Background

[PubMed]

Erythropoietin (Epo) is a heavily glycosylated chemokine that is expressed primarily in the adult kidney (during the fetal stage of development, Epo is produced mainly in the liver and not the kidneys), mediates its effects through the erythropoietin receptor (Epo-R), and is known to regulate the process of erythropoiesis (red blood cell formation from hematopoietic cells in the bone marrow) in the hematopoietic system (1). Epo-R is also found in other tissues and is believed to protect cells in the cardiac and neuronal tissues from hypoxic injury and apoptosis. The expression of Epo and Epo-R and the cell signaling pathways involved in bringing about the biological effects of this receptor-ligand complex have been described by Chateauvieux et al. (1). The use of recombinant human Epo (rhuEpo) has been approved by the United States Food and Drug Administration for the treatment of anemia in certain HIV patients and individuals undergoing chemotherapy for non-myeloid cancer or those who are experiencing chronic kidney failure. Meta-analysis of data obtained from clinical trials has shown that the use of rhuEpo in the clinic is associated with increased adverse thrombo-vascular events or neoplastic tumor progression that results in the mortality of individuals with certain types of cancer (2). In a mouse model of breast cancer it has been shown that, when rhuEpo was used in combination with chemotherapy, there was an increased incidence of cancer metastasis in the animals (3). The co-expression of Epo and Epo-R has been observed in certain neoplasms, including non-small cell lung cancer (NSCLC), and a clinical investigation has shown that expression of these genes in the NSCLC tumors correlated with a negative outcome for the patient (4). However, other studies could not establish a clear link between the expression of Epo and Epo-R and the development of cancer because the Epo-R status of a tumor cannot be determined accurately with currently used immunohistochemical methods, due to the lack of antibodies that can specifically and selectively bind to the Epo-R (5). Therefore, the development of alternative probes that specifically target the Epo-R and can be used with noninvasive imaging techniques to detect and quantify the receptor in vivo is necessary (5). Doleschel et al. coupled Cy5.5, a near-infrared dye, to rhuEpo (Epo-Cy5.5) and evaluated the fluorescent conjugate to visualize the expression of EpoR with fluorescence-mediated tomography (FMT) in human lung cancer xenografts in mice (5). The biodistribution of the probe in these animals was also investigated.

Synthesis

[PubMed]

A commercially available kit of Cy5.5 was used to couple the dye to the free carbonyl groups of the periodate-oxidized carbohydrate side chains of rhuEpo (5). Information regarding the source of rhuEpo used to produce Epo-Cy5.5, the purification, purity, and yield of the final fluorescent product, and the number of Cy5.5 molecules attached to each molecule of rhuEpo was not reported.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Epo-Cy5.5 and rhuEpo were reported to generate a similar dose-dependent proliferation curve with UT-7 cells (the proliferation of this human acute myeloid leukemia cell line is Epo dependent), indicating that the biological activity of the Cy5.5 conjugate was the same as the parent molecule (5).

NSCLC cell lines A549, H838, and H2030 were used to determine the in vitro binding specificity of Epo-Cy5.5 (5). Before performing the binding study, the expression of Epo-R mRNA and protein in these cell lines was confirmed with quantitative RT-PCR and quantitative immunoblotting, respectively (5). The expression of Epo-R was reported to be higher in the H838 cells (~2000 Epo-R protein/cell) compared with the A549 cells (~250 Epo-R protein/cell), and the H2030 cells did not express any Epo-R. The NSCLC cell lines were exposed to Epo-Cy5.5, and fluorescence microscopy of the different cells at the same exposure times revealed that the fluorescence signal generated by the H838 cells was higher than that generated by the A549 cells, and no signal was observed with the H2030 cells. This indicated that Epo-Cy5.5 had a high binding specificity for Epo-R on these cells.

To further confirm the Epo-R binding specificity of Epo-Cy5.5, Epo-R/enhanced green fluorescence protein (EGFP)-transfected HeLa cells (Epo-R/EGFP is a fusion protein that has EGFP fused to the C-terminal of the Epo-R; these cells overexpress the fusion protein) were exposed to Epo-Cy5.5 (10 nM) in the absence or presence of excess (190 nM) unlabeled rhuEpo (5). Fluorescence microscopy of the two groups of cells showed that cells incubated with Epo-Cy5.5 alone had a significantly higher (P < 0.05) signal intensity compared with cells incubated with Epo-Cy5.5 in the presence of unlabeled rhuEpo. These observations again indicated that Epo-Cy5.5 had a high specificity of binding to the Epo-R (5).

In another study, cryosections of A549 and H838 cell xenograft tumors in nude mice (see below for details) were exposed to Epo-Cy5.5 and examined under a fluorescent microscope (5). Muscle sections obtained from the same animals were used as controls because this tissue does not express Epo-R. A fluorescence signal was detected only in the tumor sections, and the H838 tumor cells generated a stronger signal compared to cells from the A549 lesions. From these observations, the investigators concluded that the level of fluorescence signal generated by the tumor cells correlated with the degree of Epo-R expression in these cells.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

Supplemental Information

[Disclaimers]

No information is currently available.

References

1.

Chateauvieux S., Grigorakaki C., Morceau F., Dicato M., Diederich M. Erythropoietin, erythropoiesis and beyond. Biochem Pharmacol. 2011;82(10):1291–303. [PubMed: 21782802]

2.

McKinney M., Arcasoy M.O. Erythropoietin for oncology supportive care. Exp Cell Res. 2011;317(9):1246–54. [PubMed: 21396935]

3.

Hedley B.D., Chu J.E., Ormond D.G., Beausoleil M.S., Boasie A., Allan A.L., Xenocostas A. Recombinant human erythropoietin in combination with chemotherapy increases breast cancer metastasis in preclinical mouse models. Clin Cancer Res. 2011;17(19):6151–62. [PubMed: 21856770]

4.

Rades D., Setter C., Dahl O., Schild S.E., Noack F. Prognostic impact of erythropoietin expression and erythropoietin receptor expression on locoregional control and survival of patients irradiated for stage II/III non-small-cell lung cancer. Int J Radiat Oncol Biol Phys. 2011;80(2):499–505. [PubMed: 20646855]

5.

Doleschel D., Mundigl O., Wessner A., Gremse F., Bachmann J., Rodriguez A., Klingmuller U., Jarsch M., Kiessling F., Lederle W. Targeted Near-Infrared Imaging of the Erythropoietin Receptor in Human Lung Cancer Xenografts. J Nucl Med. 2012;53(2):304–311. [PubMed: 22228796]