Abstract
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.
Highlights
Colorectal cancer (CRC) is one of the most common malignancies in the Western societies
We took advantage of this progress to prepare an epidermal growth factor (EGF)-Near infrared (NIR) bio-imaging agent according to this procedure, and evaluated its pharmacological properties for recognition of epidermal growth factor receptor (EGFR) in novel CRC models in vitro, resembling tumor heterogeneity, orthotopic CRC tumors in mice and CRC tissue slices ex vivo, translating results from CRC cells to human tissue specimens
Consistent with the in vitro findings, specific targeting of EGF conjugated with IRDye 800CW (EGF-NIR) to EGFR was demonstrated by analyzing the NIR images of mice bearing EGFR positive CRC orthotopic tumors
Summary
Colorectal cancer (CRC) is one of the most common malignancies in the Western societies. Long-term survival of CRC-diagnosed patients is correlated with disease stage at diagnosis. At least 40% of patients with CRC will develop either synchronous or metachronous distant metastases, most of them will succumb to their disease and die [2]. Some of characteristics of the malignant phenotype of CRC are correlated with overexpression and hyperactivation of receptor tyrosine kinases such as epidermal growth factor receptor (EGFR), which make these receptors attractive targets for cancer treatment [3]. Endoscopic polypectomy was shown to reduce CRC-related mortality [5]. This procedure requires fibro-optic colonoscopy visualization of the CRC tissue followed by histological evaluation. In addition the CRC tissues are often evaluated by RT-PCR [6], immunohistochemistry [7] and in situ hybridization [8] techniques, which showed a much higher degree of discordance between primaries and related CRC metastases [9]
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