European Radiology

, Volume 25, Issue 2, pp 472–479 | Cite as

In-vivo detection of the erythropoietin receptor in tumours using positron emission tomography

  • Felix Fuge
  • Dennis Doleschel
  • Anne Rix
  • Felix Gremse
  • Axel Wessner
  • Oliver Winz
  • Felix Mottaghy
  • Wiltrud Lederle
  • Fabian Kiessling
Molecular Imaging



Recombinant human erythropoietin (rhuEpo) is used clinically to treat anaemia. However, rhuEpo-treated cancer patients show decreased survival rates and erythropoietin receptor (EpoR) expression has been found in patient tumour tissue. Thus, rhuEpo application might promote EpoR+ tumour progression. We therefore developed the positron emission tomography (PET)-probe 68Ga-DOTA-rhuEpo and evaluated its performance in EpoR+ A549 non-small-cell lung cancer (NSCLC) xenografts.


68Ga-DOTA-rhuEpo was generated by coupling DOTA-hydrazide to carbohydrate side-chains of rhuEpo. Biodistribution was determined in tumour-bearing mice 0.5, 3, 6, and 9 h after probe injection. Competition experiments were performed by co-injecting 68Ga-DOTA-rhuEpo and rhuEpo in five-fold excess. Probe specificity was further evaluated histologically using Epo-Cy5.5 stainings.


The blood half-life of 68Ga-DOTA-rhuEpo was 2.6 h and the unbound fraction was cleared by the liver and kidney. After 6 h, the highest tumour to muscle ratio was reached. The highest 68Ga-DOTA-rhuEpo accumulation was found in liver (10.06 ± 6.26%ID/ml), followed by bone marrow (1.87 ± 0.53%ID/ml), kidney (1.58 ± 0.39 %ID/ml), and tumour (0.99 ± 0.16%ID/ml). EpoR presence in these organs was histologically confirmed. Competition experiments showed significantly (p < 0.05) lower PET-signals in tumour and bone marrow at 3 and 6 h.


68Ga-DOTA-rhuEpo shows favourable pharmacokinetic properties and detects EpoR specifically. Therefore, it might become a valuable radiotracer to monitor EpoR status in tumours and support decision-making in anaemia therapy.

Key Points

PET-probe 68 Ga-DOTA-rhuEpo was administered to assess the EpoR status in vivo

68 Ga-DOTA-rhuEpo binds specifically to EpoR positive organs in vivo

Tumour EpoR status determination might enable decision-making in anaemia therapy with rhuEpo


DOTA Anaemia Epo Lung cancer EpoR 



burst forming unit erythroid


colony forming unit erythroid


Dulbecco’s Modified Eagle’s Medium


1,4,7,10-tetraazo-cyclododecane-tetraacetic acid


diethylene-triamine-pentaacetic acid




erythropoietin receptor


fluorescence-mediated tomography


near infrared fluorescence


1,4,7-triazacyclononane-1,4,7-triacetic acid


non-small-cell lung cancer


ordered subset expectation maximization


positron emission tomography


recombinant human erythropoietin


region of interest


Standard Deviation


single photon emission computed tomography


Thin Layer Chromatography



The scientific guarantor of this publication is Fabian Kiessling. The authors of this manuscript declare relationships with the following companies: Roche Diagnostics GmbH; Philips Research. This study received funding from the German Ministry for Education and Research/Bundesministerium für Bildung und Forschung (BMBF) project numbers 0315415C and 0316042 F. Felix Gremse received funding from Philips (Philips Research, Aachen,Germany). Axel Wessner is employee of Roche Diagnostics GmbH. The authors would also like to thank Sebastian Lowins from the organic chemistry department for intensive discussions and help. No complex statistical methods were necessary for this paper. Institutional Review Board approval was not required because it was not applicable. Approval from the institutional animal care committee was obtained. No study subjects or cohorts have been previously reported. Methodology: prospective, experimental, multicenter study.


