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.