Discovery of Small Molecules That Induce Lysosomal Cell Death in Cancer Cell Lines Using an Image-Based Screening...

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Lysosomal cell death (LCD) is a caspase 3-independent cell death pathway with therapeutic potential in cancer treatment.
• Developing high-throughput (HT) assays to identify LCD inducers is crucial for advancing cancer therapies.
• Existing methods for detecting LCD have limitations, such as being affected by lysosomal pH changes.

Purpose of the Study:

• To establish a robust two-step HT screening platform for identifying lysosomal cell death (LCD) inducers.
• To overcome limitations of current methods by developing an assay unaffected by lysosomal pH.
• To gain new insights into the molecular mechanisms underlying LCD induction.

Main Methods:

• A two-step HT screening approach was employed, starting with a propidium iodide uptake assay to identify non-apoptotic cell death.
• A secondary phenotypic image-based assay utilizing a galectin-3 (Gal-3) reporter was used to confirm lysosomal permeabilization, a hallmark of LCD.
• The assay's performance was validated by screening and identifying known and novel LCD inducers.

Main Results:

• The developed platform successfully identified 24 LCD inducers, including both previously known compounds and a novel non-cationic amphiphilic drug (non-CAD) inducer.
• The image-based assay accurately identified lysosomal permeabilization independently of lysosomal pH.
• The study provided new biological insights, indicating that lysosomal accumulation and acid sphingomyelinase inhibition are not essential for LCD induction.

Conclusions:

• The validated HT platform is effective for identifying novel LCD inducers.
• This platform will facilitate further research into the molecular mechanisms of LCD.
• The findings support LCD as a promising target for developing new cancer therapeutics.