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Characterization of mammary cancer stem cells in the MMTV-PyMT mouse model

  • Research Article
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Tumor Biology

Abstract

Breast cancer stem cells, the root of tumor growth, present challenges to investigate: Primary human breast cancer cells are difficult to establish in culture and inconsistently yield tumors after transplantation into immune-deficient recipient mice. Furthermore, there is limited characterization of mammary cancer stem cells in mice, the ideal model for the study of breast cancer. We herein describe a pre-clinical breast cancer stem cell model, based on the properties of cancer stem cells, derived from transgenic MMTV-PyMT mice. Using a defined set of cell surface markers to identify cancer stem cells by flow cytometry, at least four cell populations were recovered from primary mammary cancers. Only two of the four populations, one epithelial and one mesenchymal, were able to survive and proliferate in vitro. The epithelial population exhibited tumor initiation potential with as few as 10 cells injected into syngeneic immune-competent recipients. Tumors initiated from injected cell lines recapitulated the morphological and physiological components of the primary tumor. To highlight the stemness potential of the putative cancer stem cells, B lymphoma Mo-MLV insertion region 1 homolog (Bmi-1) expression was knocked down via shRNA targeting Bmi-1. Without Bmi-1 expression, putative cancer stem cells could no longer initiate tumors, but tumor initiation was rescued with the introduction of a Bmi-1 overexpression vector in the Bmi-1 knockdown cells. In conclusion, our data show that primary mammary cancers from MMTV-PyMT mice contain putative cancer stem cells that survive in culture and can be used to create a model for study of mammary cancer stem cells.

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Abbreviations

Bmi-1:

B lymphoma Mo-MLV insertion region 1 homolog

BSA:

Bovine serum albumin

BrdU:

5-bromo-2-deoxyuridine

CK:

Cytokeratin

CSC:

Cancer stem cell

DMEM:

Dulbecco’s Modified Eagle Medium

EGFP:

Enhanced green fluorescent protein

FACS:

Fluorescence-activated cell sorting

FBS:

Fetal bovine serum

FITC:

Fluorescein isothiocyanate

FMMC:

Female mouse mammary cancer cell line

FVB/NJ:

Friend Virus B NIH Jackson mouse strain

HBSS:

Hank’s buffered salt solution

MCF:

Michigan Cancer Foundation cell line

MMMC:

Male mouse mammary cancer cell line

MMTV-PyMT:

Mouse mammary tumor virus promoter-polyoma middle T-antigen

MSCs:

Mesenchymal stem cells

PBS:

Phosphate buffered saline

RA:

All-trans retinoic acid

SMA:

Smooth muscle actin

TI:

Tumor initiation

TICs:

Tumor-initiating cells

References

  1. Sell S, Pierce GB. Maturation arrest of stem cell differentiation is a common pathway for the cellular origin of teratocarcinomas and epithelial cancers. Lab Investig. 1994;70:6–22.

    PubMed  CAS  Google Scholar 

  2. Adriaansen HJ, te Boekhorst PA, Hagemeijer AM, van der Schoot CE, Delwel HR, van Dongen JJ. Acute myeloid leukemia M4 with bone marrow eosinophilia (M4Eo) and inv(16)(p13q22) exhibits a specific immunophenotype with CD2 expression. Blood. 1993;81:3043–51.

    PubMed  CAS  Google Scholar 

  3. Auersperg N, Kruk PA, MacLaren IA, Watt FM, Mydral SE. Heterogeneous expression of keratin, involucrin, and extracellular matrix among subpopulations of a poorly differentiated human cervical carcinoma: possible relationships to patterns of invasion. Cancer Res. 1989;49:3007–14.

    PubMed  CAS  Google Scholar 

  4. Blair A, Hogge DE, Ailles LE, Lansdorp PM, Sutherland HJ. Lack of expression of Thy-1 (CD90) on acute myeloid leukemia cells with long-term proliferative ability in vitro and in vivo. Blood. 1997;89:3104–12.

