Fluorescent labeled anti-EGFR antibody for identification of regional and distant metastasis in a preclinical xenograft model

Abstract

Background: Detection of regional and distant metastatic disease has significant implications for patient management. Fluorescent imaging may be a useful technique for metastasis detection and removal.

Methods: Anti-epidermal growth factor receptor antibody (cetuximab) and isotype-matched control antibody (immunoglobulin G [IgG]) were labeled with a near-infrared fluorophore (Cy5.5), then systemically administered to mice with tumors resulting from either intraoral or intravenous injections of head and neck squamous cell carcinoma. Mice were sacrificed before undergoing fluorescent stereomicroscopy to assess pulmonary or cervical lymph node metastasis. Fluorescent areas were serially excised until wound bed demonstrated negative fluorescence.

Results: Mice bearing pulmonary metastases displayed diffuse background after IgG-Cy5.5 injection, but demonstrated a speckled fluorescent pattern across lung surface following cetuximab-Cy5.5 injection. Mice bearing cervical metastases demonstrated clear fluorescence of primary tongue tumor and bilateral cervical nodes. Fluorescence correlated with histopathology.

Conclusion: These data suggest that cetuximab-Cy5.5 may have clinical utility in the detection and guided the removal of regional and distant micrometastasis.

FIGURE 1

Cetuximab–Cy5.5 detects pulmonary metastasis in vivo. Injection of the cetuximab–Cy5.5 fluorescent bioconjugate alone in the absence of tumor cells (A–C) demonstrates some diffuse background after fluorescent imaging. Pulmonary metastases are formed after injection of 1 × 10⁶ (D–I) or 2 × 10⁶ SCC1 cells (J–L). Injection of nonspecific IgG1–Cy5.5 bioconjugate in mice bearing pulmonary metastases (D–F) does not demonstrate significant fluorescence after stereomicroscopic imaging. However, when mice bearing 1 × 10⁶ (G–I) or 2 × 10⁶ SCC1 cells (J–L) were administered cetuximab–Cy5.5, a distinctive miliary pattern of fluorescence was seen and tumor micrometastases were confirmed by pathology. Bar = 5 mm.

FIGURE 2

Fluorescent spots correlate to tumor micrometastasis. Biopsy of 2-mm fluorescent dot coincides with presence of micrometastatic disease as confirmed by pathology. Bar = 5 mm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

FIGURE 3

Cervical metastases in an orthotopic murine model were detected with systemically injected cetuximab–Cy5.5 conjugate. The primary tongue tumor (A–C) was clearly visualized under fluorescent imaging after systemic injection of cetuximab–Cy5.5. After removal of cervical skin, bilateral draining lymph nodes could be identified and confirmed by pathology. Bar = 2 mm.

FIGURE 4

Most intense lymph node fluorescence is seen as a result of specific binding. Injection of the cetuximab–Cy5.5 conjugate alone in the absence of tumor cells (A, B) demonstrates very little background fluorescence after stereomicroscopic imaging. Mice bearing primary and metastatic OSC19 tumors display diffuse fluorescence after injection of labeled isotype control IgG1–Cy5.5 (C, D). Fluorescent imaging after injection of specific cetuximab–Cy5.5 conjugate allows excellent visualization of clearly circumscribed tumors, both primary and metastatic (E, F). The difference in fluorescence intensity between the 3 groups of lymph nodes was measured and found to be very statistically significant (p = .0005). Bar = 2 mm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

FIGURE 5

Systemic injection of cetuximab–Cy5.5 conjugate enables fluorescence-guided surgical lymph node resection. Fluorescent areas were excised until fluorescence disappeared (A, C). Although tumor is not entirely clear on the hematoxylin-eosin (H&E) stain (B), due to artifact from crushing during handling of the specimen, immunohistochemical staining using cytokeratin AE1 antibody (D) emphasizes pathologic epithelial tissue within the lymph node. Only 1 node resection is shown. Bar = 2 mm.