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Updated: Mar 13, 2026
A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells
Published on: July 3, 2013
Discovery of Small Molecules That Induce Lysosomal Cell Death in Cancer Cell Lines Using an Image-Based Screening
Romina J Pagliero1, Diego S D'Astolfo1,2, Daphne Lelieveld1
11 Department of Cell Biology, University Medical Center Utrecht (UMCU) , Utrecht, the Netherlands .
Abstract:
The lysosomal cell death (LCD) pathway is a caspase 3-independent cell death pathway that has been suggested as a possible target for cancer therapy, making the development of sensitive and specific high-throughput (HT) assays to identify LCD inducers highly desirable. In this study, we report a two-step HT screening platform to reliably identify such molecules. First, using a robust HT primary screen based on propidium iodide uptake, we identified compounds that kill through nonapoptotic pathways. A phenotypic image-based assay using a galectin-3 (Gal-3) reporter was then used to further classify hits based on lysosomal permeabilization, a hallmark of LCD. The identification of permeabilized lysosomes in our image-based assay is not affected by changes in the lysosomal pH, thus resolving an important limitation in currently used methods. We have validated our platform in a screen by identifying 24 LCD inducers, some previously known to induce LCD. Although most LCD inducers were cationic amphiphilic drugs (CADs), we have also identified a non-CAD LCD inducer, which is of great interest in the field. Our data also gave new insights into the biology of LCD, suggesting that lysosomal accumulation and acid sphingomyelinase inhibition are not sufficient or necessary for the induction of LCD. Overall, our results demonstrate a robust HT platform to identify novel LCD inducers that will also be very useful for gaining deeper insights into the molecular mechanism of LCD induction.
Insights
We developed a two-step high-throughput screening platform to identify lysosomal cell death (LCD) inducers for cancer therapy. This method reliably detects molecules that trigger LCD, offering new avenues for drug discovery.
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.
