, Volume 63, Issue 1, pp 67–80 | Cite as

Long-term cultures of stem/progenitor cells from lobular and ductal breast carcinomas under non-adherent conditions

  • Agostina Nardone
  • Sara Corvigno
  • Annalisa Brescia
  • Daniel D’Andrea
  • Gennaro Limite
  • Bianca Maria VenezianiEmail author
Original Research


A small subpopulation of stem/progenitor cells can give rise to the diversity of differentiated cells that comprise the bulk of the tumor. Are proliferating cells, within the bulk of tumor, few cells with uncommon features? The cell biological approach provides a limitless model for studying the hierarchical organization of progenitor subpopulation and identifying potential therapeutic targets. Aim of the study was to expand patients’ breast cancer cells for evaluating functional cell properties, and to characterize the protein expression profile of selected cells to be compared with that of primary tumors. Breast cancer cells from estrogen receptor (ERα) positive, HER2 negative lobular (LoBS cells) and ductal (DuBS cells) histotype were cultured under non-adherent conditions to form mammospheres. Sorting of the cells by their surface expression of CD24 and CD44 gave rise to subpopulations which were propagated, enriched and characterized for the expression of epithelial and stromal markers. We found that non-adherent culture conditions generate mammospheres of slowly proliferating cells; single cells, dissociated from mammospheres, grow in soft agar; long-term cultured LoBS and DuBS cells, CD44+/CD24low, express cytokeratin 5 (CK5), α-smooth muscle actin (α-sma) and vimentin, known as markers of basal/myoepithelial cells; and ERα (only DuBS cells), HER1 (EGF-Receptor), activated HER2, and cyclinD1 as markers of luminal epithelial cell. Isolates of cells from breast cancer patients may be a tool for a marker-driven testing of targeted therapies.


Breast cancer therapy Epithelial stromal cells 



We declare no conflict of interest. This work was supported by: Associazione Italiana per la Ricerca sul Cancro, CRPO Centro Regionale di Prevenzione Oncologica, Regione Campania, Ministero dell’Universita` e Ricerca, and Ministero della Salute, Italia.


