Breast Cancer Research and Treatment

, Volume 144, Issue 3, pp 503–517 | Cite as

Primary breast tumor-derived cellular models: characterization of tumorigenic, metastatic, and cancer-associated fibroblasts in dissociated tumor (DT) cultures

  • Katherine Drews-Elger
  • Joeli A. Brinkman
  • Philip Miller
  • Sanket H. Shah
  • J. Chuck Harrell
  • Thiago G. da Silva
  • Zheng Ao
  • Amy Schlater
  • Diana J. Azzam
  • Kathleen Diehl
  • Dafydd Thomas
  • Joyce M. Slingerland
  • Charles M. Perou
  • Marc E. Lippman
  • Dorraya El-Ashry
Preclinical Study


Our goal was to establish primary cultures from dissociation of breast tumors in order to provide cellular models that may better recapitulate breast cancer pathogenesis and the metastatic process. Here, we report the characterization of six cellular models derived from the dissociation of primary breast tumor specimens, referred to as “dissociated tumor (DT) cells.” In vitro, DT cells were characterized by proliferation assays, colony formation assays, protein, and gene expression profiling, including PAM50 predictor analysis. In vivo, tumorigenic and metastatic potential of DT cultures was assessed in NOD/SCID and NSG mice. These cellular models differ from recently developed patient-derived xenograft models in that they can be used for both in vitro and in vivo studies. PAM50 predictor analysis showed DT cultures similar to their paired primary tumor and as belonging to the basal and Her2-enriched subtypes. In vivo, three DT cultures are tumorigenic in NOD/SCID and NSG mice, and one of these is metastatic to lymph nodes and lung after orthotopic inoculation into the mammary fat pad, without excision of the primary tumor. Three DT cultures comprised of cancer-associated fibroblasts (CAFs) were isolated from luminal A, Her2-enriched, and basal primary tumors. Among the DT cells are those that are tumorigenic and metastatic in immunosuppressed mice, offering novel cellular models of ER-negative breast cancer subtypes. A group of CAFs provide tumor subtype-specific components of the tumor microenvironment (TME). Altogether, these DT cultures provide closer-to-primary cellular models for the study of breast cancer pathogenesis, metastasis, and TME.


Primary cultures Tumors ER-negative breast cancer Metastatic xenograft models 



Dissociated tumor cells


Cancer-associated fibroblasts


Estrogen receptor alpha


Tumor microenvironment


Epithelial mesenchymal transition




Fibroblast activation protein


Alpha smooth muscle actin





This project was supported by the NIH Grant 1R01 CA113674 and The Bankhead Coley Cancer Research Program Pre SPORE Grant 09BW-04 to DEA. Award Number T32CA119929 from the National Cancer Institute supported KDE. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institute of Health. This project was also supported by the BCRF funds to MEL. The authors would like to thank Ms. Sonja Dean and Dr. Ayse Burcu Ergonul (University of Miami) and Dr. James Rae (University of Michigan) for valuable discussions.


