Epidermal growth factor receptor-targeted near-infrared probe cetuximab-IRDye800CW enables stable and tumor-specific fluorescence imaging in colorectal cancer models

Significance: Colorectal Cancer (CRC) remains a major cause of cancer-related mortality, with incomplete resection contributing to recurrence and poor survival. Improving intraoperative margin detection is critical to enhance surgical precision and patient outcomes.

Aim: We aim to evaluate the tumor-targeting specificity of cetuximab-IRDye800CW (Cet-IRDye800CW), an epidermal growth factor receptor (EGFR)-specific near-infrared probe, for fluorescence-guided surgery in CRC models.

Approach: Cet-IRDye800CW and IgG-IRDye800CW controls were synthesized and tested in LS174T and SW948 xenografts as well as patient-derived xenografts (PDXs). Mice received $200\mu g$ of the probe intravenously and underwent daily fluorescence imaging for 10 days. Tumor-to-background ratio (TBR) and mean fluorescence intensity (MFI) were quantified, and EGFR expression was analyzed by Western blot, reverse transcription quantitative polymerase chain reaction, and immunohistochemistry.

Results: Cetuximab-IRDye800CW significantly enhanced tumor fluorescence compared with controls in all models ( $p<0.05$ ). TBR increased progressively and peaked on day 10 (LS174T: $25.6\pm 2.94$ ; SW948: $21.6\pm 1.71$ ), whereas background MFI declined, resulting in improved tumor contrast. Tumor-specific fluorescence was detectable from day 1 and intensified over time. Similar findings were observed in PDX models, consistent with high EGFR expression.

Conclusion: Cetuximab-IRDye800CW provides stable, tumor-specific, high-contrast fluorescence imaging in EGFR-high CRC models. These findings validate its molecular targeting capability and support its translational potential for fluorescence-guided CRC surgery.