  1. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 100:3983–3988CrossRefGoogle Scholar
  2. Allred DC (2008) The utility of conventional and molecular pathology in managing breast cancer. Breast Cancer Res 10 (suppl 4):S4Google Scholar
  3. Anbazhagan R, Osin PP, Bartkova J, Nathan B, Lane EB, Gusterson BA (1998) The development of epithelial phenotypes in the human fetal and infant breast. J Pathol 184:197–206CrossRefGoogle Scholar
  4. Asselin-Labat ML, Vaillant F, Shackleton M, Bouras T, Lindeman GJ, Visvader JE (2008) Delineating the epithelial hierarchy in the mouse mammary gland. Cold Spring Harb Symp Quant Biol 73:469–478CrossRefGoogle Scholar
  5. Bartek J, Taylor-Papadimitriou J, Miller N, Millis R (1985) Patterns of expression of keratin 19 as detected with monoclonal antibodies in human breast tissues and tumours. Int J Cancer 36:299–306Google Scholar
  6. Booth BW, Smith GH (2006) Estrogen receptor-alpha and progesterone receptor are expressed in label-retaining mammary epithelial cells that divide asymmetrically and retain their template DNA strands. Breast Cancer Res 8:R49Google Scholar
  7. Brisken C, Duss S (2007) Stem cells and the stem cell niche in the breast: an integrated hormonal and developmental perspective. Stem Cell Rev 3:147–156CrossRefGoogle Scholar
  8. Campbell LL, Polyak K (2007) Breast tumor heterogeneity: cancer stem cells or clonal evolution? Cell Cycle 37:2332–2338CrossRefGoogle Scholar
  9. Cavaliere C, Corvigno S, Galgani M, Limite G, Nardone A, Veneziani BM (2010) Combined inhibitory effect of formestane and herceptin on a subpopulation of CD44+/CD24low breast cancer cells. Cancer Sci 107:1661–1669CrossRefGoogle Scholar
  10. Chen ST, Dou J, Temple R, Agarwal R, Wu KM, Walker S (2008) New therapies from old medicines. Nat Biotechnol 26:1077–1083CrossRefGoogle Scholar
  11. Clarke RB, Spence K, Anderson E, Howell A, Okano H, Potten CS (2005) A putative human breast stem cell population is enriched for steroid receptor-positive cells. Dev Biol 277:443–456CrossRefGoogle Scholar
  12. Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CHM, Jones DL, Visvader J, Weissman IL, Wahl GM (2006) Cancer stem cells-perspectives on current status and future directions: AACR workshop on cancer stem cells. Cancer Res 66:9339–9344CrossRefGoogle Scholar
  13. Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ (2005) Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res 65:10946–10951CrossRefGoogle Scholar
  14. Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, Wicha MS (2003) In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 17:1253–1270CrossRefGoogle Scholar
  15. Dowsett M, Houghton J, Iden C, Salter J, Farndon J, A’Hern R, Sainsbury R, Baum M (2006) Benefit from adjuvant tamoxifen therapy in primary breast cancer patients according oestrogen receptor, progesterone receptor, EGF receptor and HER2 status. Ann Oncol 17:818–826CrossRefGoogle Scholar
  16. Frogne T, Laenkholm AV, Lyng MB, Henriksen KL, Lykkesfeldt AE (2009) Determination of HER2 phosphorylation at tyrosine 1221/1222 improves prediction of poor survival for breast cancer patients with hormone receptor-positive tumors. Breast Cancer Res 11:R11CrossRefGoogle Scholar
  17. Garbe JC, Bhattacharya S, Merchant B, Bassett E, Swisshelm K, Feiler HS, Wyrobek AJ, Stampfer MR (2009) Molecular distinctions between stasis and telomere attrition senescence barriers shown by long-term culture of normal human mammary epithelial cells. Cancer Res 69:7557–7568CrossRefGoogle Scholar
  18. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, Jacquemier J, Viens P, Kleer CG, Liu S, Schott A, Hayes D, Birnbaum D, Wicha MS, Dontu G (2007) ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1:555–567CrossRefGoogle Scholar
  19. Gusterson BA, Warburton MJ, Mitchell D, Ellison M, Neville AM, Rudland PS (1982) Distribution of myoepithelial cells and basement membrane proteins in the normal breast and in benign and malignant breast disease. Cancer Res 42:4763–4770Google Scholar
  20. Gusterson BA, Ross DT, Heath VJ, Stein T (2005) Basal cytokeratins and their relationship to the cellular origin and functional classification of breast cancer. Breast Cancer Res 7:143–148CrossRefGoogle Scholar
  21. Hammond ME, Hayes DF, Dowsett M, Allred DC, Hagerty KL, Badve S, Fitzgibbons PL, Francis G, Goldstein NS, Hayes M, Hicks DG, Lester S, Love R, Mangu PB, McShane L, Miller K, Osborne CK, Paik S, Perlmutter J, Rhodes A, Sasano H, Schwartz JN, Sweep FC, Taube S, Torlakovic EE, Valenstein P, Viale G, Visscher D, Wheeler T, Williams RB, Wittliff JL, Wolff AC (2010) American society of clinical oncology/College of American pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer. Arch Pathol Lab Med 134:907–922Google Scholar
