Cancer Immunology, Immunotherapy

, Volume 58, Issue 11, pp 1887–1896 | Cite as

Breast tumor cells isolated from in vitro resistance to trastuzumab remain sensitive to trastuzumab anti-tumor effects in vivo and to ADCC killing

  • Timothy E. Kute
  • Lori Savage
  • John R. StehleJr
  • Jung W. Kim-Shapiro
  • Michael J. Blanks
  • James Wood
  • James P. Vaughn
Original article


An understanding of model systems of trastuzumab (Herceptin) resistance is of great importance since the humanized monoclonal antibody is now used as first line therapy with paclitaxel in patients with metastatic Her2 overexpressing breast cancer, and the majority of their tumors has innate resistance or develops acquired resistance to the treatment. Previously, we selected trastuzumab-resistant clonal cell lines in vitro from trastuzumab-sensitive parental BT-474 cells and showed that cloned trastuzumab-resistant cell lines maintain similar levels of the extracellular Her2 receptor, bind trastuzumab as efficiently as the parental cells, but continue to grow in the presence of trastuzumab and display cell cycle profiles and growth rates comparable to parental cells grown in the absence of trastuzumab (Kute et al. in Cytometry A 57:86–93, 2004). We now show that trastuzumab-resistant and trastuzumab-sensitive cells both surprisingly display trastuzumab-mediated growth inhibition in athymic nude mice. This demonstrates that resistance developed in vitro is not predictive of resistance in vivo. The observation that in vitro resistant cells are sensitive to trastuzumab in vivo could be explained by antibody dependant cellular cytotoxicity (ADCC). Therefore, both parental and trastuzumab-resistant cells were assayed for ADCC in real time on electroplates with and without trastuzumab in the presence of a natural killer cell line (NK-92), and granulocyte or mononuclear cellular fractions isolated from human peripheral blood. Mononuclear cells and NK-92 cells were more effective in killing both parental and trastuzumab-resistant cells in the presence of trastuzumab. Both trastuzumab-resistant cells and trastuzumab-sensitive cells showed similar susceptibility to ADCC despite displaying divergent growth responses to trastuzumab. The granulocyte fraction was able to kill these cells with equal efficacy in the presence or absence of trastuzumab. These results support a model of trastuzumab tumor cell killing in vivo mediated primarily by ADCC from the mononuclear fraction of innate immune cells and suggest that in the clinical setting not only should changes in signaling transduction pathways be studied in acquired tumor resistance to trastuzumab, but also mechanisms by which tumors impede immune function should be evaluated.


ADCC Trastuzumab Herceptin Breast cancer Resistance BT-474 



This work was performed from a grant from the Vaughn Jordon Foundation and from support of the pathology department and Wake Forest University Cancer Center. We would also like to thank Ms Debra Holder who help with the editing of the manuscript and Dr. Mark Willingham and Dr. Zheng Cui who provided comments and suggestions. Finally, I would like to thank Dr. Greg Russell for his help with the statistics.


