In Vivo Imaging of Xenograft Tumors Using an Epidermal Growth Factor Receptor-Specific Affibody Molecule Labeled with a Near-infrared Fluorophore

In Vivo Imaging of Xenograft Tumors Using an Epidermal Growth Factor Receptor-Specific Affibody Molecule Labeled with a Near-infrared Fluorophore

Haibiao Gong, Joy Kovar, Garrick Little, Huaxian Chen, and David Michael Olive

Introduction:
The epidermal growth factor (EGF) receptor (EGFR, HER1, ErbB1) is a transmembrane protein of the tyrosine kinase receptor family. Aberrant overexpression and/or activation of EGFR is associated with many types of cancers, including skin, breast, ovary, bladder, prostate, kidney, head and neck, and non-small cell lung cancers.
To assess the expression level of EGFR in vivo, noninvasive imaging methods are necessary. Using Affibody molecules as imaging agent is one of the approaches. Affibody molecules are a class of affinity proteins composed of 58 amino acid residues that are derived from one of the immunoglobulin G (IgG)-binding domains of staphylococcal protein A.
In this study, EGFR-specific Affibody (Eaff), which would not activate the EGFR pathway, was labeled with a NIR fluorophore for in vivo optical imaging. The NIR fluorophore labeled EGFR-specific Affibody (Eaff80) was bound specifically by EGFR-overexpressing A431 cells. They also examined the specificity of Eaff800 in vivo by imaging with both Eaff800 and an HER2-specific Affibody labeled with another NIR fluorophore (Haff682).

Material and Methods:
The Affibody molecules were provided by Affibody AB (Bromma, Sweden). The Eaff molecule is in a head-to-tail dimeric form with a molecular weight of 13.7 kDa. The HER2-specific Affibody (Haff) molecule is a fusion protein of the HER2 monomer with the album
in-binding domain (ABD) and has a molecular weight of 12.1 kDa. The Affibody molecules were conjugated with NIR dyes. The human skin epidermoid carcinoma cell line A431, ovarian adenocarcinoma cell line SK-OV-3 (SKOV3), and breast adenocarcinoma cell lines MDA-MB-231 (MDA231) and SK-BR-3 (SKBR3) were cultured. The cells were treated with Eaff or EGF, incubated at 37°C for 2 hours, cells were rinsed with PBS and lysed with radioimmunoprecipitation assay buffer. Then Western Blot were performed.

Results:

The Effect of Eaff on EGFR and ERK1/2 Phosphorylation

It is known that activation of the EGFR tyrosine kinase activity leads to the phosphorylation of a variety of target proteins, including ERK1/2 and EGFR itself . To evaluate whether Eaff stimulates EGFR-mediated signaling pathways, A431 cells were treated with 5 or 20 nM Eaff. In contrast to EGF, which stimulated the phosphorylation of both proteins in a dose-dependent manner, Eaff treatment did not change the phosphorylation level of EGFR and ERK1/2 (p44/p42 MAPK). However, a high concentration of Eaff (100 nM), when applied together with 5 nM EGF, compromised the stimulatory effect of EGF on EGFR and ERK1/2 phosphorylation(figure 1).

Figure 1

The effect of EGF and EGFR-specific Affibody (Eaff) on EGFR-mediated phosphorylation of EGFR and ERK1/2 (P44/42 MAPK) proteins. A431 cells were treated with either Eaff or EGF. Two concentrations (5 and 20 nM) for both Eaff (Eaff5 and Eaff20) and EGF (EGF5 and EGF20) were used. A combination of high concentration Eaff (100 nM, Eaff100) and 5 nM EGF was also used to treat cells. P44/42 MAPK and actin were used as internal controls. The relative expression levels were calculated by dividing the signal intensities of phospho-EGFR or phospho-P44/P42 by actin signal intensities. Note that the molecular weight markers in the second panel (phospho-P44/P42) and the third panel (P44/P42 MAPK) were the same. Ctrl indicates control without drug treatment.

