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Published on: April 3, 2018
Genome-wide RNA interference screen in cancer stem cells
Guillaume Pinna1, Marie Vandamme1, Celia Rouault2
1Plateforme ARN interférence (PARi), Université Paris Cité & Université Paris-Saclay, Inserm, iRCM/IBFJ CEA, Stabilité Génétique Cellules Souches et Radiations, Fontenay-aux-Roses, France.
Abstract:
Tumor heterogeneity represents a major hurdle for therapy. This cellular heterogeneity is mainly sustained by different subpopulations of tumorigenic cells, the so-called cancer stem cells (CSCs). CSCs burden is associated with disease progression and patient poor prognosis. In this context, deciphering molecular mechanisms regulating stemness is a key step in the development of new therapeutic strategy. Here, we provide a detailed protocol for high-throughput screening (HTS) strategy to detect modulators of CSC proportion. It is based on a miniaturized ALDEFLUOR-probed CSC assay quantitated by high-content imaging, that allows monitoring the changes in CSC proportions in response to gene silencing. Gene loss-of-function is achieved by transfecting a genome-wide RNA interference library. These genome-wide HTS strategies could lead to the identification of new therapeutic approaches in the treatment of various cancers.
Insights
This study introduces a high-throughput screening method to identify factors influencing cancer stem cells (CSCs). This approach aids in developing novel therapeutic strategies for various cancers by targeting CSCs.
Area of Science:
- Oncology
- Cancer Biology
- Stem Cell Research
Background:
- Tumor heterogeneity, driven by cancer stem cells (CSCs), is a significant obstacle in cancer therapy and is linked to poor patient prognosis.
- Understanding the molecular mechanisms that regulate stemness in CSCs is crucial for developing effective cancer treatments.
Purpose of the Study:
- To establish a high-throughput screening (HTS) strategy for identifying modulators of cancer stem cell (CSC) populations.
- To provide a detailed protocol for this HTS approach, enabling broader application in cancer research.
Main Methods:
- A miniaturized ALDEFLUOR-based CSC assay was employed, utilizing high-content imaging for quantitative analysis.
- Genome-wide RNA interference (RNAi) libraries were used to induce gene loss-of-function and assess its impact on CSC proportions.
Main Results:
- The developed HTS strategy effectively monitors changes in CSC proportions in response to gene silencing.
- The protocol allows for the quantitative assessment of CSC modulation through high-content imaging.
Conclusions:
- Genome-wide HTS strategies, like the one presented, are powerful tools for discovering novel therapeutic targets in cancer.
- This method facilitates the identification of new therapeutic approaches aimed at controlling CSC populations and improving cancer treatment outcomes.
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01:23RNA Interference
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...