The Western blotting was repeated three times, and we observed the same results each time. with fluorophore-conjugated IGF-1R antibody demonstrated fluorescent foci on the membrane of colon cancer cells. Subcutaneously- and orthotopically-transplanted HT-29-GFP and HCT 116-GFP tumors brightly fluoresced at the longer wavelengths after intravenous administration of fluorescent IGF-1R antibodies. Orthotopically-transplanted HCT 116-GFP tumors were brightly labeled by fluorescent IGF-1R antibodies CFD1 such that they could be imaged non-invasively at the longer wavelengths. In an experimental liver metastasis model, IGF-1R antibodies conjugated with PEGylated 650 nm fluorophores selectively highlighted the Indoximod (NLG-8189) liver metastases, which could then be non-invasively imaged. The IGF-1R fluorescent-antibody labeled liver metastases were very bright compared to the normal liver and the fluorescent-antibody label co-located with green fluorescent protein (GFP) expression of the colon cancer cells. The present study thus demonstrates that fluorophore-conjugated IGF-1R antibodies selectively visualize metastatic colon cancer and have clinical potential for improved diagnosis and fluorescence-guided surgery. Introduction Colorectal cancer is the second leading cause of cancer-related deaths in Western countries [1]. Development of colonoscopy enables early detection and removal of precancerous adenomatous polyps [2,3]. Complete surgical resection can cure well selected patients with liver metastasis [4,5]. Improved imaging of metastatic colon cancer should increase survival by making colonoscopy and surgery more effective. We have previously shown that targeting orthotopic metastatic colon cancer, including patient-derived orthotopic xenografts (PDOX) models, with fluorescent anti-carcinoembyronic antigen (CEA) antibodies enabled improved tumor visualization and Indoximod (NLG-8189) effective fluorescence-guided surgery (FGS) [6]. Targeting of the epidermal growth factor receptor with fluorescent antibodies improved colonoscopy [7]. Type I insulin-like growth factor receptor (IGF-1R) is a transmembrane tyrosine kinase receptor comprising two and two chains and is the major receptor for IGF-I and IGF-II. IGF-1R is Indoximod (NLG-8189) expressed in 51~100% of the colon cancers depending on the study [8C10]. The high frequency of expression in colon cancer and membrane subcellular location make IGF-1R a potential target for fluorescent antibodies to enable cancer visualization, diagnosis, and FGS [11]. Using IGF-1R also has additional clinical potential since the overexpression of IGF-1R correlates with shorter median survival of colon cancer patients who undergo surgery and adjuvant chemotherapy [10]. The present report demonstrates the feasibility of IGF-1R targeted fluorophore-conjugated antibodies to visualize metastatic colon cancer in appropriate mouse models. Materials and Methods Colon cancer cell lines The human colon cancer Indoximod (NLG-8189) cell lines HT-29 [12] and HCT 116 [13] were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), penicillin/streptomycin (Gibco-BRL, Carlsbad, CA), sodium pyruvate (Gibco-BRL), sodium bicarbonate (Cellgro, Manassas, VA), L-glutamine (Gibco-BRL), and minimal essential medium nonessential amino acids (Gibco-BRL). All cells were cultured at 37C in a 5% CO2 incubator. Construction of GFP-expressing colon cancer cell line The construction of green fluorescent protein (GFP) expressing HCT 116 is previously described [14C16]. For GFP gene transduction, 20% confluent HCT 116 cells [17] were incubated with a 1:1 mixture of retroviral supernatants of the PT67 packaging cells and RPMI 1640 (Gibco-BRL, Life Technologies, Inc.) for 72 h. The cells were harvested by trypsin/EDTA 72 h after incubation with GFP retroviral supernatants and subcultured at a ratio of 1 1:15 into selective medium that contained 200 g/ml G418. The level of G418 was increased to 800 g/ml stepwise. Clones expressing GFP were isolated with cloning cylinders (Bel-Art Products, Pequannock, NJ) by trypsin/EDTA and were amplified and transferred by conventional culture methods. High GFP expression clones were then isolated Indoximod (NLG-8189) in the absence of G418 for > 10 passages to select for stable expression of GFP [14C16]. Mice Athymic nu/nu nude mice (AntiCancer Inc., San Diego, CA), 4C6 weeks old, were used in this study. Mice were kept in a barrier facility under HEPA filtration..