Specific Cellular Binding and Uptake of Labeled Affibody Molecules

The Affibody molecules were conjugated with the maleimide dye to its C-terminal cysteine to avoid unexpecting binding of dye molecule to lysine. The incorporation of dye into the Affibody molecules was monitored by gel electrophoresis (Figure W1). The NIR fluorophore-labeled Affibody molecules were designated as Eaff800 (Eaff labeled with IRDye800CW), Eaff682 (Eaff labeled with DY-682), Haff800 (Haff labeled with IRDye800CW), and Haff682 (Haff labeled with DY-682), respectively.

To compare the protein expression levels of EGFR and HER2 in various cell lines, cell lysates of MDA231, A431, SKOV3, and SKBR3 cells were analyzed by Western blot. Both EGFR and HER2 proteins were detected in these cell lines. However, the EGFR level was much higher in A431 cells than in any other cell lines, whereas HER2 was highly expressed in SKOV3 and SKBR3 cells (Figure 2A). The binding and uptake assay was performed by incubating 5 nM Eaff800 or Haff800 with MDA231, A431, SKOV3, and SKBR3 cells. A431 cells contained high Eaff800 signal, whereas only minimal Eaff800 binding and uptake for MDA231, SKOV3, or SKBR3 cells. On the contrary, the HER2-specific Haff800 showed the strongest signal in SKOV3 and SKBR3 cells, and the signal in MDA231 and A431 cells was low (Figure 2A).

Figure 2

Specific binding and uptake of IRDye800CW-labeled Affibody molecules. (A) The protein expression levels of EGFR and HER2 in MDA-MB-231 (MDA231), A431, SKOV3, and SKBR3 cells. Actin served as an internal control. The relative expression levels were calculated by dividing the signal intensities of EGFR or HER2 by actin signal intensities. (B) The binding and uptake of EGFR-specific Eaff800 and HER2-specific Haff800 by MDA231, A431, SKOV3, and SKBR3 cells. (C) Concentration-dependent binding and uptake of Eaff800-, Haff800-, or IRDye800CW-free dye by A431 cells.

Comparison of Eaff800 with EGF800
Binding and uptake of Eaff800 was compared with EGF800. When both Eaff800 (5nM) and EGF800 (5nM) were diluted and incubated with A431 cells for different periods. The binding, uptake, and signal intensity of EGF800 were stronger than that of Eaff800 in the early stage. However binding and upatke of EGF800 decreased after 1 hour when the Eaff800 signal was still increasing. The Eaff800 signal reached maximum at 4 to 6 hours and declined thereafter (Figure 3A).

Figure 3

The comparison of cellular binding and uptake between Eaff800 and EGF800. (A) Binding and uptake time course of Eaff800 (5 nM) and EGF800 (5 nM) by A431 cells. (B) The blocking of Eaff800 (5 nM) binding by increasing concentrations of unlabeled Eaff or EGF. (C) Microscopic examination of Eaff800 (20 nM) and EGF800 (20 nM) binding and uptake by A431 cells. Sytox green was used to stain the nuclei. Scale bar, 10 µm.

Competition analysis was performed by incubating different concentrations of unlabeled Eaff or EGF with A431 cells before adding targeting agents. Both Eaff and EGF blocked the binding and uptake of Eaff800. The blocking effect of Eaff and EGF was observed at a concentration as low as 5 nM and with the increase of competitor concentrations, the Eaff800 signal declined. However, the competition effect of Eaff was more prominent than that of EGF, especially at concentrations higher than 20 nM (Figure 3B). It was also noted that both Eaff and EGF blocked the binding and uptake of EGF800 by A431 cells (data not shown).

Targeting EGFR-Overexpressing Xenograft Tumors by Eaff800
Nude mice bearing A431 tumors were injected with 0.5 nmol of Eaff800 through the tail vain. High level of Eaff800 signal was observed in the liver and kidney after 1 hour. The signal decline dramatically during the 6 to 48 hours. Most of the Eaff800 was cleared out of the body. The residual signals in the tumor and normal tissue were 8.7 ± 0.5% and 5.7 ± 1.0% of those at the highest levels (20 minutes after agent injection), respectively.

