Protein Transfection Study Using Multicellular Tumor Spheroids of Human Hepatoma Huh-7 Cells

Citation: Kato T, Tanaka M, Oba M ( Protein Transfection Study Using Multicellular Tumor Spheroids of Human Hepatoma Huh-7 Cells Takuma Kato 0 Masakazu Tanaka 0 Makoto Oba 0 0 Graduate School of Biomedical Sciences, Nagasaki University , Nagasaki , Japan Several protein transfection reagents are commercially available and are powerful tools for elucidating function of a protein in a cell. Here we described protein transfection studies of the commercially available reagents, ProDeliverIN, Xfect, and TuboFect, using Huh-7 multicellular tumor spheroid (MCTS) as a three-dimensional in vitro tumor model. A cellular uptake study using specific endocytosis inhibitors revealed that each reagent was internalized into Huh-7 MCTS by different mechanisms, which were the same as monolayer cultured Huh-7 cells. A certain amount of Pro-DeliverIN and Xfect was uptaken by Huh-7 cells through caveolae-mediated endocytosis, which may lead to transcytosis through the surface-first layered cells of MCTS. The results presented here will help in the choice and use of protein transfection reagents for evaluating anti-tumor therapeutic proteins against MCTS models. - Multicellular tumor spheroid (MCTS) is known to be a very useful three-dimensional in vitro tumor model, which represents the morphological and functional features of in vivo avascular solid tumors [13]. MCTS is characterized by actively proliferating outer cell layers and hypoxic and quiescent inner cells. Compared to monolayer cultured cells, a long-term culture can be achieved by spheroid cell cultures with the sufficient maintenance of their functions. Therefore, MCTS is a good experimental model located between an in vitro monolayer cultured cell model and in vivo animal model. This model has been widely used not only for screening ani-tumor drug candidates [4,5], but also for investigating drug delivery systems (DDS) [69]. The deep percolation of anti-tumor drugs and their DDS into tumor tissues is necessary for successful therapy, and this can be evaluated using MCTS models. Proteins are one of the most important biomacromolecules in all living cells. The application of proteins to research has ranged from biochemical experiments to drug discoveries. Proteins are easily degraded by protease and deactivated in or out of cells. A major key for the success of delivering proteins to cells directed to biochemical and drug discovery studies is the development of protein delivery systems with high efficiency and negligible cytotoxicity [10,11]. Several protein transfection reagents are commercially available due to the extensive development of excellent delivery systems [12,13]. Their reagents are powerful tools for elucidating the function of a protein in a cell and controlling cellular functions by an introduced protein. We recently reported the intracellular internalization mechanism of three different commercially available protein transfection reagents, the lipid-based Pro-DeliverIN, peptidebased Xfect, and cationic polymer-based TurboFect [14]. These reagents were internalized into monolayer cultured HeLa cells by different mechanisms, which may be helpful in choosing and using protein transfection reagents for experiments. To gain further information into the biological properties of these reagents, we reused Pro-DeliverIN, Xfect, and TurboFect in this study, and evaluated their complexes with bovine serum albumin (BSA) against human hepatoma Huh-7 MCTS models as well as monolayer cultured cell models. We have already reported that Huh-7 cells were good models for MCTS [8,9]. Less attention has been paid to studies on protein transfection reagents using MCTS models. Cellular uptake studies using specific inhibitors of endocytosis and confocal laser scanning microscope (CLSM) observations clarified the internalization routes and final localization of each complex in Huh-7 MCTS. The results obtained here may be informative for using protein transfection reagents against MCTS to screen and evaluate anti-tumor therapeutic proteins. Materials and Methods Materials Pro-DeliverIN was purchased from OZ Biosciences (Marseille, France). Xfect was obtained from Clontech Laboratories, Inc. (Palo Alto, CA, USA). TurboFect was purchased from Fermentas (Glen Burnie, MD, USA). Dulbeccos modified Eagles medium (DMEM), bovine serum albumin (BSA), fluorescein isothiocyanate conjugate BSA (FITC-BSA), filipin III from Streptomyces filipinesis, and amiloride hydrochloride were purchased from Sigma-Aldrich Co. (ST. Louis, MO, USA). Heparin, Cell lysis buffer M, and sucrose were the products of Wako Pure Chem. Co., Ltd. (Osaka, Japan). Hoechst 33342 was purchased from Dojindo Laboratories (Kumamoto, Japan). Preparation of protein transfection reagent/BSA complex Each protein transfection reagent/BSA or FITC-BSA complex was prepared according to the manufactures protocols and the previous study [14]. Briefly, 1.0 L of Pro-DeliverIN reagent, 3.0 L of 1X Xfect protein transfection reagent stock solution, and 0.8 L of TurboFect protein transfection reagent were used to prepare complexes containing 1.0 g of BSA or FITC-BSA, respectively. Dynamic light scattering (DLS) measurement The size of BSA complexes was evaluated by DLS using Nano ZS (ZEN3600, Malvern Instruments, Ltd., UK). A HeNe ion laser (633 nm) was used as the incident beam. The data obtained at a detection angle of 173 and a temperature of 37C were analyzed by a cumulant method to obtain the hydrodynamic diameters and polydispersity index (PDI) (/2) of complex. The results are presented as the mean and standard deviation obtained 3 measurements. Zeta-potential measurement The zeta-potential of BSA complexes was evaluated by the laser-Doppler electrophoresis method using Nano ZS with a HeNe ion laser (633 nm). The zeta-potential measurements were carried out at 37C. A scattering angle of 173 was used in these measurements. The results are presented as the mean and standard deviation obtained 3 measurements. Cell culture and preparation of multicellular tumor spheroid (MCTS) Human hepatoma Huh-7 cells (JCRB Cell Bank, Osaka, Japan) were maintained in DMEM supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37C. MCTS was prepared by using a 96-well culture plate designed for spheroid formation (Sumiloncelltight, Sumitomo Bakelite, Tokyo, Japan) as reported previously [8,9]. Briefly, 200 L of the cell suspension (2,500 cells/mL) was seeded onto a 96-well culture plate. After overnight incubation, Cellular uptake using monolayer cultured cells Each protein transfection reagent/FITC-BSA complex was used for these experiments. Huh-7 cells were seeded onto 96well culture plates (10,000 cells/well) and incubated overnight in 100 L of medium. The medium was replaced by fresh medium and protein transfection reagent/FITC-BSA complex solution was then applied to each well (0.25 g FITC-BSA/ well). After 3h incubation, the medium was removed, an (...truncated)