Skip to main content

Advertisement

SpringerLink
Log in

Cellular and molecular properties of 90Y-labeled cetuximab in combination with radiotherapy on human tumor cells in vitro

Zelluläre und molekulare Eigenschaften von 90Y-markiertem Cetuximab in Kombination mit Strahlentherapie bei humanen Tumorzellen in vitro

  • Original article
  • Published:
Strahlentherapie und Onkologie Aims and scope Submit manuscript

Abstract

Purpose

Anti-EGFR antibody cetuximab (C225) is used in combination with radiotherapy of head and neck squamous cell carcinoma (HNSCC) patients. We investigated whether conjugation of cetuximab with trans-cyclohexyl-diethylene-triamine-pentaacetic acid (CHX-A″-DTPA) and radiolabeling with 90Yttrium affect the molecular and cellular function of cetuximab and improve its combined effect with external-beam irradiation (EBI).

Methods

The following cell lines were used: HNSCC UT5, SAS, FaDu, as well as A43, Chinese hamster ovary cells (CHO), and human skin fibroblast HSF7. Binding affinity and kinetics, specificity, retention, and the combination of 90Y-cetuximab with EBI were evaluated.

Results

Control cetuximab and CHX-A″-DTPA-cetuximab blocked the proliferation activity of UT5 cells. In combination with EBI, CHX-A″-DTPA-cetuximab increased the radiosensitivity of UT5 to a similar degree as control cetuximab did. In contrast, in SAS and HSF7 cells neither proliferation nor radiosensitivity was affected by either of the antibodies. Binding [90Y]Y-CHX-A″-DTPA-cetuximab (90Y-cetuximab) to EGFR in HNSCC cells occurred time dependently with a maximum binding at 24 h. Retention of 90Y-cetuximab was similar in both HNSCC cell lines; 24 h after treatment, approximately 90% of bound activity remained in the cell layer. Competition assays, using cell membranes in the absence of an internalized fraction of cetuximab, showed that the cetuximab affinity is not lost as a result of conjugation with CHX-A″-DTPA. Cetuximab and CHX-A″-DTPA-cetuximab blocked EGF-induced Y1068 phosphorylation of EGFR. The lack of an effect of cetuximab on EGF-induced Akt and ERK1/2 phosphorylation and the inhibition of irradiation (IR)-induced Akt and ERK1/2 phosphorylation by cetuximab were not affected by DTPA conjugation. 90Y-cetuximab in combination with EBI resulted in a pronounced inhibition of colony formation of HNSCC cells.

Conclusions

Conjugation of CHX-A″-DTPA to cetuximab does not alter the cellular and biological function of cetuximab. 90Y-labeling of cetuximab in combination with EBI may improve radiotherapy outcome.

Zusammenfassung

Ziel

Der Anti-EGFR-Antikörper Cetuximab (C225) ist in der Strahlentherapie von Kopf-Hals-Tumoren etabliert. Ziel der Untersuchung war zu erfassen, inwiefern die Konjugation von Cetuximab mit trans-Cyclohexyl-Diethylen-Triamin-Pentaessigsäure (CHX-A″-DTPA) und Radiomarkierung mit 90Yttrium (90Y) die molekularen und zellulären Eigenschaften von Cetuximab beeinflusst und dadurch zu einer Verstärkung der Wirkung von externer Bestrahlung führt.

Methode

Humane Kopf-Hals-Tumor-Zelllinien UT5, SAS, FaDu sowie A341-Zellen, CHO-Zellen („Chinese hamster ovary cells“) und humane Hautfibroblasten (HSF1) wurden für die Untersuchungen verwendet. Die Spezifität der Membranbindung und Retention sowie deren Kinetik und die Kombinationswirkung von 90Y-markiertem Cetuximab mit externer Strahlentherapie wurden evaluiert.

