Characterizing breast cancer xenograft epidermal growth factor receptor expression by using near-infrared optical imaging

Abstract

Background: Epidermal growth factor receptor (EGFR) overexpression is associated with several key features of cancer development and growth. Therefore, EGFR is a very promising biological target for tumor diagnosis and anticancer therapy. Characterization of EGFR expression is important for clinicians to select patients for EGFR-targeted therapy and evaluate therapeutic effects.

Purpose: To investigate whether near-infrared (NIR) fluorescent dye Cy5.5-labeled anti-EGFR monoclonal antibody Erbitux can characterize EGFR expression level in MDA-MB-231 and MCF-7 breast cancer xenografts using an in vivo NIR imaging method.

Material and methods: A fluorochrome probe was designed by coupling Cy5.5 to Erbitux through acidylation, and the fluorescence property of the Erbitux-Cy5.5 conjugate was characterized by fluorospectroscopy. Flow cytometry and laser confocal microscopy were used to test the EGFR specificity of the antibody probe in vitro. Erbitux-Cy5.5 was also injected intravenously into immune-deficient mice bearing MDA-MB-231 or MCF-7 tumors. Whole-body and region-of-interest fluorescence images were acquired and analyzed. The EGFR expression was also analyzed and confirmed by immunohistochemical assay.

Results: The maximum excitation/emission wavelength for the Erbitux-Cy5.5 probe was 674/697 nm, similar to that of free Cy5.5 (674/712 nm). Confocal microscopy confirmed receptor-specific uptake in MDA-MB-231 and MCF-7 cells. In flow cytometry probe specificity assay, Erbitux-Cy5.5 showed a 9.32-fold higher affinity for MDA-MB-231 than MCF-7 cells. In vivo NIR imaging also indicated specific uptake in EGFR-positive tumors. Probe uptake rate and maximum intake dose in MDA-MB-231 were significantly higher than those in MCF-7 xenografts (P < 0.001). Immunohistochemical staining confirmed the in vivo imaging results, showing differentiated EGFR expression in MDA-MB-231 (+ + +) and MCF-7 (+) tumor tissues.

Conclusion: Erbitux-Cy5.5 may be used as a specific NIR contrast agent for the noninvasive characterization of EGFR expression level in breast cancer xenografts.

Fig. 1

In vitro probe binding studies by flow cytometry and confocal microscopy. A. Flow cytometry results for MDA-MB-231 and MCF-7 cell lines incubation with ligand linked to a fluorescent dye. Five nM Erbitux-Cy5.5 showed strong binding to EGFR-overexpressing MDA-MB-231 cells and weak binding to low-EGFR-expressing MCF-7 cells. B. Confocal differential interference contrast (DIC) and fluorescence images of MDA-MB-231 and MCF-7 cells in the presence of Erbitux-Cy5.5. The first and third rows display the results from the cells incubated with 5 nM Erbitux-Cy5.5. The second and fourth rows display cells pretreated with Erbitux before incubation with Erbitux-Cy5.5. All images were acquired under the same conditions and are displayed on the same scale (scale bar, 10 μm).

Fig. 2

In vivo near-infrared (NIR) imaging. MDA-MB-231 or MCF-7 cells were injected into the left chest mammary gland fat pad of female athymic nude mice. Representative whole-body NIR images of MDA-MB-231 and MCF-7 xenograft mice at 24 hours post injection of Erbitux-Cy5.5 are shown. Fluorescence signal is clearly visualized in the left thoracic tumor region of MDA-MB-231 and MCF-7 xenografts. Blocking experiment indicates an apparent decrease of fluorescent signals by pre-injection of excess Erbitux (tumors indicated by arrows).

Fig. 3

In vivo probe kinetics comparison in MDA-MB-231 and MCF-7 tumor models. Fluorescence intensity in the tumor regions after injection of Erbitux-Cy5.5 in mice with MDA-MB-231 tumors (A) and with MCF-7 tumors (C) was significantly higher than that in normal regions (P <0.001) and changed with time (P <0.001 both for MDA-MB-231 and MCF-7, B and D). In mice with MDA-MB-231 tumors, the rate of Erbitux-Cy5.5 uptake was higher than that of MCF-7 tumors, with maximum probe uptake dose 1.65-fold higher in MDA-MB-231 than MCF-7 tumors (P <0.001). All plots are representative of results from the group of mice treated under the same experimental conditions (n = 5/group).

Fig. 4

EGFR immunohistochemical assay. A. The strong red-brownish membrane-bound immunostaining on the tumor cells reflects the abundance of EGFR in MDA-MB-231 (+ + +) (400×). B. The moderate brownish membrane of the MCF-7 tumor cells reflects weak-to-normal EGFR expression (+) (400×). C. Negative EGFR expression control showed no membranous staining (−) (400×).