MULTIPLEX GPCR INTERNALIZATION ASSAY USING REVERSE TRANSDUCTION ON ADENOVIRAL VECTOR IMMOBILIZED MICROPARTICLES S. Han1,2, H.J. Bae1,2, W. Park3 and S. Kwon1,2* 1Department of Electrical and Computer Engineering, Inter-university Semiconductor Research Center (ISRC), Seoul National University, SOUTH KOREA 2Center for Nanoparticle Research, Institute for Basic Science (IBS), SOUTH KOREA and 3Department of Electronics and Radio Engineering, Institute for Laser Engineering, Kyung Hee University, SOUTH KOREA ABSTRACT We present a new multiplexing method for high-throughput cell-based assays in a microtiter well based on reverse transduction of cells by adenoviral vectors immobilized on encoded microparticles. Our particle-based approach spatially confines the gene delivery to cells seeded on the particles and provides the code for identifying the delivered gene, thus easily achieving a multiplex cell microarray in a microtiter well by means of a single pipetting without the cross- expression of genes and the positional identification. Utilizing this method with adenoviral vectors having a G-protein coupled recpeptor (GPCR) gene, we demonstrated 3-plex GPCR internalization assay. KEYWORDS: Multiplex GPCR assay, Reverse transduction, Adenovirus, Encoded microparticle INTRODUCTION G-protein coupled receptors (GPCRs) in the cell membrane are major drug targets in pharmaceutical industry since they interact with a huge variety of endogenous ligands and trigger intracellular functions related to many physiological processes or diseases [1]. Many cell-based assay strategies have been developed to identify GPCR-targeted drugs with more biologically relevant data. Since typical cell-based assays are performed in the microtiter wells and it allows only one type of receptor for each well, multiplex cellular assay technologies have emerged to run high-throughput compound screening with over several hundreds of GPCRs. The reverse transfection technology provides an efficient method to cultivate heterogeneous cell types in one assay site because it uses an array of discrete plasmid DNA spots on a glass slide so that cellular uptake of the gene and its expression occur locally as cells grow on the spots [2]. However, a bunch of sequential spotting process are required using an expensive printing machine to compose heterogeneous spots on one slide. Here, we present a new reverse transduction technology utilizing encoded microparticles, immobilization of adenoviral vectors on them, and heterogeneous dispensing of them into a microtiter well by means of a single pipetting instead of a repetitive spotting process. Figure 1: (a) A sequential illustration of multiplex gene expression in a microtiter well using reverse transduction of cells on adenoviral vector immobilized encoded microparticles. (b) Fabrication of shape-coded microparticles using OFML and immobilization of adenoviral vectors on the particles. (c) Verification of virus immobilization by SEM. 978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 642 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences 27-31 October 2013, Freiburg, Germany THEORY Recombinant adenoviral vectors have become popular gene transfer agents into living cells with high transduction efficiency and are also suitable for preparing cells with an expression of a specific GPCR for cell-based GPCR assays. However, since they are almost always used free in solution, it is hard to accomplish localized or site-specific delivery of them. To address this challenge, we use shape-coded microparticles as a solid substrate for immobilizing viral vectors in order to prevent them from diffusing freely in solution (Fig. 1a). After dispensing the particles heterogeneously into a microtiter well with one pipetting, cells are seeded and cultivated on the particles. Then, only cells on the particles are infected and express the protein encoded by adenoviral vectors without cross-expression between particles because viral vectors are immobilized on the particles. In addition, the expressed protein can be recognized by the shape code of the micropaticles, so multiplex cellular composition with different types of GPCRs is easily achieved for multiplex GPCR assays. GPCR internalization is the process that redistributes the receptor proteins from cell membrane into intracellular compartments when they are activated ligands [3]. This process results in activation or inhibition of a certain intracellular function, thus monitoring of ligand-induced internalization is one of methods to identify GPCR-targeted drugs. The receptor internalization in living cells can be visualized by using fluorescent reagents such as green fluorescent protein (GFP) and it is evaluated quantitatively by image processing of the fluorescent images. In this study, we used GFP- tagged GPCR adenoviral vectors to visualize the GPCR internalization in living cells and the internalization was evaluated quantitatively by measuring the formation of clusters of GFP-tagged receptors using open-source cell image