  1. 1.
    Barok M, Isola J, Palyi-Krekk Z, Nagy P, Juhasz I, Vereb G, Kauraniemi P, Kapanen A, Tanner M, Vereb G, Szollosi J (2007) Trastuzumab causes antibody-dependent cellular cytotoxicity-mediated growth inhibition of submacroscopic JIMT-1 breast cancer xenografts despite intrinsic drug resistance. Mol Cancer Ther 6:2065–2072PubMedCrossRefGoogle Scholar
  2. 2.
    Burris HIII, Yardley D, Jones S, Houston G, Broome C, Thompson D, Greco FA, White M, Hainsworth J (2004) Phase II trial of trastuzumab followed by weekly paclitaxel/carboplatin as first-line treatment for patients with metastatic breast cancer. J Clin Oncol 22:1621–1629PubMedCrossRefGoogle Scholar
  3. 3.
    Burstein HJ, Harris LN, Gelman R, Lester SC, Nunes RA, Kaelin CM, Parker LM, Ellisen LW, Kuter I, Gadd MA, Christian RL, Kennedy PR, Borges VF, Bunnell CA, Younger J, Smith BL, Winer EP (2003) Preoperative therapy with trastuzumab and paclitaxel followed by sequential adjuvant doxorubicin/cyclophosphamide for HER2 overexpressing stage II or III breast cancer: a pilot study. J Clin Oncol 21:46–53PubMedCrossRefGoogle Scholar
  4. 4.
    Carter P, Presta L, Gorman CM, Ridgway JB, Henner D, Wong WL, Rowland AM, Kotts C, Carver ME, Shepard HM (1992) Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci USA 89:4285–4289PubMedCrossRefGoogle Scholar
  5. 5.
    Cartron G, Zhao-Yang L, Baudard M, Kanouni T, Rouille V, Quittet P, Klein B, Rossi JF (2008) Granulocyte-macrophage colony-stimulating factor potentiates rituximab in patients with relapsed follicular lymphoma: results of a phase II study. J Clin Oncol 26:2725–2731PubMedCrossRefGoogle Scholar
  6. 6.
    Citri A, Yarden Y (2006) EGF-ERBB signalling: towards the systems level. Nat Rev Mol Cell Biol 7:505–516PubMedCrossRefGoogle Scholar
  7. 7.
    Clynes RA, Towers TL, Presta LG, Ravetch JV (2000) Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat Med 6:443–446PubMedCrossRefGoogle Scholar
  8. 8.
    Drugger DL, Hollingshead PG, Wong D, Romero M, Erickson SL, RH S (2002) Acquistion of Herceptin resistance by HER2 mutation in the HER2 transgenic mouse breast cancer model. Proceedings of the 93rd Annual Meeting of the American Association Cancer Research, San FranciscoGoogle Scholar
  9. 9.
    Gennari R, Menard S, Fagnoni F, Ponchio L, Scelsi M, Tagliabue E, Castiglioni F, Villani L, Magalotti C, Gibelli N, Oliviero B, Ballardini B, Da Prada G, Zambelli A, Costa A (2004) Pilot study of the mechanism of action of preoperative trastuzumab in patients with primary operable breast tumors overexpressing HER2. Clin Cancer Res 10:5650–5655PubMedCrossRefGoogle Scholar
  10. 10.
    Glamann J, Hansen AJ (2006) Dynamic detection of natural killer cell-mediated cytotoxicity and cell adhesion by electrical impedance measurements. Assay Drug Dev Technol 4:555–563PubMedCrossRefGoogle Scholar
  11. 11.
    Klapper LN, Waterman H, Sela M, Yarden Y (2000) Tumor-inhibitory antibodies to HER-2/ErbB-2 may act by recruiting c-Cbl and enhancing ubiquitination of HER-2. Cancer Res 60:3384–3388PubMedGoogle Scholar
  12. 12.
    Klos KS, Zhou X, Lee S, Zhang L, Yang W, Nagata Y, Yu D (2003) Combined trastuzumab and paclitaxel treatment better inhibits ErbB-2-mediated angiogenesis in breast carcinoma through a more effective inhibition of Akt than either treatment alone. Cancer 98:1377–1385PubMedCrossRefGoogle Scholar
  13. 13.
    Kostler WJ, Schwab B, Singer CF, Neumann R, Rucklinger E, Brodowicz T, Tomek S, Niedermayr M, Hejna M, Steger GG, Krainer M, Wiltschke C, Zielinski CC (2004) Monitoring of serum Her-2/neu predicts response and progression-free survival to trastuzumab-based treatment in patients with metastatic breast cancer. Clin Cancer Res 10:1618–1624PubMedCrossRefGoogle Scholar
  14. 14.
    Kute T, Lack CM, Willingham M, Bishwokama B, Williams H, Barrett K, Mitchell T, Vaughn JP (2004) Development of Herceptin resistance in breast cancer cells. Cytometry A 57:86–93PubMedCrossRefGoogle Scholar
  15. 15.
    Mayfield S, Vaughn JP, Kute TE (2001) DNA strand breaks and cell cycle perturbation in herceptin treated breast cancer cell lines. Breast Cancer Res Treat 70:123–129PubMedCrossRefGoogle Scholar
  16. 16.
    Nagata Y, Lan KH, Zhou X, Tan M, Esteva FJ, Sahin AA, Klos KS, Li P, Monia BP, Nguyen NT, Hortobagyi GN, Hung MC, Yu D (2004) PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients. Cancer Cell 6:117–127PubMedCrossRefGoogle Scholar
  17. 17.