  22. Honeth G, Bendahl PO, Ringnér M, Saal LH, Gruvberger-Saal SK, Lövgren K, Grabau D, Fernö M, Borg A, Hegardt C (2008) The CD44+/CD24- phenotype is enriched in basal-like breast tumors. Breast Cancer Res 10:R53CrossRefGoogle Scholar
  23. Johnston S, Pippen J Jr, Pivot X, Lichinitser M, Sadeghi S, Dieras V, Gomez HL, Romieu G, Manikhas A, Kennedy MJ, Press MF, Maltzman J, Florance A, O’Rourke L, Oliva C, Stein S, Pegram M (2009) Lapatinib combined with letrozole versus letrozole and placebo as first-line therapy for postmenopausal hormone receptor-positive metastatic breast cancer. J Clin Oncol 27:5538–5546CrossRefGoogle Scholar
  24. Kaufman B, Mackey JR, Clemens MR, Bapsy PP, Vaid A, Wardley A, Tjulandin S, Jahn M, Lehle M, Feyereislova A, Révil C, Jones A (2009) Trastuzumab plus anastrozole versus anastrozole alone for the treatment of postmenopausal women with human epidermal growth factor receptor 2-positive, hormone receptor-positive metastatic breast cancer: results from the randomized phase III TAnDEM study. J Clin Oncol 27:5529–5537CrossRefGoogle Scholar
  25. Knowlden JM, Hutcheson IR, Jones HE, Madden T, Gee JM, Harper ME, Barrow D, Wakeling AE, Nicholson RI (2003) Elevated levels of epidermal growth factor receptor/c-erbB2 heterodimers mediate an autocrine growth regulatory pathway in tamoxifen-resistant MCF-7 cells. Endocrinology 144:1032–1044CrossRefGoogle Scholar
  26. Korkaya H, Paulson A, Iovino F, Wicha MS (2008) HER2 regulates the mammary stem/progenitor cell population driving tumorigenesis and invasion. Oncogene 27:6120–6130CrossRefGoogle Scholar
  27. Lim E, Vaillant F, Wu D, Forrest NC, Pal B, Hart AH, Asselin-Labat ML, Gyorki DE, Ward T, Partanen A, Feleppa F, Huschtscha LI, Thorne HJ, ConFab FoxSB, Yan M, French JD, Brown MA, Smyth GK, Visvader JE, Lindeman GJ (2009) Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers. Nat Med 15:907–913CrossRefGoogle Scholar
  28. Massarweh S, Osborne CK, Creighton CJ, Qin L, Tsimelzon A, Huang S, Weiss H, Rimawi M, Schiff R (2008) Tamoxifen resistance in breast tumors is driven by growth factor receptor signaling with repression of classic estrogen receptor genomic function. Cancer Res 68:826–833CrossRefGoogle Scholar
  29. Moll R, Franke WW, Schiller DL (1982) The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31:11–24CrossRefGoogle Scholar
  30. Nagle RB, Bocker W, Davis JR, Heid HW, Kaufmann M, Lucas DO, Jarasch ED (1986) Characterization of breast carcinomas by two monoclonal antibodies distinguishing myoepithelial from luminal epithelial cells. J Histochem Cytochem 34:869–881Google Scholar
  31. Nardone A, Cavaliere C, Corvigno S, Limite G, De Placido S, Veneziani BM (2009) A banking strategy toward customized therapy in breast cancer. Cell Tissue Bank 10:301–308CrossRefGoogle Scholar
  32. Oliveira LR, Jeffrey SS, Ribeiro-Silva A (2010) Stem cells in human breast cancer. Histol Histopathol 25:371–385Google Scholar
  33. Osborne CK, Shou J, Massarweh S, Schiff R (2005) Crosstalk between estrogen receptor and growth factor receptor pathways as a cause for endocrine therapy resistance in breast cancer. Clin Cancer Res 11:865s–870sGoogle Scholar
  34. Paik S, Kim C, Wolmark N (2008) HER2 status and benefit from adjuvant trastuzumab in breast cancer. N Engl J Med 358:1409–1411CrossRefGoogle Scholar
  35. Park SY, Gönen M, Kim HJ, Michor F, Polyak K (2010) Cellular genetic diversity in the progression of in situ human breast carcinomas to an invasive phenotype. J Clin Invest 120:636–644CrossRefGoogle Scholar
  36. Pece S, Tosoni D, Confalonieri S, Mazzarol G, Vecchi M, Ronzoni S, Bernard L, Viale G, Pelicci PG, Di Fiore PP (2010) Biological and molecular heterogeneity of breast cancers correlates with their cancer stem cell content. Cell 140:62–73CrossRefGoogle Scholar
  37. Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, Fluge O, Pergamenschikov A, Williams C, Zhu SX, Lonning PE, Borresen-Dale AL, Brown PO, Botstein D (2000) Molecular portraits of human breast tumours. Nature 406:747–752CrossRefGoogle Scholar
  38. Petersen OW, Polyak K (2010) Stem cells in the human breast. Cold Spring Harb Perspect Biol 2. doi:  10.1101/cshperspect.a003160
  39. Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D, Pilotti S, Pierotti MA, Daidone MG (2005) Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 65:5506–5511CrossRefGoogle Scholar