  1. 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 100:3983–3988PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Amano H, Hayashi I, Endo H, Kitasato H, Yamashina S, Maruyama T et al (2003) Host prostaglandin E(2)-EP3 signaling regulates tumor-associated angiogenesis and tumor growth. J Exp Med 197:221–232PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Augsten M, Hagglof C, Olsson E, Stolz C, Tsagozis P, Levchenko T et al (2009) CXCL14 is an autocrine growth factor for fibroblasts and acts as a multi-modal stimulator of prostate tumor growth. Proc Natl Acad Sci USA 106:3414–3419PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Bastien RR, Rodriguez-Lescure A, Ebbert MT, Prat A, Munarriz B, Rowe L et al (2012) PAM50 breast cancer subtyping by RT-qPCR and concordance with standard clinical molecular markers. BMC Med Genomics 5:44PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Battula VL, Evans KW, Hollier BG, Shi Y, Marini FC, Ayyanan A et al (2010) Epithelial–mesenchymal transition-derived cells exhibit multilineage differentiation potential similar to mesenchymal stem cells. Stem Cells 28:1435–1445PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Bayliss J, Hilger A, Vishnu P, Diehl K, El-Ashry D (2007) Reversal of the estrogen receptor negative phenotype in breast cancer and restoration of antiestrogen response. Clin Cancer Res 13:7029–7036PubMedCrossRefGoogle Scholar
  7. 7.
    Bayraktar UD, Kim TK, Drews-Elger K, Benjamin C, El-Ashry D, Wieder E et al (2011) Simultaneous measurement of ERalpha, HER2, and phosphoERK1/2 in breast cancer cell lines by flow cytometry. Breast Cancer Res Treat 129:623–628PubMedCrossRefGoogle Scholar
  8. 8.
    Bhowmick NA, Neilson EG, Moses HL (2004) Stromal fibroblasts in cancer initiation and progression. Nature 432:332–337PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Boire A, Covic L, Agarwal A, Jacques S, Sherifi S, Kuliopulos A (2005) PAR1 is a matrix metalloprotease-1 receptor that promotes invasion and tumorigenesis of breast cancer cells. Cell 120:303–313PubMedCrossRefGoogle Scholar
  10. 10.
    Bos PD, Zhang XH, Nadal C, Shu W, Gomis RR, Nguyen DX et al (2009) Genes that mediate breast cancer metastasis to the brain. Nature 459:1005–1009PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Burdall SE, Hanby AM, Lansdown MR, Speirs V (2003) Breast cancer cell lines: friend or foe? Breast Cancer Res 5:89–95PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Cailleau R, Young R, Olive M, Reeves WJ Jr (1974) Breast tumor cell lines from pleural effusions. J Natl Cancer Inst 53:661–674PubMedGoogle Scholar
  13. 13.
    Carey LA, Dees EC, Sawyer L, Gatti L, Moore DT, Collichio F et al (2007) The triple negative paradox: primary tumor chemosensitivity of breast cancer subtypes. Clin Cancer Res 13:2329–2334PubMedCrossRefGoogle Scholar
  14. 14.
    Cheang MC, Voduc KD, Tu D, Jiang S, Leung S, Chia SK et al (2012) Responsiveness of intrinsic subtypes to adjuvant anthracycline substitution in the NCIC.CTG MA.5 randomized trial. Clin Cancer Res 18:2402–2412PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Fan C, Oh DS, Wessels L, Weigelt B, Nuyten DS, Nobel AB et al (2006) Concordance among gene-expression-based predictors for breast cancer. N Engl J Med 355:560–569PubMedCrossRefGoogle Scholar
  16. 16.
    Fantozzi A, Christofori G (2006) Mouse models of breast cancer metastasis. Breast Cancer Res 8:212PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Francia G, Cruz-Munoz W, Man S, Xu P, Kerbel RS (2011) Mouse models of advanced spontaneous metastasis for experimental therapeutics. Nat Rev Cancer 11:135–141PubMedCrossRefGoogle Scholar
  18. 18.
    Garin-Chesa P, Old LJ, Rettig WJ (1990) Cell surface glycoprotein of reactive stromal fibroblasts as a potential antibody target in human epithelial cancers. Proc Natl Acad Sci USA 87:7235–7239PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Hanahan D, Coussens LM (2012) Accessories to the crime: functions of cells recruited to the tumor microenvironment. Cancer Cell 21:309–322PubMedCrossRefGoogle Scholar
  20. 20.
    Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674PubMedCrossRefGoogle Scholar
  21. 21.
    Herschkowitz JI, Simin K, Weigman VJ, Mikaelian I, Usary J, Hu Z et al (2007) Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors. Genome Biol 8:R76PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Hess KR, Pusztai L, Buzdar AU, Hortobagyi GN (2003) Estrogen receptors and distinct patterns of breast cancer relapse. Breast Cancer Res Treat 78:105–118PubMedCrossRefGoogle Scholar
