Related Experiment Video
Updated: May 19, 2026
Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening
Published on: September 5, 2018
A 1536-well quantitative high-throughput screen to identify compounds targeting cancer stem cells
Lesley A Mathews1, Jonathan M Keller, Bonnie L Goodwin
1Division of Preclinical Innovation, National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Rockville, MD 20850, USA. mathewsla@mail.nih.gov
Abstract:
Tumor cell subpopulations called cancer stem cells (CSCs) or tumor-initiating cells (TICs) have self-renewal potential and are thought to drive metastasis and tumor formation. Data suggest that these cells are resistant to current chemotherapy and radiation therapy treatments, leading to cancer recurrence. Therefore, finding new drugs and/or drug combinations that cause death of both the differentiated tumor cells as well as CSC populations is a critical unmet medical need. Here, we describe how cancer-derived CSCs are generated from cancer cell lines using stem cell growth media and nonadherent conditions in quantities that enable high-throughput screening (HTS). A cell growth assay in a 1536-well microplate format was developed with these CSCs and used to screen a focused collection of oncology drugs and clinical candidates to find compounds that are cytotoxic against these highly aggressive cells. A hit selection process that included potency and efficacy measurements during the primary screen allowed us to efficiently identify compounds with potent cytotoxic effects against spheroid-derived CSCs. Overall, this research demonstrates one of the first miniaturized HTS assays using CSCs. The procedures described here should enable further testing of the effect of compounds on CSCs and help determine which pathways need to be targeted to kill them.
Insights
This study developed a high-throughput screening method to identify drugs targeting cancer stem cells (CSCs), which are resistant to conventional therapies and drive tumor recurrence. The new assay effectively screened compounds for cytotoxicity against these aggressive tumor-initiating cells.
Area of Science:
- Oncology
- Cancer Stem Cell Biology
- Drug Discovery
Background:
- Cancer stem cells (CSCs) possess self-renewal capabilities, driving tumor formation, metastasis, and recurrence.
- CSCs exhibit resistance to standard chemotherapy and radiation, representing a critical challenge in cancer treatment.
- Developing therapeutic strategies targeting both differentiated tumor cells and CSCs is an unmet medical need.
Purpose of the Study:
- To establish a miniaturized high-throughput screening (HTS) assay for evaluating drug cytotoxicity against cancer stem cells (CSCs).
- To identify novel compounds and drug combinations effective against CSC populations.
- To facilitate the discovery of new therapeutic targets for eliminating aggressive cancer cells.
Main Methods:
- Generation of cancer-derived CSCs from cell lines using stem cell media and nonadherent culture conditions.
- Development of a miniaturized cell growth assay in a 1536-well microplate format for CSCs.
- Screening of a focused collection of oncology drugs and clinical candidates using the developed HTS assay.
Main Results:
- Successfully established a CSC model amenable to high-throughput screening.
- Identified compounds exhibiting potent cytotoxic effects against spheroid-derived CSCs through a rigorous hit selection process.
- Demonstrated the efficacy of the miniaturized HTS assay in identifying effective anti-CSC agents.
Conclusions:
- The developed HTS assay provides a scalable platform for drug discovery targeting cancer stem cells.
- This approach enables efficient identification of cytotoxic compounds against aggressive CSC populations.
- Further research using this platform can guide the development of novel cancer therapies targeting CSC-specific pathways.