analysis software (CellProfiler). EXPERIMENTAL In order to fabricate the shape-coded microparticles, we used optofluidic maskless lithography system (OFML) [4] composed of a microfluidic channel and an optical lithography (Fig. 1b). Poly(ethylene glycol) diacrylate was copolymerized with acrylic acid in the microchannel by an exposure to patterned UV light to produce carboxyl group on the particle. Then, the adenoviral vectors containing a certain gene were immobilized on the corresponding particles via EDC/NHS coupling. After washing out unbound vectors, particles were gathered together to generate the library of viral vector immobilized particles. The immobilization of adenoviral vectors on the particles was confirmed by SEM observation after OsO4 vapor fixation (Fig. 1c). Adenovirus was recognized by its typical size, about 100 nm in diameter. For GPCR internalization assay, three different kinds of GFP-tagged GPCR viral vectors were immobilized on three different shapes of particles respectively. These particles were dispensed to microtiter wells simultaneously with a single pipetting. Then, U-2 OS cells were seeded at a density of 20,000 cells per a well and incubated for 40 hours for localized heterogeneous transduction. Infected cells were washed with PBS three times and incubated in serum-free medium for 1 hour. The cells were stimulated by ligand for 1 hour. After fixation with formaldehyde solution and nuclei staining with Hoechst 33342 dye, images were acquired. RESULTS AND DISCUSSION In Fig. 2a, GFP and mCherry adenoviral vectors were immobilized on the circular and square shaped particles, respectively. Then, U-2 OS cells were seeded on the particles to produce different types of cells in one well. The overlay of green and red channel images shows multiplex fluorescent gene expression of cells on the particles without cross-expression between particles. With this confirmation of localized reverse transduction on the particles, we generated heterogeneous cell configuration with three different GPCR proteins in one microtiter well using GFP-tagged GPCR adenoviral vectors immobilized particles: cholecystokinin B receptor (CCKBR) on circle, galanin receptor 1 (GALR1) on square, and histamine receptor H1 (HRH1) on star (Fig. 2b). The type of expressed receptor in cells on the particle was identified by recognizing the shape code of particles in the bright-field image. GPCR internalization assay with three kinds of ligands was performed with this microtiter plate. Cholecystokinin octapeptide (CCK-8), human galanin, and 2-(4-Imidazolyl)ethylamine (Histamine) were used and they are well-known ligands for CCKBR, GALR1, and HRH1 respectively. Representative images for each reaction after 1 hour stimulation are shown in Fig. 3. As shown in the images, clear granular formation of internalized receptors in cells was observed only for their ligands. We measured the internalization quantitatively by detecting this granular formation based on its intensity and size. The quantitative value was calculated by dividing sum of pixel values of granules by sum of pixel values of a cell. In the graphs, each receptor has the highest value against its ligand among three ligands. Figure 2: Multiplex expression of (a) fluorescent proteins (GFP, mCherry) and GFP-tagged GPCR proteins using the viral vector immobilized microparticles. 643 Figure 3: Multiplex GPCR internalization assay using adenoviral vector immobilized microparticles with 3 receptors (CCKBR, GALR1, and HRH1) and 3 ligands (CCK-8, Galanin, and Histamine). The receptor internalization was visualized using GFP-tagged GPCRs and the graphs show quantitative measurements of the internalization. CONCLUSION In conclusion, we developed reverse transduction method using viral vector immobilized encoded microparticles for multiplex cell-based GPCR internalization assays. The assay result shows the feasibility of our method as a high- throughput screening method to identify correct ligands for GPCRs. We envision that our technology could apply the multiplexing to many kinds of cell-based assays for increasing their throughput. ACKNOWLEDGEMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea govern- ment (MEST) (No. 2010-0017860 and NRF-2011-35B-D00015). REFERENCES [1] W. Thomsen, J. Frazer and D. Unett, "Functional assays for screening GPCR targets," Curr. Opin. Biotech., vol. 16, pp. 655-665, 2005. [2] J. Ziauddin and D. M. Sabatini, "Microarrays of cells expressing defined cDNAs," Nature, vol. 411, pp. 107-110, 2001. [3] S. Fukunaga, S. Setoguchi, A. Hirasawa and G. Tsujimoto, "Monitoring ligand-mediated internalization of G pro- tein-coupled receptor as a novel pharmacological approach," Life Sci., vol. 80, pp. 17-23, 2006. [4] S. E. Chung, W. Park, H. Park, K. Yu, N. Park and S. Kwon, "Optofluidic maskless lithography system for real- time synthesis of photopolymerized microstructures in microfluidic channels," Appl. Phy. Lett., vol. 91, pp. 041106- 041106-3, 2007. CONTACT *S. Kwon, tel: +82-2-880-1736; [email protected] 644.