Bio-imaging of colorectal cancer models using near infrared labeled epidermal growth factor.

Novel strategies that target the epidermal growth factor receptor (EGFR) have led to the clinical development of monoclonal antibodies, which treat metastatic colorectal cancer (mCRC) but only subgroups of patients with increased wild type KRAS and EGFR gene copy, respond to these agents. Furthermore, resistance to EGFR blockade inevitably occurred, making future therapy difficult. Novel bio-imaging (BOI) methods may assist in quantization of EGFR in mCRC tissue thus complementing the immunohistochemistry methodology, in guiding the future treatment of these patients. The aim of the present study was to explore the usefulness of near infrared-labeled EGF (EGF-NIR) for bio-imaging of CRC using in vitro and in vivo orthotopic tumor CRC models and ex vivo human CRC tissues. We describe the preparation and characterization of EGF-NIR and investigate binding, using BOI of a panel of CRC cell culture models resembling heterogeneity of human CRC tissues. EGF-NIR was specifically and selectively bound by EGFR expressing CRC cells, the intensity of EGF-NIR signal to background ratio (SBR) reflected EGFR levels, dose-response and time course imaging experiments provided optimal conditions for quantization of EGFR levels by BOI. EGF-NIR imaging of mice with HT-29 orthotopic CRC tumor indicated that EGF-NIR is more slowly cleared from the tumor and the highest SBR between tumor and normal adjacent tissue was achieved two days post-injection. Furthermore, images of dissected tissues demonstrated accumulation of EGF-NIR in the tumor and liver. EGF-NIR specifically and strongly labeled EGFR positive human CRC tissues while adjacent CRC tissue and EGFR negative tissues expressed weak NIR signals. This study emphasizes the use of EGF-NIR for preclinical studies. Combined with other methods, EGF-NIR could provide an additional bio-imaging specific tool in the standardization of measurements of EGFR expression in CRC tissues

Whole body <i>in vivo</i> and isolated tissue <i>ex vivo</i> EGF-NIR BOI of mice with HT-29 orthotopic tumors.

<p>(A) Photograph of an orthotopic tumor and EGFR protein expression in the tumors; (B) Time course of EGF-NIR accumulation in tissues of tumor-bearing mice. The mice were injected i.v. with 1 nmol of EGF-NIR in untreated mice (upper row, n = 6) or mice pre-injected with 1 µg/ml cetuximab (lower row, n = 4); high resolution BOI of a mouse injected with EGF-NIR and circles indicate ROI measurements (C) Time course of tissue accumulation of EGF-NIR at 48 hours from mice presented in B; Signal intensity at 800 nm were normalized to background fluorescence using an arbitrary tumor circle (10–20 ROIs/mouse) compared to an identical area on the flank (adjacent muscle); * p<0.05 compared to EGF-NIR 4 hours; ** p<0.05 compared to mice injected with EGF-NIR; (D) EGF-NIR signal/background ratio in isolated tissue from the tumor-bearing mice 48 hours after injection. * p<0.05 compared to muscle, ** p<0.05 compared to liver; Insert: Upper-photographs of tissues in the dish; Middle-NIR images; Lower-spectral intensity maps; Intensity scale-red-brown (5) high expression; blue (3) very low expression.</p