  1. 1.
    Spivak JL (2005) The anaemia of cancer: death by a thousand cuts. Nat Rev Cancer 5:543–555PubMedCrossRefGoogle Scholar
  2. 2.
    Sato Y, Yanagita M (2013) Renal anemia: from incurable to curable. Am J Physiol Renal Physiol 305:F1239–F1248PubMedCrossRefGoogle Scholar
  3. 3.
    Henke M, Mattern D, Pepe M et al (2006) Do erythropoietin receptors on cancer cells explain unexpected clinical findings? J Clin Oncol 24:4708–4713PubMedCrossRefGoogle Scholar
  4. 4.
    Wright JR, Ung YC, Julian JA et al (2007) Randomized, double-blind, placebo-controlled trial of erythropoietin in non-small-cell lung cancer with disease-related anemia. J Clin Oncol 25:1027–1032PubMedCrossRefGoogle Scholar
  5. 5.
    Bohlius J, Schmidlin K, Brillant C et al (2009) Recombinant human erythropoiesis-stimulating agents and mortality in patients with cancer: a meta-analysis of randomised trials. Lancet 373:1532–1542PubMedCrossRefGoogle Scholar
  6. 6.
    Bohlius J, Wilson J, Seidenfeld J et al (2006) Recombinant human erythropoietins and cancer patients: updated meta-analysis of 57 studies including 9353 patients. J Natl Cancer Inst 98:708–714PubMedCrossRefGoogle Scholar
  7. 7.
    Bennett CL, Silver SM, Djulbegovic B et al (2008) Venous thromboembolism and mortality associated with recombinant erythropoietin and darbepoetin administration for the treatment of cancer-associated anemia. J Am Med Assoc 299:914–924CrossRefGoogle Scholar
  8. 8.
    Okazaki T, Ebihara S, Asada M, Yamanda S, Niu K, Arai H (2008) Erythropoietin promotes the growth of tumors lacking its receptor and decreases survival of tumor-bearing mice by enhancing angiogenesis. Neoplasia 10:932–939PubMedCentralPubMedGoogle Scholar
  9. 9.
    Belenkov AI, Shenouda G, Rizhevskaya E et al (2004) Erythropoietin induces cancer cell resistance to ionizing radiation and to cisplatin. Mol Cancer Ther 3:1525–1532PubMedGoogle Scholar
  10. 10.
    Shiozawa Y, McGee S, Pienta MJ et al (2013) Erythropoietin supports the survival of prostate cancer, but not growth and bone metastasis. J Cell Biochem 114:2471–2478PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Todaro M, Turdo A, Bartucci M et al (2013) Erythropoietin activates cell survival pathways in breast cancer stem-like cells to protect them from chemotherapy. Cancer Res 73:6393–6400PubMedCrossRefGoogle Scholar
  12. 12.
    Rózsás A, Berta J, Rojkó L et al (2013) Erythropoietin receptor expression is a potential prognostic factor in human lung adenocarcinoma. PLoS ONE 8:e77459PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Baltaziak M, Wincewicz A, Kanczuga-Koda L et al (2013) The relationships between hypoxia-dependent markers: HIF-1alpha, EPO and EPOR in colorectal cancer. Folia Histochem Cytobiol Pol Acad Sci Pol Histochem Cytochem Soc 51:320–325CrossRefGoogle Scholar
  14. 14.
    Ferracin M, Bassi C, Pedriali M et al (2013) miR-125b targets erythropoietin and its receptor and their expression correlates with metastatic potential and ERBB2/HER2 expression. Mol Cancer 12:130PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Miyake M, Goodison S, Lawton A, Zhang G, Gomes-Giacoia E, Rosser CJ (2013) Erythropoietin is a JAK2 and ERK1/2 effector that can promote renal tumor cell proliferation under hypoxic conditions. J Hematol Oncol 6:65PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Elliott S, Sinclair A, Collins H, Rice L, Jelkmann W (2014) Progress in detecting cell-surface protein receptors: the erythropoietin receptor example. Ann Hematol 93:181–192PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Doleschel D, Mundigl O, Wessner A et al (2012) Targeted Near-Infrared Imaging of the Erythropoietin Receptor in Human Lung Cancer Xenografts. J Nucl Med 53:304–311PubMedCrossRefGoogle Scholar
  18. 18.
    Ntziachristos V, Ripoll J, Wang LV, Weissleder R (2005) Looking and listening to light: the evolution of whole-body photonic imaging. Nat Biotechnol 23:313–320PubMedCrossRefGoogle Scholar
  19. 19.
    Yoshioka E, Kato K, Shindo H, Mitsuoka C, S.-I. Kitajima S-I, Ogata H et al: Pharmacokinetic study of darbepoetin alfa (2007) Absorption, distribution, and excretion after a single intravenous and subcutaneous administration to rats. Xenobiotica. 37(1)Google Scholar
