The identification of functional driver events in
cancer is central to furthering our understanding of
cancer biology and indispensable for the discovery of the next generation of novel
drug targets. It is becoming apparent that more complex models of
cancer are required to fully appreciate the contributing factors that drive
tumorigenesis in vivo and increase the efficacy of novel
therapies that make the transition from pre-clinical models to clinical trials. Here we present a methodology for generating uniform and reproducible
tumor spheroids that can be subjected to
siRNA functional screening. These spheroids display many characteristics that are found in solid
tumors that are not present in traditional two-dimension culture. We show that several commonly used
breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the
breast cancer cell line BT474, confirming their dependency on amplification of the
epidermal growth factor receptor HER2 and mutation of
phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as
tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.