Abstract Whole animal optical imaging provides new ways to detect tumor cells and other biological activities in the system. Bioluminescent imaging has advantages over fluorescent imaging since animal tissues have less autoluminescence. Due to high signal to background ratio, bioluminescent imaging can detect subtle changes of light emission in the animals. Luciferases are the most popular enzymes for optical imaging. Lights are produced by enzymatic reactions in the presence of substrates. Commonly used luciferases were cloned from firefly (Photinus pyralis) and Sea Pansy (Renilla reniformis). Firefly and Renilla luciferase uses D-luciferin and coelenterazine as a substrate, respectively. Since these luciferases use different kinds of substrates, they can be used in a reporter assay simultaneously. Bioluminescent reporter assays can be achieved by conjugating transcription factor responding elements and a luciferase reporter gene. Upon signals from the outside of the cells, luciferase expression can be regulated. Using multiple responding elements for unique transcription factors, one can identify signaling pathways that are responsive to drug treatments. We have tested 10 different lentivirus reporters that carry destabilized firefly luciferase along with basal promoter element joined to tandem repeats of distinct transcription responding element. PC3M human prostate cancer cells were permanently transfected and stable cell lines were generated. As a control, constitutively active Renilla luciferase reporter was co-transfected to each reporter line. In order to identify signaling pathways that are responding to drug treatment, we plated equal number of reporter cells in well plates and treated cells with various compounds. Cisplatin, lipopolysaccaride (LPS), Paclitaxel, PMA, SB203580 or PD98059 was used for different duration. Bioluminescent images were taken at multiple time points and light emissions were quantitated. Among the compound tested, LPS generated more than 50 fold increase of bioluminescence in NF-kB luciferase (NFkB-luc) reporter cell line in vitro. In addition, the assay showed dose-dependent response. Renilla luciferase activities remained same in all reporter cell lines. To validate in vivo response, PC3M NFkB-luc cells were implanted into male nu/nu mice subcutaneously. As controls, we also implanted a non-responding reporter upon LPS treatment (TGFb-luc) and negative control without responding elements (Neg-luc). Animals were administered with LPS and in vivo bioluminescence images were taken using a cooled CCD camera. Our results showed that animals implanted with NFkB-luc mice showed 6 fold increase of luciferase activity while TGFb-luc or Neg-luc implanted animals showed little change in luciferase activity. Present study demonstrates that bioluminescent reporter assays can be applied to elucidate the mode of action of a drug. 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 5228.

Whole animal in vivo imaging has contributed significantly to the detection of disease progression and drug efficacy for the past several years (1). With the introduction of sensitive imaging instruments, applications in fluorescent imaging have expanded to monitoring tumor cell growth and gene expression. The new fluorescent proteins have improved brightness, photostability and better tissue penetration of signals at a far red wavelength. In addition, there has been an explosion of innovation in a variety of fluorescent proteins developed from existing red fluorescent proteins. Previously, we developed brighter bioluminescent cancer cell lines using enhanced firefly luciferase 2 (luc2) (2). Because 4T1-luc2 cells are so bright, we were able to detect a single bioluminescent cell in vivo. To expand the applications of genetically labeled fluorescent tumor cells, we have developed single and dual labeled 4T1, PC3M and MDA-MB-231 cell lines using tdTomato fluorescent protein and luc2. First, we engineered a vector encoding the tdTomato protein in which expression is under the control of human ubiquitin C promoter (3). Parental cells were stably transfected with the tdTomato vector and antibiotic resistant cells were selected. In parallel, luc2 labeled cells were transfected with the tdTomato vector to develop dual labeled cell lines (4T1-luc2-tdTomato, PC3M-luc2-tdTomato and MDA-MB-231-luc2-tdTomato). Our initial analyses showed that the expression level of tdTomato protein was unstable over time in both single and dual labeled cells. Therefore, we attempted single cell cloning and isolated individual clones of both single and dual labeled cells. Positive clones were subjected to fluorescent activated cell sorting (FACS). Once cells were isolated, luciferase expression was characterized in the dual labeled cell lines. Cultures were maintained to test the stability of tdTomato and luc2 expression levels over a 4 week time period. The results indicated that the cloned populations were stably expressing both reporters. We confirmed that the growth rates of both single and dually labeled cells were comparable to those of the parental cell line. To monitor tumor growth in vivo, cells were implanted subcutaneously and orthotopically into nu/nu mice. Tumor progressions were monitored non-invasively and in real time using an enclosed imaging system containing a cooled CCD camera. Our data demonstrates that we could follow primary tumor growth and metastases in vivo using fluorescent and bioluminescent imaging. These dual reporter systems enable the monitoring of pathway specific signaling in vivo with exquisite sensitivity using both bioluminescence and fluorescence. Furthermore, histological sections can later be interrogated with fluorescent detection.