IRDye 800CW-Epidermal growth factor


Leung K.

Publication Details



In vitro Rodents



Epidermal growth factor (EGF) is a 53–amino acid cytokine (6.2 kDa) that is secreted by ectodermic cells, monocytes, kidneys, and duodenal glands (1). EGF stimulates growth of epidermal and epithelial cells. EGF and at least seven other growth factors and their transmembrane receptor kinases play important roles in cell proliferation, survival, adhesion, migration, and differentiation. The EGF receptor (EGFR) family consists of four transmembrane receptors, which include EGFR (HER1/erbB-1), HER2 (erbB-2/neu), HER3 (erbB-3), and HER4 (erbB-4) (2). HER1, HER3, and HER4 comprise three major functional domains: an extracellular ligand-binding domain, a hydrophobic transmembrane domain, and a cytoplasm tyrosine kinase domain. No ligand has been clearly identified for HER2. However, HER2 can be activated as a result of ligand binding to other HER receptors with the formation of receptor homodimers and/or heterodimers (3). HER1 as well as HER2 are overexpressed on many solid tumor cells such as breast, non–small-cell lung, head and neck, and colon cancers (4-6). The high levels of HER1 and HER2 expression on cancer cells are associated with a poor prognosis (7-10).

Optical fluorescence imaging is increasingly being used to monitor biological functions of specific targets (11-13). However, the intrinsic fluorescence of biomolecules poses a problem when fluorophores that absorb visible light (350–700 nm) are used. Near-infrared (NIR) fluorescence (700–1,000 nm) detection avoids the natural background fluorescence interference of biomolecules, which provides a high contrast between target and background tissues in small animals. NIR fluorophores have a wider dynamic range and minimal background fluorescence as a result of reduced scattering compared with visible fluorescence detection. NIR fluorophores also have high sensitivity, attributable to low background fluorescence, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components such as diode lasers and silicon detectors. NIR fluorescence imaging is a non-invasive complement to radionuclide imaging in small animals.

Trastuzumab, a humanized immunoglobulin G1 (IgG1) monoclonal antibody against the extracellular domain of recombinant HER2 (14), and C225, an anti-EGFR chimeric monoclonal antibody, have been labeled as 111In-trastuzumab (15-17) and 99mTc-EC-C225 (C225 is also known as cetuximab) (18, 19), respectively, for imaging EGFR expression on solid tumors using single-photon emission computed tomography (SPECT). However, antibodies that are approximately 25-fold larger than EGF may not be easily transported to cells within solid tumors. 111In- and 68Ga-labeled trastuzumab F(ab) antibody fragments as well as 111In-EGF and 99mTc-HYNIC-EGF have been used to partially overcome this permeability barrier (20, 21). EGF has also been successfully coupled with Cy5.5 NIR dye (22) and IRDye 800CW (23) for optical imaging of EGFR density in tumors in mice using non-radioactive materials.



Commercially available IRDye 800CW-N-hydroxysuccinimide ester (LI-COR, Lincoln, NE) was used to conjugate EGF to form IRDye 800CW-EGF, which was purified by dialysis (23). The molar ratio of IRDye 800CW to EGF and yield were not reported. IRDye 800CW (molecular weight, 962 Da) has an extinction coefficient of 237,000 M-1cm-1 and a quantum yield of 0.12, with a maximum excitation wavelength of 783 nm and a maximum emission wavelength of 801 nm in fetal bovine serum.

In Vitro Studies: Testing in Cells and Tissues


Kovar et al. (23) performed cell-binding assays with IRDye 800CW-EGF using human prostate cancer cell lines PC3M-LN4 (EGFR-positive, metastatic) and 22Rv1 (EGFR-positive, non-metastatic). Incubation of IRDye 800CW-EGF (1–70 nM) for 2 min at 37°C resulted in high fluorescence intensity in both cell lines in a dose-dependent manner. On the other hand, IRDye 800CW binding in both cell types was negligible. Binding of IRDye 800CW-EGF was completely blocked by C225 (100 μg) and EGF (1 μM) with inhibition constant (IC50) values of 10 μg and 0.1 μM, respectively.

Animal Studies



Kovar et al. (23) studied the accumulation of IRDye 800CW-EGF using a whole-body fluorescence detection system in nude mice bearing PC3M-LN4 tumors. IRDye 800CW-EGF (1 nmol/mouse) was injected intravenously into mice that showed distinct fluorescence signals in the tumors and lymph nodes at 72–96 h after injection. The fluorescence intensity started to decrease after 96 h. Pretreatment with C225 (13 nmol/mouse 60 min before IRDye 800CW-EGF injection) inhibited the IRDye 800CW-EGF binding in the tumors and lymph nodes by 34–46% (nonspecific IgG antibody pretreatment was not performed). Other experiments showed that the accumulation of IRDye 800CW-EGF was distinctly higher in the 22Rv1 tumors than in the PC3M-LN4 tumors as the tumors grew in size. However, all mice with PC3M-LN4 tumors (n = 4) were node-positive, and all 22Rv1 tumor-bearing mice (n = 4) were node-negative, which confirmed that PC3M-LN4 is metastatic and 22Rv1 is non-metastatic.

Adams et al. (24) performed in vivo whole-body fluorescence imaging of IRDye 800CW-EGF (1 nmol) in nude mice (n = 5) bearing EGFR-positive MDA-MB-468 and EGFR-negative MDA-MB-435 human breast cancer cells at 24 h after injection. Accumulation into MDA-MB-468 tumors was higher than MDA-MB-435 tumors. Pretreatment with C225 (13 nmol/mouse 24 h before IRDye 800CW-EGF injection) inhibited the IRDye 800CW-EGF binding in the tumors by ~50%.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

NIH Support

R01 CA106584-01A1, R01 CA106584-02, R01 EB003132, R01 EB005173


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