title: A Novel Cellular Spheroid-Based Autophagy Screen Applying Live Fluorescence Microscopy Identifies Nonactin as a Strong Inducer of Autophagosomal Turnover.
abstract: Dysregulation of the basal autophagic flux has been linked to several pathological conditions, including neurodegenerative diseases and cancer. In addition, autophagy has profound effects on the response of tumor cells to therapy. Hence, the search for pharmacological modulators of autophagy is of great clinical relevance. We established a drug screening assay in which the autophagic flux is measured by recording the fluorescence emission of the tandem fusion protein mRFP-GFP-LC3 by dynamic live-cell imaging. We optimized the assay for the identification of autophagy modulators in three dimensions with U343 glioma cell spheroids, which represent a more realistic cancer model than conventional 2D cell cultures. We validated the assay by screening a library of known autophagy modulators. As the first application, a small library of 94 natural compounds was screened for its impact on autophagy. We discovered the cyclic ionophore nonactin as a new and potent autophagy inducer. This novel autophagy screening assay based on 3D tumor spheroids is robust, reproducible, and scalable. It provides a valuable tool for both basic research and drug screening campaigns.
authors.affiliation: 1 Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe Universität Frankfurt am Main, Frankfurt am Main, Germany.
authors.forename: Francesco
authors.initials: F
authors.lastname: Pampaloni
authors.affiliation: 1 Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe Universität Frankfurt am Main, Frankfurt am Main, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Mayer
authors.affiliation: 1 Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe Universität Frankfurt am Main, Frankfurt am Main, Germany.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Kabat Vel-Job
authors.affiliation: 1 Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe Universität Frankfurt am Main, Frankfurt am Main, Germany.
authors.forename: Nariman
authors.initials: N
authors.lastname: Ansari
authors.affiliation: 1 Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe Universität Frankfurt am Main, Frankfurt am Main, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Hötte
authors.affiliation: 2 Experimental Neurosurgery, Department of Neurosurgery, Goethe Universität Frankfurt am MainFrankfurt am Main, Hessen, Germany.
authors.forename: Donat
authors.initials: D
authors.lastname: Kögel
authors.affiliation: 1 Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe Universität Frankfurt am Main, Frankfurt am Main, Germany.
authors.forename: Ernst H K
authors.initials: EHK
authors.lastname: Stelzer
mesh_terms: D001343:Autophagy / Q000187:drug effects*; D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D005910:Glioma / Q000188:drug therapy; D006801:Humans; D018942:Macrolides / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000379:methods; D018874:Spheroids, Cellular / Q000187:drug effects*
chemical_list: D018942:Macrolides; C050476:nonactin
keywords: autophagy; high-throughput screening; live fluorescence microscopy; multicellular spheroids