Ergebnisse

Unmarkiertes Kontroll-Cetuximab und CHX-A″-DTPA-Cetuximab führten zu einer Proliferationsblockade von UT5-Zellen. In Kombination mit externer Bestrahlung zeigt das CHX-A″-DTPA-Cetuximab bei UT5-Zellen eine gleichartige Radiosensivitierung wie nicht markiertes Kontroll-Cetuximab. Im Gegensatz dazu waren weder SAS-Tumorzellen noch HSF7-Fibroblasten durch beide Antikörper in ihrer Proliferationsrate und Radiosensitivität zu beeinflussen. Die Bindung von [90Y]Y-CHX-A″-DTPA-Cetuximab (90Y-Cetuximab) an EGFR („epidermal growth factor receptor“) in Kopf-Hals-Tumorzellen erfolgte zeitabhängig und erreichte ein Maximum nach 24 h. Ebenso erfolgte die Retention in beiden Kopf-Hals-Tumor-Zelllinien, 24 h nach Behandlungsbeginn verblieben etwa 90% der gebundenen Aktivität in der Zellmembran. Mittels Kompetitionstests mit gereinigten Zellmembranen konnte dargestellt werden, dass die EGFR-Affinität von Cetuximab durch die Konjugation des Chelators CHX-A″-DTPA nicht beeinträchtigt wurde. Cetuximab und CHX-A″-DTPA-Cetuximab führten zu einer Blockade der EGF-induzierten Phosphorylierung von EGFR an der Tyrosinposition Y1068. Durch die DTPA-Konjugation des Antikörpers wurde weder die EGF- noch die strahleninduzierte Phosphorylierung von Akt und ERK1/2 noch die Inhibition der strahleninduzierten Akt- und ERK1/2-Phosphorylierung durch Cetuximab beeinflusst. 90Y-Cetuximab bewirkte in Kombination mit externer Bestrahlung eine deutliche Inhibition des klonogenen Potenzials von Kopf-Hals-Tumorzellen.

Schlussfolgerungen

Aufgrund der CHX-A″-DTPA-Konjugation kommt es zu keiner Veränderung der zellulären und radiobiologischen Funktion von Cetuximab. 90Y-markiertes Cetuximab bewirkt in Kombination mit externer Bestrahlung eine differenzielle und zelltypabhängige Verbesserung der Radiotherapiewirkung bei Kopf-Hals-Tumorzelllinien.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

References

  1. Johnson KA, Brown PH (2010) Drug development for cancer chemoprevention: focus on molecular targets. Semin Oncol 37:345–358

    Article  PubMed  CAS  Google Scholar 

  2. Riesterer O, Mason KA, Raju U et al (2009) Enhanced response to C225 of A431 tumor xenografts growing in irradiated tumor bed. Radiother Oncol 92:383–387

    Article  PubMed  CAS  Google Scholar 

  3. Herbst RS, Shin DM (2002) Monoclonal antibodies to target epidermal growth factor receptor-positive tumors: a new paradigm for cancer therapy. Cancer 94:1593–1611

    Article  PubMed  CAS  Google Scholar 

  4. Sunada H, Magun BE, Mendelsohn J, MacLeod CL (1986) Monoclonal antibody against epidermal growth factor receptor is internalized without stimulating receptor phosphorylation. Proc Natl Acad Sci U S A 83:3825–3829

    Article  PubMed  CAS  Google Scholar 

  5. Fan Z, Lu Y, Wu X, Mendelsohn J (1994) Antibody-induced epidermal growth factor receptor dimerization mediates inhibition of autocrine proliferation of A431 squamous carcinoma cells. J Biol Chem 269:27595–27602

    PubMed  CAS  Google Scholar 

  6. Gerber DE, Choy H (2010) Cetuximab in combination therapy: from bench to clinic. Cancer Metastasis Rev 29:171–180

    Article  PubMed  CAS  Google Scholar 

  7. Krause M, Gurtner K, Deuse Y, Baumann M (2009) Heterogeneity of tumour response to combined radiotherapy and EGFR inhibitors: differences between antibodies and TK inhibitors. Int J Radiat Biol 85:943–954