    PubMed  CAS  Google Scholar 

  5. Grabowski P, Schonfelder J, Ahnert-Hilger G, Foss HD, Stein H, Berger G, et al. Heterogeneous expression of neuroendocrine marker proteins in human undifferentiated carcinoma of the colon and rectum. Ann N Y Acad Sci. 2004;1014:270–4.

    Article  PubMed  CAS  Google Scholar 

  6. Sutherland HJ, Blair A, Zapf RW. Characterization of a hierarchy in human acute myeloid leukemia progenitor cells. Blood. 1996;87:4754–61.

    PubMed  CAS  Google Scholar 

  7. Tokar EJ, Ancrile BB, Cunha GR, Webber MM. Stem/progenitor and intermediate cell types and the origin of human prostate cancer. Differentiation. 2005;73:463–73.

    Article  PubMed  CAS  Google Scholar 

  8. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature. 2001;414:105–11.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  11. Cho RW, Wang X, Diehn M, Shedden K, Chen GY, Sherlock G, et al. Isolation and molecular characterization of cancer stem cells in MMTV-Wnt-1 murine breast tumors. Stem Cells. 2008;26:364–71.

    Article  PubMed  CAS  Google Scholar 

  12. Holyoake T, Jiang X, Eaves C, Eaves A. Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia. Blood. 1999;94:2056–64.

    PubMed  CAS  Google Scholar 

  13. Hotfilder M, Rottgers S, Rosemann A, Schrauder A, Schrappe M, Pieters R, et al. Leukemic stem cells in childhood high-risk ALL/t(9;22) and t(4;11) are present in primitive lymphoid-restricted CD34 + CD19- cells. Cancer Res. 2005;65:1442–9.

    Article  PubMed  CAS  Google Scholar 

  14. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 1994;367:645–8.

    Article  PubMed  CAS  Google Scholar 

  15. Lawson DA, Xin L, Lukacs RU, Cheng D, Witte ON. Isolation and functional characterization of murine prostate stem cells. Proc Natl Acad Sci U S A. 2007;104:181–6.

    Article  PubMed  CAS  Google Scholar 

  16. Matsui W, Huff CA, Wang Q, Malehorn MT, Barber J, Tanhehco Y, et al. Characterization of clonogenic multiple myeloma cells. Blood. 2004;103:2332–6.

    Article  PubMed  CAS  Google Scholar 

  17. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, et al. Identification of a cancer stem cell in human brain tumors. Cancer Res. 2003;63:5821–8.

    PubMed  CAS  Google Scholar 

  18. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, et al. Identification of human brain tumour initiating cells. Nature. 2004;432:396–401.

    Article  PubMed  CAS  Google Scholar 

  19. Sell S. Potential gene therapy strategies for cancer stem cells. Curr Gene Ther. 2006;6:579–91.

    Article  PubMed  CAS  Google Scholar 

  20. Pardal R, Clarke MF, Morrison SJ. Applying the principles of stem-cell biology to cancer. Nat Rev Cancer. 2003;3:895–902.

    Article  PubMed  CAS  Google Scholar 

  21. Sell S, Glinsky GV. Preventive and therapeutic strategies for cancer stem cells. In: Farrar W, editor. Cancer stem cells. Cambridge: Cambridge University Press; 2010. p. 68–92.

    Google Scholar 

  22. Wang JC, Dick JE. Cancer stem cells: lessons from leukemia. Trends Cell Biol. 2005;15:494–501.

    Article  PubMed  CAS  Google Scholar 

  23. Hamburger AW, Salmon SE. Primary bioassay of human tumor stem cells. Science. 1977;197:461–3.

    Article  PubMed  CAS  Google Scholar 

  24. Guy CT, Cardiff RD, Muller WJ. Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Mol Cell Biol. 1992;12:954–61.

    PubMed  CAS  Google Scholar 

  25. Lessard J, Sauvageau G. Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature. 2003;423:255–60.

    Article  PubMed  CAS  Google Scholar 

  26. Liu S, Dontu G, Mantle ID, Patel S, Ahn NS, Jackson KW, et al. Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res. 2006;66:6063–71.

