessed by expression of a dominant-negative receptor. Cancer Res 56:959–963

    CAS  PubMed  Google Scholar 

  18. Chaicharoenaudomrung N, Kunhorm P, Noisa P (2019) Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling. World J Stem Cells 11:1065–1083

    Article  Google Scholar 

  19. Goldhammer N, Kim J, Timmermans-Wielenga V, Petersen OW (2019) Characterization of organoid cultured human breast cancer. Breast Cancer Res 21:141

    Article  CAS  Google Scholar 

  20. Dekkers JF, Alieva M, Wellens LM et al (2019) High-resolution 3D imaging of fixed and cleared organoids. Nat Protoc 14:1756–1771

    Article  CAS  Google Scholar 

  21. Roelofs C, Hollande F, Redvers R et al (2019) Breast tumour organoids: promising models for the genomic and functional characterisation of breast cancer. Biochem Soc Trans 47:109–117

    Article  CAS  Google Scholar 

  22. Smart CE, Morrison BJ, Saunus JM et al (2013) In vitro analysis of breast cancer cell line tumourspheres and primary human breast epithelia mammospheres demonstrates inter- and intrasphere heterogeneity. PLoS One 8:e64388

    Article  CAS  Google Scholar 

  23. Casbas-Hernandez P, Fleming JM, Troester MA (2011) Gene expression analysis of in vitro cocultures to study interactions between breast epithelium and stroma. J Biomed Biotechnol 2011:520987

    Article  Google Scholar 

  24. Shackleton M, Vaillant F, Simpson KJ et al (2006) Generation of a functional mammary gland from a single stem cell. Nature 439:84–88

    Article  CAS  Google Scholar 

  25. 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 17:111–117

    Article  Google Scholar 

  26. Kumar S, Srivastav RK, Wilkes DW et al (2019) Estrogen-dependent DLL1-mediated Notch signaling promotes luminal breast cancer. Oncogene 38:2092–2107

    Article  CAS  Google Scholar 

  27. DeRose YS, Wang G, Lin YC et al (2011) Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes. Nat Med 17:1514–1520

    Article  CAS  Google Scholar 

  28. Chakrabarti R, Wei Y, Romano RA et al (2012) Elf5 regulates mammary gland stem/progenitor cell fate by influencing notch signaling. Stem Cells 30:1496–1508

    Article  CAS  Google Scholar 

  29. Chakrabarti R, Wei Y, Hwang J et al (2014) DeltaNp63 promotes stem cell activity in mammary gland development and basal-like breast cancer by enhancing Fzd7 expression and Wnt signalling. Nat Cell Biol 16:1004–1015. 1001–1013

    Article  CAS  Google Scholar 

  30. Wu MH, Huang SB, Lee GB (2010) Microfluidic cell culture systems for drug research. Lab Chip 10:939–956

    Article  CAS  Google Scholar 

  31. Chakrabarti R, Celia-Terrassa T, Kumar S et al (2018) Notch ligand Dll1 mediates cross-talk between mammary stem cells and the macrophageal niche. Science 360

    Google Scholar 

  32. Kumar S, Wilkes DW, Samuel N et al (2018) DeltaNp63-driven recruitment of myeloid-derived suppressor cells promotes metastasis in triple-negative breast cancer. J Clin Invest 128:5095–5109

    Article  Google Scholar 

  33. Kuen J, Darowski D, Kluge T, Majety M (2017) Pancreatic cancer cell/fibroblast co-culture induces M2 like macrophages that influence therapeutic response in a 3D model. PLoS One 12:e0182039

    Article  Google Scholar 

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Acknowledgments

We thank Dr. Snahlata Singh for critical reading of the manuscript and helpful discussions. This work was supported by grants from American Cancer Society and NCI-R01 (R01CA237243) grant to R.C.

Author information

Authors and Affiliations

  1. Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Ajeya Nandi & Rumela Chakrabarti

Authors
  1. Ajeya Nandi
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  2. Rumela Chakrabarti
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Corresponding author

Correspondence to Rumela Chakrabarti .

Editor information

Editors and Affiliations

  1. Stem Cell and Cancer Biology Laboratory, Division of Experimental Medicine and Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA

    Nagarajan Kannan

  2. Hull York Medical School, York, UK

    Philip Beer

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Nandi, A., Chakrabarti, R. (2022). Assessment of Breast Cancer Stem Cell Activity Using a Spheroid Formation Assay. In: Kannan, N., Beer, P. (eds) Stem Cell Assays. Methods in Molecular Biology, vol 2429. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1979-7_33

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  • DOI: https://doi.org/10.1007/978-1-0716-1979-7_33

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1978-0

  • Online ISBN: 978-1-0716-1979-7

  • eBook Packages: Springer Protocols

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