Near-Infrared Fluorescence Imaging of EGFR-Overexpressing Tumors in the Mouse Xenograft Model Using scFv-IRDye800CW and Cetuximab-IRDye800CW

Abstract

EGFR (epidermal growth factor receptor) is overexpressed in a variety of human cancers (including squamous cell carcinoma of head and neck, colon cancer, and some breast cancers) and therefore is regarded as an ideal target for cancer therapy or imaging purposes. In the current study, we produced a scFv-based near-infrared probe (called cet.Hum.scFv-IRDye-800CW) and evaluated its ability in recognizing and imaging of EGFR-overexpressing tumors in a mouse model. Like the molecular probe consisting of its parental antibody (cetuximab, an FDA-approved monoclonal antibody) and IRD800CW, cet.Hum.scFv-IRDye-800CW was able to recognize EGFR-overexpressing tumors in mice. cet.Hum.scFv-IRDye-800CW was found to be superior to the cetuximab-based probe in imaging of mouse tumors. The tumor-to-background ratio and blood clearance rate were higher when cet.Hum.scFv-IRDye-800CW was used as an imaging probe.

Figure 1

SDS-PAGE and western blotting results. (a) His-tag affinity chromatography purified cet.Hum.scFv. Protein bands of the same molecular weight (27 kDa) appeared in 1 lane. (b) The results of western blotting with the antibodies and A-431 and U-87 MG cells. Alpha-tubulin was used as the loading control. Both cet.Hum.scFv and cetuximab are able to form thick protein bands of approximately 175 kDa with A-431 cells, but not with U-87 MG cells.

Figure 2

Characterization of IRDye800CW-labeled cet.Hum.scFv and cetuximab. SDS-PAGE image after Coomassie blue staining (lanes 1, 2, and 3 are protein ladder, cetuximab, and cet.Hum.scFv, respectively). The SDS-PAGE under near-infrared filter-equipped animal imaging system (lanes 4 and 5 are cetuximab and cet.Hum.scFv, respectively).

Figure 3

The saturation binding curves of IRDye800CW-labeled and unlabeled antibodies when reacting with A-431 and U-87 cells. All experiments were done in triplicate.

Figure 4

NIR fluorescence images of cet.Hum.scFv-IRDye800CW and cetuximab-IRDye800CW conjugates in mice bearing A-431 tumor xenografts. Images were obtained at noninjection and at 1, 4, 24, 48, 72, and 96 hours postinjection of 100 μL for the cet.Hum.scFv-IRDye800CW and cetuximab-IRDye800CW (75 μg). Scale bar changes are shown on the top. The degree of labeling or D/P ratio of cet.Hum.scFv-IRDye800CW and cetuximab-IRDye800CW was 1.983 and 2.128, respectively.

Figure 5

NIR fluorescence analysis of IRDye800CW-conjugated molecules after applying on A-431 tumor-bearing mice. (a) Intensity of fluorescent signals emitting from tumor tissues. (b) Tumor-to-background ratio analysis. Error bars represent mean ± standard deviation. The asterisks indicate significant differences between groups (^∗p < 0.05, ^∗∗p < 0.01).

Figure 6

NIR fluorescence imaging of U-87 MG-bearing mice after injection of IRDye800CW-conjugated antibodies. Degree of labeling and amount of injection are the same as described in the Figure 4 caption.

Figure 7

NIR fluorescence analysis of U-87 MG tumors receiving IRDye800CW-conjugated antibodies. (a) Signal intensity 24 h after injection; both antibodies raised the same signal intensity. The maximum difference in signal intensity occurred 48 h after injection; a significant difference at statistical level of 5% (^∗p < 0.05). (b) Tumor-to-background ratio. There were no significant differences in the ratios of cet.Hum.scFv-IRDye800CW and cetuximab-IRDye800CW.

Figure 8

Molecular structure of IRDye800CW NHS ester (https://www.licor.com/bio/reagents/irdye-800cw-nhs-ester).