    Article  PubMed  CAS  Google Scholar 

  27. Molofsky AV, Pardal R, Iwashita T, Park IK, Clarke MF, Morrison SJ. Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation. Nature. 2003;425:962–7.

    Article  PubMed  CAS  Google Scholar 

  28. Park IK, Qian D, Kiel M, Becker MW, Pihalja M, Weissman IL, et al. Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem cells. Nature. 2003;423:302–5.

    Article  PubMed  CAS  Google Scholar 

  29. Raaphorst FM. Self-renewal of hematopoietic and leukemic stem cells: a central role for the Polycomb-group gene Bmi-1. Trends Immunol. 2003;24:522–4.

    Article  PubMed  CAS  Google Scholar 

  30. Park I-K, Morrison SJ, Clarke MF. Bmi1, stem cells, and senescence regulation. J Clin Invest. 2004;113:175–9.

    PubMed  CAS  Google Scholar 

  31. Kim JH, Yoon SY, Jeong SH, Kim SY, Moon SK, Joo JH, et al. Overexpression of Bmi-1 oncoprotein correlates with axillary lymph node metastases in invasive ductal breast cancer. Breast. 2004;13:383–8.

    Article  PubMed  Google Scholar 

  32. Pietersen AM, Evers B, Prasad AA, Tanger E, Cornelissen-Steijger P, Jonkers J, et al. Bmi1 regulates stem cells and proliferation and differentiation of committed cells in mammary epithelium. Curr Biol. 2008;18:1094–9.

    Article  PubMed  CAS  Google Scholar 

  33. Saeki M, Kobayashi D, Tsuji N, Kuribayashi K, Watanabe N. Diagnostic importance of overexpression of Bmi-1 mRNA in early breast cancers. Int J Oncol. 2009;35:511–5.

    PubMed  CAS  Google Scholar 

  34. Guo BH, Feng Y, Zhang R, Xu LH, Li MZ, Kung HF, et al. Bmi-1 promotes invasion and metastasis, and its elevated expression is correlated with an advanced stage of breast cancer. Mol Cancer. 2011;10:10.

    Article  PubMed  CAS  Google Scholar 

  35. Riis ML, Luders T, Nesbakken AJ, Vollan HS, Kristensen V, Bukholm IR. Expression of BMI-1 and Mel-18 in breast tissue—a diagnostic marker in patients with breast cancer. BMC Cancer. 2010;10:686.

    Article  PubMed  Google Scholar 

  36. Shackleton M, Vaillant F, Simpson KJ, Stingl J, Smyth GK, Asselin-Labat ML, et al. Generation of a functional mammary gland from a single stem cell. Nature. 2006;439:84–8.

    Article  PubMed  CAS  Google Scholar 

  37. Stingl J, Eirew P, Ricketson I, Shackleton M, Vaillant F, Choi D, et al. Purification and unique properties of mammary epithelial stem cells. Nature. 2006;439:993–7.

    PubMed  CAS  Google Scholar 

  38. Buggiano V, Schere-Levy C, Abe K, Vanzulli S, Piazzon I, Smith GH, et al. Impairment of mammary lobular development induced by expression of TGFbeta1 under the control of WAP promoter does not suppress tumorigenesis in MMTV-infected transgenic mice. Int J Cancer. 2001;92:568–76.

    Article  PubMed  CAS  Google Scholar 

  39. Marquardt D. An algorithm for least-squares estimation of nonlinear parameters. SIAM J Appl Math. 1963;11:431–41.

    Article  Google Scholar 

  40. Bishop YM, Fienberg SE, Holland PW. Discrete multivariate analysis: theory and practice. New York: Springer Science + Business Media; 2007.

    Google Scholar 

  41. Sleeman KE, Kendrick H, Ashworth A, Isacke CM, Smalley MJ. CD24 staining of mouse mammary gland cells defines luminal epithelial, myoepithelial/basal and non-epithelial cells. Breast Cancer Res. 2006;8:R7.

    Article  PubMed  Google Scholar 

  42. Feng B, Chen L. Review of mesenchymal stem cells and tumors: executioner or coconspirator? Cancer Biother Radiopharm. 2009;24:717–21.

