Targeted near-infrared imaging of the erythropoietin receptor in human lung cancer xenografts.
The putative presence of the erythropoietin receptor (EpoR) on human cancer cells has given rise to controversial discussion about the use of recombinant human erythropoietin (rhuEpo) for treatment of patients with chemotherapy-induced anemia. In vivo analysis of the EpoR status in tumors could help in elucidating the role of erythropoietin in cancer. Thus, the aim of this study was to develop a targeted EpoR probe for the investigation of EpoR expression in human lung cancer xenografts by fluorescence-mediated tomography.


METHODS
Epo-Cy5.5 was generated by coupling Cy5.5 to rhuEpo. In vitro binding assays were performed using the EpoR-positive non-small cell lung cancer (NSCLC) cell lines A549 (lower EpoR expression) and H838 (higher EpoR expression), the EpoR-negative cell line H2030, and EpoR/EGFP-overexpressing HeLa cells. In vivo specificity of Epo-Cy5.5 was confirmed by competition analyses using micro-CT/fluorescence-mediated tomography fusion imaging. Biodistribution was analyzed over 50 h after injection. Binding of Epo-Cy5.5 was validated on tumor cryosections.


RESULTS
After intravenous injection, the probe was rapidly cleared from the circulation. An accumulation was observed in liver and kidneys, with a maximum at 7 h after injection followed by a decline, indicating renal excretion. Almost constant accumulation of Epo-Cy5.5 was found in bone marrow and tumors, indicating specific receptor binding. The probe allowed the discrimination between H838 with higher EpoR expression (89.54 ± 15.91 nM at 25 h) and A549 tumors with lower EpoR expression (60.45 ± 14.59 nM at 25 h, P < 0.05). Tumor accumulation of Epo-Cy5.5 could be significantly reduced by adding unlabeled rhuEpo (P < 0.05 at 4, 7, and 24 h). In vitro validation confirmed specific binding of Epo-Cy5.5 to the tumor cells, and this binding correlated with the EpoR expression level. Binding was also observed on endothelial cells. Vessel density and Epo-Cy5.5 binding on endothelial cells were comparable.


CONCLUSIONS
Epo-Cy5.5 allows the longitudinal analysis of EpoR expression in tumors and thereby can investigate the influence of erythropoietin on EpoR expression, tumor growth, and angiogenesis.
Department of Experimental Molecular Imaging, Medical Faculty, RWTH-Aachen University, Aachen, Germany.
Dennis
D
Doleschel
Olaf
O
Mundigl
Axel
A
Wessner
Felix
F
Gremse
Julie
J
Bachmann
Agustin
A
Rodriguez
Ursula
U
Klingmüller
Michael
M
Jarsch
Fabian
F
Kiessling
Wiltrud
W
Lederle
D000818:Animals; D001853:Bone Marrow / Q000378:metabolism; D002232:Carbocyanines / Q000378:metabolism; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D042783:Endothelial Cells / Q000378:metabolism; D004921:Erythropoietin / Q000378:metabolism / Q000493:pharmacokinetics; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007259:Infrared Rays; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology*; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D015335:Molecular Probes / Q000378:metabolism* / Q000493:pharmacokinetics; D017467:Receptors, Erythropoietin / Q000378:metabolism*; D013379:Substrate Specificity
C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D015335:Molecular Probes; D017467:Receptors, Erythropoietin; D004921:Erythropoietin