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We aimed to develop a far-red luminescence imaging technology for visualization of disease specific antigens on cell surfaces in a living body. First, we conjugated a far-red fluorescent indocyanine derivative to biotinylated Cypridina luciferase. This conjugate produced a bimodal spectrum that has long-wavelength bioluminescence emission in the far-red region as a result of bioluminescence resonance energy transfer. To generate a far-red luminescent probe with targeting and imaging capabilities of tumors, we then linked this conjugate to an anti-human Dlk-1 monoclonal antibody via the biotin-avidin interaction. This far-red luminescent probe enabled us to obtain high-resolution microscopic images of live, Dlk-1-expressing Huh-7 cells without an external light source, and to monitor the accumulation of this probe in tumor-bearing mice. Thus this far-red luminescent probe is a convenient analytical tool for the evaluations of monoclonal antibody localization in a living body.Cypridina luciferase ͉ far-red luminescent probe ͉ luciferin ͉ tumor A n increasing number of monoclonal antibodies have been used to target antigens on cancer cells for clinical diagnosis and therapy, based on the fact that some antigens expressed on cancer cells surface reflect malignant behaviors invasion, metastasis, and neo-vascularization (1-5). Molecular imaging of antibodies in the whole body will enable us to prescribe the appropriate antibody therapy in terms of dose and the timing of administration. Fluorescence imaging (FLI) and bioluminescence imaging (BLI) have played an important role in molecular imaging in small animals (6-8). Photon detection is affordable and easy to use compared with radioisotope imaging. BLI is achieved with a luciferin-luciferase reaction in the presence of molecular oxygen. However, most bioluminescence spectra are in the visible region, overlapping with the absorption spectrum of hemoglobin, attenuating the bioluminescence intensity in live animals. Recently, a ''self-illuminating quantum dot probe'' was developed to improve the light penetration based on bioluminescence resonance energy transfer (BRET) between the bioluminescence of Renilla luciferase and quantum dots (9). The multivalent conjugation of Renilla luciferase to single dots allowed for highly efficient BRET between luciferase and quantum dots. However, the large size of the conjugate may cause problems in metabolism and localization in vivo (10).BRET is a natural phenomenon observed in marine organisms. Green fluorescent protein, for example, is a well-known energy acceptor in the bioluminescence of Renilla luciferase and aequorin. BRET between the bioluminescence of Renilla luciferase and green fluorescent protein mutants has been used to study protein interactions (11). Recently several far-red fluorescent protein variants showing emission maxima around 650 nm were developed for in vivo imaging (12), but have not been well characterized as energy acceptors for BRET systems. On the other hand, the organic dyes indocyanine and its derivat...

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Purpose: Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that induces immune tolerance in mice. Our prior study showed that high tumoral IDO expression in endometrial cancer tissues correlates with disease progression and impaired patient survival.The purpose of the present study was to clarify the functional role of IDO in human endometrial cancer cells and to investigate the therapeutic potential of IDO inhibitors. Experimental Design: IDO cDNA was transfected into the human endometrial carcinoma cell line AMEC, resulting in the establishment of stable clones of IDO-overexpressing AMEC cells (AMEC-IDO). AMEC-IDO cells were characterized in vitro as well as in vivo using a mouse xenograft model. Results: There was no significant difference in in vitro cell proliferation, migration, or chemosensitivity to paclitaxel between AMEC-IDO and control vector^transfected cells (AMEC-pcDNA). However, in vivo tumor growth was markedly enhanced in AMEC-IDO^xenografted nude mice when compared with AMEC-pcDNA^xenografted mice. Splenic natural killer (NK) cell counts in AMEC-IDO^xenografted mice were significantly decreased when compared with control mice. Furthermore, conditioned medium obtained from AMEC-IDO cell cultures markedly reduced the NK lysis activity of nude mice. Finally, oral administration of the IDO inhibitor 1-methyl-Dtryptophan in combination with paclitaxel in AMEC-IDO^xenografted mice strongly potentiated the antitumor effect of paclitaxel, resulting in significantly prolonged survival. Conclusions: This is the first evidence showing that IDO overexpression in human cancer cells contributes to tumor progression in vivo with suppression of NK cells. Our data suggest that targeting IDO may be a novel therapeutic strategy for endometrial cancer.