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The cancer stem cell hypothesis posits that a subpopulation of cancer stem cells is frequently responsible for a tumor's progression and resistance to treatment. The differential cellular morphology and gene expression between cancer stem cells and the majority of the tumor is becoming a point of attack for research into the next generation of therapeutic agents that may work through an induction of differentiation rather than apoptosis. Advances in the field of high-content imaging (HCI), combined with modern shRNA technology and subpopulation analysis tools, have created an ideal screening system to detect these morphological changes in a subset of cells upon gene knockdown. The authors examined several glioblastoma stem cell isolates pre- and postdifferentiation to elucidate the phenotypic effects caused by both serum differentiation and gene knockdown. Neural markers were first characterized in these cells at varying states of differentiation using HCI and immunoblots. The authors then chose one of these isolates, in both the pre- and postdifferentiated forms, for further analysis and screened for morphological changes upon shRNA knockdown of a panel of cancer testis antigens (CTAs). CTAs are a family of proteins that are normally expressed in male germ cells as well as heterogeneously expressed in some metastatic tumors. This gene family has also been implicated in the differentiation of normal human stem cells, therefore making it an ideal candidate for modulation in tumor stem cells. Using their approach, the authors identified the differential effects of gene knockdown in both cell types leading to either changes in neural stem cell marker expression or a decreased cell density likely due to growth arrest or cell death. The resolution that HCI brings to a screen at the subpopulation level makes it an excellent tool for the analysis of phenotypic changes induced by shRNA knockdown in a variety of tumor stem cells.

Knockdown of Cancer Testis Antigens Modulates Neural Stem Cell Marker Expression in Glioblastoma Tumor Stem Cells