title: Biosensor-expressing spheroid cultures for imaging of drug-induced effects in three dimensions.
abstract: In the past, the majority of antitumor compound-screening approaches had been performed in two-dimensional (2D) cell cultures. Although easy to standardize, this method provides results of limited significance because cells are surrounded by an artificial microenvironment, are not exposed to hypoxia gradients, and lack cell-cell contacts. These nonphysiological conditions directly affect relevant parameters such as the resistance to anticancer drugs. Multicellular tumor spheroids more closely resemble the in vivo situation in avascularized tumors. To monitor cellular reactions within this three-dimensional model system, we stably transfected a spheroid-forming glioblastoma cell line with Grx1-roGFP2, a green fluorescent protein (GFP)-based glutathione-specific redox sensor that detects alterations in the glutathione redox potential. Functionality and temporal dynamics of the sensor were verified with redox-active substances in 2D cell culture. Based on structured illumination microscopy using nonphototoxic light doses, ratio imaging was then applied to monitor the response of the glutathione system to exogenous hydrogen peroxide in optical sections of a tumor spheroid. Our approach provides a proof of concept for biosensor-based imaging in 3D cell cultures.
authors.affiliation: Institut für Lasertechnologien in der Medizin und Messtechnik an der Universität Ulm, Ulm, Germany. rainer.wittig@ilm.uni-ulm.de
authors.forename: Rainer
authors.initials: R
authors.lastname: Wittig
authors.forename: Verena
authors.initials: V
authors.lastname: Richter
authors.forename: Stephanie
authors.initials: S
authors.lastname: Wittig-Blaich
authors.forename: Petra
authors.initials: P
authors.lastname: Weber
authors.forename: Wolfgang S L
authors.initials: WS
authors.lastname: Strauss
authors.forename: Thomas
authors.initials: T
authors.lastname: Bruns
authors.forename: Tobias P
authors.initials: TP
authors.lastname: Dick
authors.forename: Herbert
authors.initials: H
authors.lastname: Schneckenburger
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D015374:Biosensing Techniques / Q000379:methods*; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005909:Glioblastoma / Q000188:drug therapy / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008853:Microscopy / Q000379:methods; D010084:Oxidation-Reduction; D018874:Spheroids, Cellular / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D049452:Green Fluorescent Proteins; D006861:Hydrogen Peroxide; D005978:Glutathione
keywords: Grx1-roGFP2; biosensor; glutathione-specific redox sensor; phototoxicity; spheroid imaging