    Article  PubMed  CAS  Google Scholar 

  8. Gurtner K, Deuse Y, Butof R et al (2011) Diverse effects of combined radiotherapy and EGFR inhibition with antibodies or TK inhibitors on local tumour control and correlation with EGFR gene expression. Radiother Oncol 99:323–330

    Article  PubMed  CAS  Google Scholar 

  9. Bonner JA, Harari PM, Giralt J et al (2010) Radiotherapy plus cetuximab for locoregionally advanced head and neck cancer: 5-year survival data from a phase 3 randomised trial, and relation between cetuximab-induced rash and survival. Lancet Oncol 11:21–28

    Article  PubMed  CAS  Google Scholar 

  10. Bonner JA, Harari PM, Giralt J et al (2006) Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med 354:567–578

    Article  PubMed  CAS  Google Scholar 

  11. Curran D, Giralt J, Harari PM et al (2007) Quality of life in head and neck cancer patients after treatment with high-dose radiotherapy alone or in combination with cetuximab. J Clin Oncol 25:2191–2197

    Article  PubMed  CAS  Google Scholar 

  12. Zhang N, Erjala K, Kulmala J et al (2009) Concurrent cetuximab, cisplatin, and radiation for squamous cell carcinoma of the head and neck in vitro. Radiother Oncol 92:388–392

    Article  PubMed  CAS  Google Scholar 

  13. Huang SM, Harari PM (2000) Modulation of radiation response after epidermal growth factor receptor blockade in squamous cell carcinomas: inhibition of damage repair, cell cycle kinetics, and tumor angiogenesis. Clin Cancer Res 6:2166–2174

    PubMed  CAS  Google Scholar 

  14. Dittmann K, Mayer C, Rodemann HP (2005) Inhibition of radiation-induced EGFR nuclear import by C225 (Cetuximab) suppresses DNA-PK activity. Radiother Oncol 76:157–161

    Article  PubMed  CAS  Google Scholar 

  15. Dittmann K, Mayer C, Rodemann HP (2010) Nuclear EGFR as novel therapeutic target: insights into nuclear translocation and function. Strahlenther Onkol 186:1–6

    Article  PubMed  Google Scholar 

  16. Dittmann K, Mayer C, Fehrenbacher B et al (2005) Radiation-induced epidermal growth factor receptor nuclear import is linked to activation of DNA-dependent protein kinase. J Biol Chem 280:31182–1189

    Article  PubMed  CAS  Google Scholar 

  17. Toulany M, Kasten-Pisula U, Brammer I et al (2006) Blockage of epidermal growth factor receptor-phosphatidylinositol 3-kinase-AKT signaling increases radiosensitivity of K-RAS mutated human tumor cells in vitro by affecting DNA repair. Clin Cancer Res 12:4119–4126

    Article  PubMed  CAS  Google Scholar 

  18. Kriegs M, Kasten-Pisula U, Rieckmann T et al (2010) The epidermal growth factor receptor modulates DNA double-strand break repair by regulating non-homologous end-joining. DNA Repair (Amst) 9:889–897

    Google Scholar 

  19. Toulany M, Minjgee M, Kehlbach R et al (2010) ErbB2 expression through heterodimerization with erbB1 is necessary for ionizing radiation- but not EGF-induced activation of Akt survival pathway. Radiother Oncol 97:338–345

    Article  PubMed  CAS  Google Scholar 

  20. Nieder C (2012) RTOG study 0324: Cetuximab in combination with chemoradiation in patients with stage IIIA/B non-small-cell lung cancer. Strahlenther Onkol 188:194–195

    Article  PubMed  CAS  Google Scholar 

  21. Alongi F, Bignardi M, Garassino I et al (2012) Prospective phase II trial of cetuximab plus VMAT-SIB in locally advanced head and neck squamous cell carcinoma. Feasibility and tolerability in elderly and chemotherapy-ineligible patients. Strahlenther Onkol 188:49–55

    Article  PubMed  CAS  Google Scholar 

  22. Selzer E, Liederer S, Lemaire C et al (2011) Incidence of dermatitis in head and neck cancer patients treated with primary radiotherapy and cetuximab. Strahlenther Onkol 187:373–377