    Nagy P, Friedlander E, Tanner M, Kapanen AI, Carraway KL, Isola J, Jovin TM (2005) Decreased accessibility and lack of activation of ErbB2 in JIMT-1, a herceptin-resistant, MUC4-expressing breast cancer cell line. Cancer Res 65:473–482PubMedGoogle Scholar
  18. 18.
    Nahta R, Esteva FJ (2007) Trastuzumab: triumphs and tribulations. Oncogene 26:3637–3643PubMedCrossRefGoogle Scholar
  19. 19.
    Nauseef WM (2007) Isolation of human neutrophils from venous blood. Methods Mol Biol 412:15–20PubMedCrossRefGoogle Scholar
  20. 20.
    Piccart-Gebhart MJ, Procter M, Leyland-Jones B, Goldhirsch A, Untch M, Smith I, Gianni L, Baselga J, Bell R, Jackisch C, Cameron D, Dowsett M, Barrios CH, Steger G, Huang CS, Andersson M, Inbar M, Lichinitser M, Lang I, Nitz U, Iwata H, Thomssen C, Lohrisch C, Suter TM, Ruschoff J, Suto T, Greatorex V, Ward C, Straehle C, McFadden E, Dolci MS, Gelber RD (2005) Trastuzumab after adjuvant chemotherapy in HER2-positive breast cancer. N Engl J Med 353:1659–1672PubMedCrossRefGoogle Scholar
  21. 21.
    Price-Schiavi SA, Jepson S, Li P, Arango M, Rudland PS, Yee L, Carraway KL (2002) Rat Muc4 (sialomucin complex) reduces binding of anti-ErbB2 antibodies to tumor cell surfaces, a potential mechanism for herceptin resistance. Int J Cancer 99:783–791PubMedCrossRefGoogle Scholar
  22. 22.
    Repka T, Chiorean EG, Gay J, Herwig KE, Kohl VK, Yee D, Miller JS (2003) Trastuzumab and interleukin-2 in HER2-positive metastatic breast cancer: a pilot study. Clin Cancer Res 9:2440–2446PubMedGoogle Scholar
  23. 23.
    Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram M, Baselga J, Norton L (2001) Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 344:783–792PubMedCrossRefGoogle Scholar
  24. 24.
    Solly K, Wang X, Xu X, Strulovici B, Zheng W (2004) Application of real-time cell electronic sensing (RT-CES) technology to cell-based assays. Assay Drug Dev Technol 2:363–372PubMedCrossRefGoogle Scholar
  25. 25.
    Spiridon CI, Ghetie MA, Uhr J, Marches R, Li JL, Shen GL, Vitetta ES (2002) Targeting multiple Her-2 epitopes with monoclonal antibodies results in improved antigrowth activity of a human breast cancer cell line in vitro and in vivo. Clin Cancer Res 8:1720–1730PubMedGoogle Scholar
  26. 26.
    Spiridon CI, Guinn S, Vitetta ES (2004) A comparison of the in vitro and in vivo activities of IgG and F(ab′)2 fragments of a mixture of three monoclonal anti-Her-2 antibodies. Clin Cancer Res 10:3542–3551PubMedCrossRefGoogle Scholar
  27. 27.
    Tanner M, Kapanen AI, Junttila T, Raheem O, Grenman S, Elo J, Elenius K, Isola J (2004) Characterization of a novel cell line established from a patient with Herceptin-resistant breast cancer. Mol Cancer Ther 3:1585–1592PubMedGoogle Scholar
  28. 28.
    Varchetta S, Gibelli N, Oliviero B, Nardini E, Gennari R, Gatti G, Silva LS, Villani L, Tagliabue E, Menard S, Costa A, Fagnoni FF (2007) Elements related to heterogeneity of antibody-dependent cell cytotoxicity in patients under trastuzumab therapy for primary operable breast cancer overexpressing Her2. Cancer Res 67:11991–11999PubMedCrossRefGoogle Scholar
  29. 29.
    Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L, Slamon DJ, Murphy M, Novotny WF, Burchmore M, Shak S, Stewart SJ, Press M (2002) Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER2-overexpressing metastatic breast cancer. J Clin Oncol 20:719–726PubMedCrossRefGoogle Scholar
  30. 30.
    Xing JZ, Zhu L, Jackson JA, Gabos S, Sun XJ, Wang XB, Xu X (2005) Dynamic monitoring of cytotoxicity on microelectronic sensors. Chem Res Toxicol 18:154–161PubMedCrossRefGoogle Scholar
  31. 31.
    Yakes FM, Chinratanalab W, Ritter CA, King W, Seelig S, Arteaga CL (2002) Herceptin-induced inhibition of phosphatidylinositol-3 kinase and Akt Is required for antibody-mediated effects on p27, cyclin D1, and antitumor action. Cancer Res 62:4132–4141PubMedGoogle Scholar
  32. 32.
    Zhu J, Wang X, Xu X, Abassi YA (2006) Dynamic and label-free monitoring of natural killer cell cytotoxic activity using electronic cell sensor arrays. J Immunol Methods 309:25–33PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Timothy E. Kute
    • 1
  • Lori Savage
    • 1
  • John R. StehleJr
    • 1
  • Jung W. Kim-Shapiro
    • 1
  • Michael J. Blanks
    • 1
  • James Wood
    • 2
  • James P. Vaughn
    • 2
  1. 1.Department of PathologyWake Forest UniversityWinston-SalemUSA
  2. 2.Department of Cancer BiologyWake Forest University School of MedicineWinston-SalemUSA

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