  23. 23.
    Honeth G, Bendahl PO, Ringner M, Saal LH, Gruvberger-Saal SK, Lovgren K et al (2008) The CD44+/CD24 phenotype is enriched in basal-like breast tumors. Breast Cancer Res 10:R53PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Hu Z, Fan C, Oh DS, Marron JS, He X, Qaqish BF et al (2006) The molecular portraits of breast tumors are conserved across microarray platforms. BMC Genom 7:96CrossRefGoogle Scholar
  25. 25.
    Ikeda Y, Hayashi I, Kamoshita E, Yamazaki A, Endo H, Ishihara K et al (2004) Host stromal bradykinin B2 receptor signaling facilitates tumor-associated angiogenesis and tumor growth. Cancer Res 64:5178–5185PubMedCrossRefGoogle Scholar
  26. 26.
    Iorns E, Drews-Elger K, Ward TM, Dean S, Clarke J, Berry D et al (2012) A new mouse model for the study of human breast cancer metastasis. PLoS ONE 7:e47995PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Jeong H, Ryu YJ, An J, Lee Y, Kim A (2012) Epithelial–mesenchymal transition in breast cancer correlates with high histological grade and triple-negative phenotype. Histopathology 60:E87–E95PubMedCrossRefGoogle Scholar
  28. 28.
    Jessani N, Humphrey M, McDonald WH, Niessen S, Masuda K, Gangadharan B et al (2004) Carcinoma and stromal enzyme activity profiles associated with breast tumor growth in vivo. Proc Natl Acad Sci USA 101:13756–13761PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Kalluri R, Zeisberg M (2006) Fibroblasts in cancer. Nat Rev Cancer 6:392–401PubMedCrossRefGoogle Scholar
  30. 30.
    Kao J, Salari K, Bocanegra M, Choi YL, Girard L, Gandhi J et al (2009) Molecular profiling of breast cancer cell lines defines relevant tumor models and provides a resource for cancer gene discovery. PLoS ONE 4:e6146PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell GW et al (2007) Mesenchymal stem cells within tumour stroma promote breast cancer metastasis. Nature 449:557–563PubMedCrossRefGoogle Scholar
  32. 32.
    Kokkinos MI, Wafai R, Wong MK, Newgreen DF, Thompson EW, Waltham M (2007) Vimentin and epithelial–mesenchymal transition in human breast cancer—observations in vitro and in vivo. Cells Tissues Organs 185:191–203PubMedCrossRefGoogle Scholar
  33. 33.
    Kuperwasser C, Chavarria T, Wu M, Magrane G, Gray JW, Carey L et al (2004) Reconstruction of functionally normal and malignant human breast tissues in mice. Proc Natl Acad Sci USA 101:4966–4971PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD et al (2005) Genes that mediate breast cancer metastasis to lung. Nature 436:518–524PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Minn AJ, Kang Y, Serganova I, Gupta GP, Giri DD, Doubrovin M et al (2005) Distinct organ-specific metastatic potential of individual breast cancer cells and primary tumors. J Clin Invest 115:44–55PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Minn AJ, Gupta GP, Padua D, Bos P, Nguyen DX, Nuyten D et al (2007) Lung metastasis genes couple breast tumor size and metastatic spread. Proc Natl Acad Sci USA 104:6740–6745PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Mueller MM, Fusenig NE (2004) Friends or foes—bipolar effects of the tumour stroma in cancer. Nat Rev Cancer 4:839–849PubMedCrossRefGoogle Scholar
  38. 38.
    Nanni P, Nicoletti G, Palladini A, Croci S, Murgo A, Ianzano ML et al (2012) Multiorgan metastasis of human HER-2+ breast cancer in Rag2−/−; Il2rg−/− mice and treatment with PI3K inhibitor. PLoS ONE 7:e39626PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Orimo A, Weinberg RA (2006) Stromal fibroblasts in cancer: a novel tumor-promoting cell type. Cell Cycle 5:1597–1601PubMedCrossRefGoogle Scholar
  40. 40.
    Orimo A, Gupta PB, Sgroi DC, Arenzana-Seisdedos F, Delaunay T, Naeem R et al (2005) Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 121:335–348PubMedCrossRefGoogle Scholar
  41. 41.
    O’Toole SA, Beith JM, Millar EK, West R, McLean A, Cazet A et al (2013) Therapeutic targets in triple negative breast cancer. J Clin Pathol 66:530–542PubMedCrossRefGoogle Scholar
  42. 42.
    Parker JS, Mullins M, Cheang MC, Leung S, Voduc D, Vickery T et al (2009) Supervised risk predictor of breast cancer based on intrinsic subtypes. J Clin Oncol 27:1160–1167PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA et al (2000) Molecular portraits of human breast tumours. Nature 406:747–752PubMedCrossRefGoogle Scholar
  44. 44.
