Abstract 4323: ATP binding cassette transporters modulate both D-luciferin- and coelenterazine-based bioluminescence imaging

Abstract

Abstract Bioluminescence imaging (BLI) of luciferase reporters provides a relatively simple, cost-effective, and extremely sensitive means to image fundamental biological processes in cancer research, including tumor growth, angiogenesis and metastasis. Translocation of D-luciferin (the substrate for Click Beetle Red (cLuc) and Firefly (fLuc) luciferase), and coelenterazine (the substrate for Renilla (rLuc), and Gaussia (gLuc) luciferase), across the cellular membrane could affect BLI intensity in vivo. ATP-binding cassette (ABC) transporters are membrane-bound efflux pumps utilizing the energy from ATP hydrolysis. They are implicated in multidrug resistant phenotypes of tumor cells and may be cancer stem cell markers. To investigate the effect of ABC transporters on luciferase-based BLI, we generated HEK-293 reporter cells with selective overexpression of different ABC transporters (ABCB1, ABCC1 and ABCG2) and different luciferases (cLuc, fLuc, rLuc and gLuc). In vitro studies showed that BLI signal intensity of intact cells compared to cell lysates decreased 89%, 77% and 75% when ABCG2 was overexpressed in HEK-293 cLuc, fLuc and rLuc reporter cells, respectively. No significant BLI change was observed in HEK-293 gLuc reporter cells with high expression of ABCG2. Overexpression of ABCB1 reduced BLI intensity only in HEK-293 rLuc reporter cells by 51%. ABCC1 overexpression did not affect BLI intensity in any of the HEK-293 reporter cells. These data indicate that ABCG2 affects BLI signal intensity generated from both D-luciferin and coelenterazine, and that ABCB1 affects coelenterazine (but not D-luciferin) signal intensity. Selective ABC transporter inhibitors, Reversin 121 against ABCB1, MK-571 against ABCC1, and fumitremorgin C (FTC) against ABCG2, were applied in HEK-293 reporter cells with ABC transporter overexpression. Compared with vehicle treatment, FTC increased BLI intensity more than 2-fold in HEK-293 cLuc, fLuc and rLuc reporter cells; Reversin 121 elevated BLI intensity 2.1 fold only in HEK-293 rLuc reporter cells. BLI of xenografts derived from HEK-293 reporter cells with overexpression of ABC transporters confirmed the results of inhibitor treatment in vivo. These findings demonstrate that ABC transporters can significantly modulate intracellular delivery of luciferase-based substrates, and that endogenous expression of ABC transporters can significantly affect BLI readout, both in vitro and in vivo. In this context, the BLI-based assay can provide a high throughout platform to screen the novel ABC transporter inhibitors for the improvement of antitumor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4323.