    Article  PubMed  Google Scholar 

  23. Niyazi M, Siefert A, Schwarz SB et al (2011) Therapeutic options for recurrent malignant glioma. Radiother Oncol 98:1–14

    Article  PubMed  Google Scholar 

  24. Liu Z, Liu Y, Jia B et al (2010) Epidermal growth factor receptor-targeted radioimmunotherapy of human head and neck cancer xenografts using 90Y-labeled fully human antibody panitumumab. Mol Cancer Ther 9:2297–2308

    Article  PubMed  CAS  Google Scholar 

  25. Gemmete JJ, Mukherji SK (2011) Panitumumab (Vectibix). AJNR Am J Neuroradiol

  26. Rades D, Wolff C, Nadrowitz R et al (2009) Radioactive EGFR antibody cetuximab in multimodal cancer treatment: stability and synergistic effects with radiotherapy. Int J Radiat Oncol Biol Phys 75:1226–1231

    Article  PubMed  CAS  Google Scholar 

  27. Rades D, Nadrowitz R, Buchmann I et al (2010) Radiolabeled cetuximab plus whole-brain irradiation (WBI) for the treatment of brain metastases from non-small cell lung cancer (NSCLC). Strahlenther Onkol 186:458–462

    Article  PubMed  Google Scholar 

  28. Wheeler DL, Dunn EF, Harari PM (2010) Understanding resistance to EGFR inhibitors-impact on future treatment strategies. Nat Rev Clin Oncol 7:493–507

    Article  PubMed  CAS  Google Scholar 

  29. Toulany M, Dittmann K, Baumann M, Rodemann HP (2005) Radiosensitization of Ras-mutated human tumor cells in vitro by the specific EGF receptor antagonist BIBX1382BS. Radiother Oncol 74:117–129

    Article  PubMed  CAS  Google Scholar 

  30. Debucquoy A, Machiels JP, McBride WH, Haustermans K (2010) Integration of epidermal growth factor receptor inhibitors with preoperative chemoradiation. Clin Cancer Res 16:2709–2714

    Article  PubMed  CAS  Google Scholar 

  31. Rodemann HP, Dittmann K, Toulany M (2007) Radiation-induced EGFR-signaling and control of DNA-damage repair. Int J Radiat Biol 83:781–791

    Article  PubMed  CAS  Google Scholar 

  32. Meyn RE, Munshi A, Haymach JV et al (2009) Receptor signaling as a regulatory mechanism of DNA repair. Radiother Oncol 92:316–322

    Article  PubMed  CAS  Google Scholar 

  33. Wang M, Morsbach F, Sander D et al (2011) EGF receptor inhibition radiosensitizes NSCLC cells by inducing senescence in cells sustaining DNA double-strand breaks. Cancer Res 71:6261–6269

    Article  PubMed  CAS  Google Scholar 

  34. Bonner JA, Yang ES, Trummell HQ et al (2011) Inhibition of STAT-3 results in greater cetuximab sensitivity in head and neck squamous cell carcinoma. Radiother Oncol 99:339–343

    Article  PubMed  CAS  Google Scholar 

  35. Milenic DE, Wong KJ, Baidoo KE et al (2008) Cetuximab: preclinical evaluation of a monoclonal antibody targeting EGFR for radioimmunodiagnostic and radioimmunotherapeutic applications. Cancer Biother Radiopharm 23:619–631

    Article  PubMed  CAS  Google Scholar 

  36. Aerts HJ, Dubois L, Perk L et al (2009) Disparity between in vivo EGFR expression and 89Zr-labeled cetuximab uptake assessed with PET. J Nucl Med 50:123–131

    Article  PubMed  CAS  Google Scholar 

  37. Liao HJ, Carpenter G (2009) Cetuximab/C225-induced intracellular trafficking of epidermal growth factor receptor. Cancer Res 69:6179–6183

