Genome-wide RNA interference screen in cancer stem cells

Methods Cell Biol. 2022;170:81-99. doi: 10.1016/bs.mcb.2022.02.008. Epub 2022 Apr 12.


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.

Keywords: ALDH; Breast cancer; Cancer stem cells; Genome-wide RNA interference library; High-content imaging; High-throughput screening; Therapeutic resistance; Tumor heterogeneity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Humans
  • Neoplasms* / genetics
  • Neoplasms* / pathology
  • Neoplastic Stem Cells* / pathology
  • RNA Interference