    Article  PubMed  CAS  Google Scholar 

  43. Kode JA, Mukherjee S, Joglekar MV, Hardikar AA. Mesenchymal stem cells: immunobiology and role in immunomodulation and tissue regeneration. Cytotherapy. 2009;11:377–91.

    Article  PubMed  CAS  Google Scholar 

  44. 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 

  45. Reinhard MC, Goltz HL, Warner SG, Mirand EA. Growth rate and percentage takes following inoculation of known numbers of viable mouse tumor cells. Exp Med Surg. 1952;10:254–8.

    PubMed  CAS  Google Scholar 

  46. Li A, Simmons PJ, Kaur P. Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci U S A. 1998;95:3902–7.

    Article  PubMed  CAS  Google Scholar 

  47. Stingl J, Caldas C. Molecular heterogeneity of breast carcinomas and the cancer stem cell hypothesis. Nat Rev Cancer. 2007;7:791–9.

    Article  PubMed  CAS  Google Scholar 

  48. Tani H, Morris RJ, Kaur P. Enrichment for murine keratinocyte stem cells based on cell surface phenotype. Proc Natl Acad Sci U S A. 2000;97:10960–5.

    Article  PubMed  CAS  Google Scholar 

  49. Ying M, Wang S, Sang Y, Sun P, Lal B, Goodwin CR, Guerrero-Cazares H, Quinones-Hinojosa A, Laterra J, Xia S. Regulation of glioblastoma stem cells by retinoic acid: role for Notch pathway inhibition. Oncogene. 2011.

  50. Redova M, Chlapek P, Loja T, Zitterbart K, Hermanova M, Sterba J, et al. Influence of LOX/COX inhibitors on cell differentiation induced by all-trans retinoic acid in neuroblastoma cell lines. Int J Mol Med. 2010;25:271–80.

    PubMed  CAS  Google Scholar 

  51. Nishioka C, Ikezoe T, Yang J, Gery S, Koeffler HP, Yokoyama A. Inhibition of mammalian target of rapamycin signaling potentiates the effects of all-trans retinoic acid to induce growth arrest and differentiation of human acute myelogenous leukemia cells. Int J Cancer. 2009;125:1710–20.

    Article  PubMed  CAS  Google Scholar 

  52. Li RJ, Ying X, Zhang Y, Ju RJ, Wang XX, Yao HJ, et al. All-trans retinoic acid stealth liposomes prevent the relapse of breast cancer arising from the cancer stem cells. J Control Release. 2011;149:281–91.

    Article  PubMed  CAS  Google Scholar 

  53. Levkoff LH, Marshall 2nd GP, Ross HH, Caldeira M, Reynolds BA, Cakiroglu M, et al. Bromodeoxyuridine inhibits cancer cell proliferation in vitro and in vivo. Neoplasia. 2008;10:804–16.

    PubMed  CAS  Google Scholar 

  54. Ross HH, Levkoff LH, Marshall 2nd GP, Caldeira M, Steindler DA, Reynolds BA, et al. Bromodeoxyuridine induces senescence in neural stem and progenitor cells. Stem Cells. 2008;26:3218–27.

    Article  PubMed  CAS  Google Scholar 

  55. Quintana E, Shackleton M, Sabel MS, Fullen DR, Johnson TM, Morrison SJ. Efficient tumour formation by single human melanoma cells. Nature. 2008;456:593–8.

    Article  PubMed  CAS  Google Scholar 

  56. Boiko AD, Razorenova OV, van de Rijn M, Swetter SM, Johnson DL, Ly DP, et al. Human melanoma-initiating cells express neural crest nerve growth factor receptor CD271. Nature. 2010;466:133–7.

    Article  PubMed  CAS  Google Scholar 

  57. Schatton T, Murphy GF, Frank NY, Yamaura K, Waaga-Gasser AM, Gasser M, et al. Identification of cells initiating human melanomas. Nature. 2008;451:345–9.

    Article  PubMed  CAS  Google Scholar 

  58. Callahan R, Smith GH. MMTV-induced mammary tumorigenesis: gene discovery, progression to malignancy and cellular pathways. Oncogene. 2000;19:992–1001.