    Article  PubMed  CAS  Google Scholar 

  38. Jaramillo ML, Leon Z, Grothe S et al (2006) Effect of the anti-receptor ligand-blocking 225 monoclonal antibody on EGF receptor endocytosis and sorting. Exp Cell Res 312:2778–2790

    Article  PubMed  CAS  Google Scholar 

  39. Eiblmaier M, Meyer LA, Watson MA et al (2008) Correlating EGFR expression with receptor-binding properties and internalization of 64Cu-DOTA-cetuximab in 5 cervical cancer cell lines. J Nucl Med 49:1472–1479

    Article  PubMed  CAS  Google Scholar 

  40. Nordberg E, Friedman M, Gostring L et al (2007) Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule. Nucl Med Biol 34:609–618

    Article  PubMed  CAS  Google Scholar 

  41. Gostring L, Chew MT, Orlova A et al (2010) Quantification of internalization of EGFR-binding Affibody molecules: Methodological aspects. Int J Oncol 36:757–763

    Article  PubMed  Google Scholar 

  42. Koi L, Bruechner K, Helbig L et al (2011) Combination of external and internal irradiation using radionuclide-labelled Cetuximab in FaDu human squamous cell carcinomas xenografted in nude mice. Wolsberg meeting series 9:60

    Google Scholar 

  43. Eke I, Cordes N (2011) Dual targeting of EGFR and focal adhesion kinase in 3D grown HNSCC cell cultures. Radiother Oncol 99:279–286

    Article  PubMed  CAS  Google Scholar 

  44. Berger C, Krengel U, Stang E et al (2011) Nimotuzumab and cetuximab block ligand-independent EGF receptor signaling efficiently at different concentrations. J Immunother 34:550–555

    Article  PubMed  CAS  Google Scholar 

  45. Yoshida T, Okamoto I, Okabe T et al (2008) Matuzumab and cetuximab activate the epidermal growth factor receptor but fail to trigger downstream signaling by Akt or Erk. Int J Cancer 122:1530–1538

    Article  PubMed  CAS  Google Scholar 

  46. Li S, Schmitz KR, Jeffrey PD et al (2005) Structural basis for inhibition of the epidermal growth factor receptor by cetuximab. Cancer Cell 7:301–311

    Article  PubMed  CAS  Google Scholar 

  47. Patel D, Bassi R, Hooper A et al (2009) Anti-epidermal growth factor receptor monoclonal antibody cetuximab inhibits EGFR/HER-2 heterodimerization and activation. Int J Oncol 34:25–32

    PubMed  CAS  Google Scholar 

Download references

Acknowledgment

This work was supported by grants from the German Federal Ministry of Research and Education (BMBF, grant nos. 02NUK006 and 03NUK006).

Author information

Authors and Affiliations

  1. Division of Radiobiology & Molecular Environmental Research, Department of Radiation Oncology, University of Tuebingen, Roentgenweg 11, 72076, Tuebingen, Germany

    M. Saki, M. Toulany &  H.P. Rodemann

  2. Institute of Radiopharmacy, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany

    W. Sihver, M. Zenker, J.-M. Heldt, B. Mosch, H.-J. Pietzsch & J. Steinbach

  3. Department of Radiation Oncology, OncoRay-National Center for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Technische Universität, Dresden, Dresden, Germany

    M. Baumann

Authors
  1. M. Saki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Toulany
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. W. Sihver
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M. Zenker
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J.-M. Heldt
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. B. Mosch
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. H.-J. Pietzsch
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. M. Baumann
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. J. Steinbach
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. H.P. Rodemann
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to H.P. Rodemann.

Additional information

M. Saki and M. Toulany shared first authorship of this paper.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Saki, M., Toulany, M., Sihver, W. et al. Cellular and molecular properties of 90Y-labeled cetuximab in combination with radiotherapy on human tumor cells in vitro. Strahlenther Onkol 188, 823–832 (2012). https://doi.org/10.1007/s00066-012-0121-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00066-012-0121-4

Keywords

Schlüsselwörter

Search

Navigation