Figure 4

In vivo optical imaging of nude mice bearing A431 tumors using Eaff800. (A) A representative series of whole body images (dorsal view) acquired at different time points after injection of 0.5 nmol of Eaff800. The tumors were indicated with arrows. (B) Clearance of Eaff800 from the tumor and normal tissue. Average signal intensities were quantified using ROIs of equivalent-sized areas from the tumor sites and contralateral sites at indicated time points. Data were presented as mean ± SD of three individual mice. (C) TBR at different time points after probe injection. TBR was calculated by dividing the mean tumor signal by the mean background signal of the contralateral site.

The mice were killed 1 day after injection to analysis the distribution of Eaff800 in different organs. Quantification of tissue section revealed the strongest signal in the liver, followed by the kidney and tumor. All other tissues contained only a low level of signal (Figure 5, B and C). Interestingly, unlike the even distribution of Eaff800 in the liver, a higher Eaff800 signal was located in the renal cortex of the kidney compared with other regions (Figure 5B).

Figure 5

Tissue distribution of Eaff800. (A) Nude mice bearing A431 tumors were killed 1 day after Eaff800 injection. The organs were collected and rinsed in PBS before imaging. Ht indicates heart; In, intestine; Kn, kidney; Ln, lung; Lv, liver; Ms, muscle; Tm, tumor. Note that the liver was imaged separately and merged to the picture because the liver signal was so strong that it illuminated the surrounding tissues if imaged together. (B) Fluorescence images of cryosections of dissected organs. The organs were snap-frozen in OCT compound and sectioned at 8-µm thickness. (C) Quantification of signal intensities of tissue sections. Average signal intensities were calculated using ROIs from different tissue sections.
Two-color In Vivo Imaging Using Eaff800 and Haff682
0.5 nmol of Eaff800 and Haff682 were addeed together to A431 cells or SKOV3 cells. Eaff800 binding was stronger in A431 when Haff682 level was stronger in SKOV3. As illustrated in figure 6, The A431 tumor on the left side is green, which represented predominant Eaff800 signal, and the SKOV3 tumor on the right side is red, which represented predominant Haff682 signal. The TBRs for A431 tumor and SKOV3 tumor were 1.7 ± 0.2 and 2.1 ± 0.3, respectively. Because pseudocolored images can distinguish different intensities easier, the mouse images with Eaff800 signal or Haff682 signal were also presented in pseudo color (Figure W2). Images of tumor sections also demonstrated that Eaff800 and Haff682 accumulated preferably in A431 tumors and SKOV3 tumors, respectively (Figure 6B).

Figure 6

Two-color in vivo optical imaging with Eaff800 and Haff682. (A) Nude mice bearing A431 and SKOV3 tumors on the left and right sides, respectively, were injected with 100 µl of PBS containing 0.5 nmol of Eaff800 and 0.5 nmol of Haff682. Whole body images (dorsal view) were acquired 1 day after agent injection. Green and red represent IRDye800CW and DY-682 fluorescence signals, respectively. The tumors were indicated with arrows. (B) Fluorescence images of cryosections of A431 and SKOV3 tumors. Mice bearing A431 and SKOV3 tumors were killed 1 day after agent injection. The tumors were snap-frozen in OCT compound and sectioned at 8-µm thickness.

Discussion
In this experiment Eaff800 had shown its potential as a powerful tool for optical imaging. This agent was based on an Affibody molecule specifically binding to EGFR and labeled with a NIR fluorophore. The specificity of Eaff800 was examined by in vitro cell binding and uptake analysis and confirmed by targeting EGFR-overexpressing tumors in xenograft mouse models. Moreover, in combination with an HER2-specific probe Haff682, Eaff800 could be used to distinguish between EGFR- and HER2-overexpressing tumors.