Orosphere assay: a method for propagation of head and neck cancer stem cells.

BACKGROUND Recent evidence suggests that head and neck squamous cell carcinomas (HNSCCs) harbor a small subpopulation of highly tumorigenic cells, designated cancer stem cells. A limiting factor in cancer stem cell research is the intrinsic difficulty of expanding cells in an undifferentiated state in vitro. METHODS Here, we describe the development of the orosphere assay, a method for the study of putative head and neck cancer stem cells. An orosphere is defined as a nonadherent colony of cells sorted from primary HNSCCs or from HNSCC cell lines and cultured in 3-dimensional soft agar or ultralow attachment plates. Aldehyde dehydrogenase activity and CD44 expression were used here as stem cell markers. RESULTS This assay allowed for the propagation of head and neck cancer cells that retained stemness and self-renewal. CONCLUSION The orosphere assay is well suited for studies designed to understand the pathobiology of head and neck cancer stem cells.