  40. Ronnov-Jessen L, Petersen OW, Koteliansky VE, Bissell MJ (1995) The origin of the myofibroblasts in breast cancer. Recapitulation of tumor environment in culture unravels diversity and implicates converted fibroblasts and recruited smooth muscle cells. J Clin Invest 95:859–873CrossRefGoogle Scholar
  41. Schiff R, Osborne CK (2005) Endocrinology and hormone therapy in breast cancer: new insight into estrogen receptor-alpha function and its implication for endocrine therapy resistance in breast cancer. Breast Cancer Res 7:205–211CrossRefGoogle Scholar
  42. Shackleton M, Vaillant F, Simpson KJ, Stingl J, Smyth GK, Asselin-Labat ML, Wu L, Lindeman GJ, Visvader JE (2006) Generation of a functional mammary gland from a single stem cell. Nature 439:84–88CrossRefGoogle Scholar
  43. Shipitsin M, Campbell LL, Argani P, Weremowicz S, Bloushtain-Qimron N, Yao J, Nikolskaya T, Serebryiskaya T, Beroukhim R, Hu M, Halushka MK, Sukumar S, Parker LM, Anderson KS, Harris LN, Garber JE, Richardson AL, Schnitt SJ, Nikolsky Y, Gelman RS, Polyak K (2007) Molecular definition of breast tumor heterogeneity. Cancer Cell 11:259–273CrossRefGoogle Scholar
  44. Singer CF, Gschwantler-Kaulich D, Fink-Retter A, Pfeiler G, Walter I, Hudelist G, Helmy S, Spiess AC, Lamm W, Kubista E (2009) HER2 overexpression and activation, and tamoxifen efficacy in receptor-positive early breast cancer. J Cancer Res Clin Oncol 135:807–813CrossRefGoogle Scholar
  45. Sleeman KE, Kendrick H, Ashworth A, Isacke CM, Smalley MJ (2006) CD24 staining of mouse mammary gland cells defines luminal epithelial, myoepithelial/basal and non-epithelial cells. Breast Cancer Res 8:R7CrossRefGoogle Scholar
  46. Sleeman KE, Kendrick H, Robertson D, Isacke CM, Ashworth A, Smalley MJ (2007) Dissociation of estrogen receptor expression and in vivo stem cell activity in the mammary gland. J Cell Biol 176:19–26CrossRefGoogle Scholar
  47. Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van de Rijn M, Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein D, Eystein Lonning P, Borresen-Dale AL (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 98:10869–10874CrossRefGoogle Scholar
  48. Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, Deng S, Johnsen H, Pesich R, Geisler S, Demeter J, Perou CM, Lønning PE, Brown PO, Børresen-Dale AL, Botstein D (2003) Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci USA 100:8418–8423 Google Scholar
  49. Sotiriou C, Neo SY, McShane LM, Korn EL, Long PM, Jazaeri A, Martiat P, Fox SB, Harris AL, Liu ET (2003) Breast cancer classification and prognosis based on gene expression profiles from a population-based study. Proc Natl Acad Sci USA 100:10393–10398 Google Scholar
  50. Stingl J, Eirew P, Ricketson I, Shackleton M, Vaillant F, Choi D, Li HI, Eaves CJ (2006) Purification and unique properties of mammary epithelial stem cells. Nature 439:993–997Google Scholar
  51. Suzuki T, Miki Y, Ohuchi N, Sasano H (2008) Intratumoral estrogen production in breast carcinoma: significance of aromatase. Breast Cancer 15:270–277CrossRefGoogle Scholar
  52. van de Vijver MJ, He YD, van’t Veer LJ, Dai H, Hart AA, Voskuil DW, Schreiber GJ, Peterse JL, Roberts C, Marton MJ, Parrish M, Atsma D, Witteveen A, Glas A, Delahaye L, van der Velde T, Bartelink H, Rodenhuis S, Rutgers ET, Friend SH, Bernards R (2002) A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med 347:1999–2009Google Scholar
  53. Veneziani BM, Criniti V, Cavaliere C, Corvigno S, Nardone A, Picarelli S, Tortora G, Ciardiello F, Limite G, De Placido S (2007) In vitro expansion of human breast cancer epithelial and mesenchymal stromal cells: optimization of a coculture model for personalized therapy approaches. Mol Cancer Ther 6:3091–3100CrossRefGoogle Scholar
  54. Villadsen R, Fridriksdottir AJ, Rønnov-Jessen L, Gudjonsson T, Rank F, Labarge MA, Bissell MJ, Petersen OW (2007) Evidence for a stem cell hierachy in the adult human breast. J Cell Biol 177:87–101CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Agostina Nardone
    • 1
  • Sara Corvigno
    • 1
  • Annalisa Brescia
    • 1
  • Daniel D’Andrea
    • 1
  • Gennaro Limite
    • 2
  • Bianca Maria Veneziani
    • 1
    • 3
    Email author
  1. 1.Dipartimento di Biologia e Patologia Cellulare e Molecolare “L. Califano”Università di Napoli Federico IINaplesItaly
  2. 2.Dipartimento di Chirurgia Generale Oncologica e Tecnologie AvanzateUniversità degli Studi di Napoli Federico IINaplesItaly
  3. 3.Oncotech, Facoltà di Medicina e ChirurgiaUniversità degli Studi di Napoli Federico IINaplesItaly

Personalised recommendations