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A Novel Cellular Spheroid-Based Autophagy Screen Applying Live Fluorescence Microscopy Identifies Nonactin as a
Francesco Pampaloni1, Benjamin Mayer1, Konstantin Kabat Vel-Job1
11 Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe Universität Frankfurt am Main, Frankfurt am Main, Germany.
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.
Insights
Researchers developed a new 3D cell culture assay to screen for drugs that modulate autophagy, a key process in cancer and neurodegenerative diseases. They identified nonactin as a potent autophagy inducer.
Area of Science:
- Cell Biology
- Pharmacology
- Oncology
Background:
- Autophagic flux dysregulation is implicated in neurodegenerative diseases and cancer.
- Autophagy significantly influences tumor cell response to therapies.
- Identifying pharmacological autophagy modulators is crucial for clinical applications.
Purpose of the Study:
- To establish and optimize a novel drug screening assay for autophagy modulators.
- To utilize a 3D glioma cell spheroid model for more realistic cancer studies.
- To discover new compounds that impact autophagic flux.
Main Methods:
- Developed a live-cell imaging assay measuring autophagic flux via mRFP-GFP-LC3 tandem fusion protein fluorescence.
- Optimized the assay using U343 glioma cell spheroids (3D model).
- Validated the assay by screening known autophagy modulators and a library of natural compounds.
Main Results:
- Successfully established a robust and reproducible 3D autophagy screening assay.
- Screened 94 natural compounds and identified the cyclic ionophore nonactin as a potent autophagy inducer.
- Demonstrated the assay's scalability and suitability for drug discovery.
Conclusions:
- The novel 3D spheroid-based autophagy screening assay is a valuable tool for basic research and drug discovery.
- Nonactin represents a newly identified potent inducer of autophagy.
- This assay provides a more realistic platform for identifying therapeutic agents targeting autophagy.