  20. 20.
    dos Clemente GS, Duarte VLS (2011) Synthesis and biological evaluation of 125I-erythropoietin as a potential radiopharmaceutical agent for tumours. Braz J Pharm Sci 47:83–88Google Scholar
  21. 21.
    Ehrenreich H, Degner D, Meller J et al (2004) Erythropoietin: a candidate compound for neuroprotection in schizophrenia. Mol Psychiatry 9:42–54PubMedGoogle Scholar
  22. 22.
    Fuge F, Weiler M, Gätjens J, Lammers T, Kiessling F (2013) Comparison and systematic optimization of synthetic protocols for DOTA–hydrazide generation. Tetrahedron Lett 54:918–920CrossRefGoogle Scholar
  23. 23.
    Bauwens M, Chekol R, Vanbilloen H, Bormans G, Verbruggen A (2010) Optimal buffer choice of the radiosynthesis of 68Ga–Dotatoc for clinical application. Nucl Med Commun 31:753–758PubMedCrossRefGoogle Scholar
  24. 24.
    Narhi LO, Arakawa T, Aoki KH, Elmore R, Rohde MF, Boone T et al (1991) The effect of carbohydrate on the structure and stability of erythropoietin. J Biol Chem 266:23022–23026PubMedGoogle Scholar
  25. 25.
    Tsuda E, Kawanishi G, Ueda M, Masuda S, Sasaki R (1990) The role of carbohydrate in recombinant human erythropoietin. Eur J Biochem 188:405–411PubMedCrossRefGoogle Scholar
  26. 26.
    Kunjachan S, Gremse F, Theek B et al (2013) Noninvasive optical imaging of nanomedicine biodistribution. ACS Nano 7:252–262PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Agoram B, Aoki K, Doshi S, Gegg C, Jang G, Molineux G et al (2009) Investigation of the effects of altered receptor binding activity on the clearance of erythropoiesis-stimulating proteins: Nonerythropoietin receptor-mediated pathways may play a major role. J Pharm Sci 98:2198–2211PubMedCrossRefGoogle Scholar
  28. 28.
    Blom E, Velikyan I, Estrada S, Hall H, Muhammad T, Ding C et al (2012) 68Ga-Labeling of RGD peptides and biodistribution. Int J Clin Exp Med 5:165–172PubMedCentralPubMedGoogle Scholar
  29. 29.
    Ranyuk E, Lebel R, Bérubé-Lauzière Y, Klarskov K, Lecomte R, van Lier JE et al (2013) 68Ga/DOTA- and 64Cu/NOTA-Phthalocyanine Conjugates as Fluorescent/PET Bimodal Imaging Probes. Bioconjug Chem 24:1624–1633PubMedCrossRefGoogle Scholar
  30. 30.
    Knetsch PA, Petrik M, Griessinger CM, Rangger C, Fani M, Kesenheimer C et al (2011) [68Ga]NODAGA-RGD for imaging αvβ3 integrin expression. Eur J Nucl Med Mol Imaging 38:1303–1312PubMedCrossRefGoogle Scholar
  31. 31.
    Zhang Y, Hong H, Engle JW, Bean J, Yang Y, Leigh BR et al (2011) Positron Emission Tomography Imaging of CD105 Expression with a 64Cu-Labeled Monoclonal Antibody: NOTA Is Superior to DOTA. PLoS ONE 6:e28005PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Jelkmann W (2002) The enigma of the metabolic fate of circulating erythropoietin (Epo) in view of the pharmacokinetics of the recombinant drugs rhEpo and NESP. Eur J Haematol 69:265–274PubMedCrossRefGoogle Scholar
  33. 33.
    Oxboel J, Brandt-Larsen M, Schjoeth-Eskesen C et al (2014) Comparison of two new angiogenesis PET tracers 68Ga-NODAGA-E[c(RGDyK)]2 and (64)Cu-NODAGA-E[c(RGDyK)]2; in vivo imaging studies in human xenograft tumors. Nucl Med Biol 41:259–267PubMedCrossRefGoogle Scholar
  34. 34.
    Kang CM, Kim S-M, Koo H-J et al (2013) In vivo characterization of 68Ga-NOTA-VEGF 121 for the imaging of VEGF receptor expression in U87MG tumor xenograft models. Eur J Nucl Med Mol Imaging 40:198–206PubMedCrossRefGoogle Scholar

Copyright information

© European Society of Radiology 2014

Authors and Affiliations

  • Felix Fuge
    • 1
  • Dennis Doleschel
    • 1
  • Anne Rix
    • 1
  • Felix Gremse
    • 1
  • Axel Wessner
    • 2
  • Oliver Winz
    • 3
  • Felix Mottaghy
    • 3
  • Wiltrud Lederle
    • 1
  • Fabian Kiessling
    • 1
  1. 1.Department for Experimental Molecular Imaging (ExMI), Medical FacultyRWTH Aachen UniversityAachenGermany
  2. 2.Roche Diagnostics GmbH, R&D RPD Protein ChemistryPenzbergGermany
  3. 3.Clinic for Nuclear MedicineUniversity Clinic RWTH AachenAachenGermany

Personalised recommendations