    Prat A, Parker JS, Karginova O, Fan C, Livasy C, Herschkowitz JI et al (2010) Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res 12:R68PubMedCentralPubMedCrossRefGoogle Scholar
  45. 45.
    Price JE, Polyzos A, Zhang RD, Daniels LM (1990) Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice. Cancer Res 50:717–721PubMedGoogle Scholar
  46. 46.
    Radisky DC, Kenny PA, Bissell MJ (2007) Fibrosis and cancer: do myofibroblasts come also from epithelial cells via EMT? J Cell Biochem 101:830–839PubMedCentralPubMedCrossRefGoogle Scholar
  47. 47.
    R-Core-Team (2013) R: a language and environment for statistical computing. R Foundation for Statistical Computing, ViennaGoogle Scholar
  48. 48.
    Rouzier R, Perou CM, Symmans WF, Ibrahim N, Cristofanilli M, Anderson K et al (2005) Breast cancer molecular subtypes respond differently to preoperative chemotherapy. Clin Cancer Res 11:5678–5685PubMedCrossRefGoogle Scholar
  49. 49.
    Sarrio D, Rodriguez-Pinilla SM, Hardisson D, Cano A, Moreno-Bueno G, Palacios J (2008) Epithelial–mesenchymal transition in breast cancer relates to the basal-like phenotype. Cancer Res 68:989–997PubMedCrossRefGoogle Scholar
  50. 50.
    Shekhar MP, Santner S, Carolin KA, Tait L (2007) Direct involvement of breast tumor fibroblasts in the modulation of tamoxifen sensitivity. Am J Pathol 170:1546–1560PubMedCentralPubMedCrossRefGoogle Scholar
  51. 51.
    Siegel R, Naishadham D, Jemal A (2012) Cancer statistics, 2012. CA Cancer J Clin 62:10–29PubMedCrossRefGoogle Scholar
  52. 52.
    Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H et al (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 98:10869–10874PubMedCentralPubMedCrossRefGoogle Scholar
  53. 53.
    Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A et al (2003) Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci USA 100:8418–8423PubMedCentralPubMedCrossRefGoogle Scholar
  54. 54.
    Spaeth EL, Labaff AM, Toole BP, Klopp A, Andreeff M, Marini FC (2013) Mesenchymal CD44 expression contributes to the acquisition of an activated fibroblast phenotype via TWIST activation in the tumor microenvironment. Cancer Res 73:5347–5359PubMedCrossRefGoogle Scholar
  55. 55.
    Speirs V, Green AR, Walton DS, Kerin MJ, Fox JN, Carleton PJ et al (1998) Short-term primary culture of epithelial cells derived from human breast tumours. Br J Cancer 78:1421–1429PubMedCentralPubMedCrossRefGoogle Scholar
  56. 56.
    Sternlicht MD, Lochter A, Sympson CJ, Huey B, Rougier JP, Gray JW et al (1999) The stromal proteinase MMP3/stromelysin-1 promotes mammary carcinogenesis. Cell 98:137–146PubMedCentralPubMedCrossRefGoogle Scholar
  57. 57.
    Straussman R, Morikawa T, Shee K, Barzily-Rokni M, Qian ZR, Du J et al (2012) Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature 487:500–504PubMedCentralPubMedCrossRefGoogle Scholar
  58. 58.
    Tomaskovic-Crook E, Thompson EW, Thiery JP (2009) Epithelial to mesenchymal transition and breast cancer. Breast Cancer Res 11:213PubMedCentralPubMedCrossRefGoogle Scholar
  59. 59.
    Weigelt B, Peterse JL, van’t Veer LJ (2005) Breast cancer metastasis: markers and models. Nat Rev Cancer 5:591–602PubMedCrossRefGoogle Scholar
  60. 60.
    Williams CS, Tsujii M, Reese J, Dey SK, DuBois RN (2000) Host cyclooxygenase-2 modulates carcinoma growth. J Clin Invest 105:1589–1594PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Katherine Drews-Elger
    • 1
  • Joeli A. Brinkman
    • 1
  • Philip Miller
    • 1
    • 2
  • Sanket H. Shah
    • 1
    • 2
  • J. Chuck Harrell
    • 3
  • Thiago G. da Silva
    • 1
    • 4
  • Zheng Ao
    • 1
  • Amy Schlater
    • 5
  • Diana J. Azzam
    • 1
  • Kathleen Diehl
    • 6
  • Dafydd Thomas
    • 7
  • Joyce M. Slingerland
    • 1
  • Charles M. Perou
    • 3
  • Marc E. Lippman
    • 1
  • Dorraya El-Ashry
    • 1
  1. 1.Sylvester Comprehensive Cancer Center, Department of MedicineUniversity of Miami Miller School of MedicineMiamiUSA
  2. 2.Sheila and David Fuente Graduate Program in Cancer BiologyUniversity of Miami Miller School of MedicineMiamiUSA
  3. 3.Department of GeneticsUniversity of North Carolina at Chapel HillChapel HillUSA
  4. 4.Department of SurgeryUniversity of Miami Miller School of MedicineMiamiUSA
  5. 5.Division of Hematology/Oncology, Department of Internal MedicineUniversity of Michigan Medical CenterAnn ArborUSA
  6. 6.Department of SurgeryUniversity of Michigan Medical CenterAnn ArborUSA
  7. 7.Department of PathologyUniversity of Michigan Medical CenterAnn ArborUSA

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