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Breast cancer stem cells are suspected to be responsible for tumour recurrence, metastasis formation as well as chemoresistance. Consequently, great efforts have been made to understand the molecular mechanisms underlying cancer stem cell maintenance. In order to study these rare cells  in-vitro , they are typically enriched via mammosphere culture. Here we developed a mammosphere-based negative selection shRNAi screening system suitable to analyse the involvement of thousands of genes in the survival of cells with cancer stem cell properties.    Methods  We describe a sub-population expressing the stem-like marker CD44 + /CD24 -/low  in SUM149 that were enriched in mammospheres. To identify genes functionally involved in the maintenance of the sub-population with cancer stem cell properties, we targeted over 5000 genes by RNAi and tested their ability to grow as mammospheres. The identified candidate ATG4 A  was validated in mammosphere and soft agar colony formation assays. Further, we evaluated the influence of ATG4 A  expression on the sub-population expressing the stem-like marker CD44 + /CD24 low . Next, the tumorigenic potential of SUM149 after up- or down-regulation of ATG4 A  was examined by xenograft experiments.    Results  Using this method, Jak-STAT as well as cytokine signalling were identified to be involved in mammosphere formation. Furthermore, the autophagy regulator ATG4A was found to be essential for the maintenance of a sub-population with cancer stem cell properties and to regulate breast cancer cell tumourigenicity  in vivo.Conclusion  In summary, we present a high-throughput screening system to identify genes involved in cancer stem cell maintenance and demonstrate its utility by means of ATG4 A .

A mammosphere formation RNAi screen reveals that ATG4A promotes a breast cancer stem-like phenotype