    Article  PubMed  CAS  Google Scholar 

  59. Cardiff RD, Anver MR, Gusterson BA, Hennighausen L, Jensen RA, Merino MJ, et al. The mammary pathology of genetically engineered mice: the consensus report and recommendations from the Annapolis meeting. Oncogene. 2000;19:968–88.

    Article  PubMed  CAS  Google Scholar 

  60. Maglione JE, Moghanaki D, Young LJ, Manner CK, Ellies LG, Joseph SO, et al. Transgenic Polyoma middle-T mice model premalignant mammary disease. Cancer Res. 2001;61:8298–305.

    PubMed  CAS  Google Scholar 

  61. Glinsky GV, Berezovska O, Glinskii AB. Microarray analysis identifies a death-from-cancer signature predicting therapy failure in patients with multiple types of cancer. J Clin Invest. 2005;115:1503–21.

    Article  PubMed  CAS  Google Scholar 

  62. Wang Q, Li WL, You P, Su J, Zhu MH, Xie DF, et al. Oncoprotein BMI-1 induces the malignant transformation of HaCaT cells. J Cell Biochem. 2009;106:16–24.

    Article  PubMed  CAS  Google Scholar 

  63. Lukacs RU, Memarzadeh S, Wu H, Witte ON. Bmi-1 is a crucial regulator of prostate stem cell self-renewal and malignant transformation. Cell Stem Cell. 2010;7:682–93.

    Article  PubMed  CAS  Google Scholar 

  64. Cui H, Hu B, Li T, Ma J, Alam G, Gunning WT, et al. Bmi-1 is essential for the tumorigenicity of neuroblastoma cells. Am J Pathol. 2007;170:1370–8.

    Article  PubMed  CAS  Google Scholar 

  65. Jagani Z, Wiederschain D, Loo A, He D, Mosher R, Fordjour P, et al. The Polycomb group protein Bmi-1 is essential for the growth of multiple myeloma cells. Cancer Res. 2010;70:5528–38.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors would like to thank Andrew Reilly for his statistical analysis of the TI data.

Conflicts of interest

None

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Authors and Affiliations

  1. Translational and Functional Genomics Laboratory, Ordway Research Institute, Albany, NY, 12208, USA

    Jun Ma, Denise Grant Lanza, Anna Glinskii & Gennadi Glinsky

  2. Laboratory of Adult and Cancer Stem Cells, Ordway Research Institute, Albany, NY, 12208, USA

    Jun Ma, Denise Grant Lanza, Ian Guest & Stewart Sell

  3. Ordway Cancer Center and Flow Cytometry Core Facility, Ordway Research Institute, Albany, NY, 12208, USA

    Chang Uk-Lim

  4. Wadsworth Center, New York State Department of Health, Albany, NY, 12201, USA

    Stewart Sell

  5. Department of Biomedical Sciences, School of Public Health, University at Albany, State University of New York, Albany, NY, 12201, USA

    Denise Grant Lanza & Stewart Sell

  6. Department of Pathology and Laboratory Medicine, Albany Medical College, Albany, NY, 12208, USA

    Gennadi Glinsky

  7. Division of Urology, Department of Surgery, Albany Medical College, Albany, NY, 12208, USA

    Gennadi Glinsky

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  1. Jun Ma
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  2. Denise Grant Lanza
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  3. Ian Guest
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  4. Chang Uk-Lim
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  5. Anna Glinskii
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  6. Gennadi Glinsky
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  7. Stewart Sell
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Corresponding author

Correspondence to Stewart Sell.

Additional information

This study was supported by NIH R01 CA 112481 (Dr. Sell) and the Ordway Research Institute (Dr. Glinsky).

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Ma, J., Lanza, D.G., Guest, I. et al. Characterization of mammary cancer stem cells in the MMTV-PyMT mouse model. Tumor Biol. 33, 1983–1996 (2012). https://doi.org/10.1007/s13277-012-0458-4

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  • DOI: https://doi.org/10.1007/s13277-012-0458-4

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