Identifying cancer stem cells in tumourspheres

JP Schoning, M Monteiro and G Wenyi

Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland (UQ), Corner College and Cooper Roads, St Lucia, Brisbane QLD, Australia

Effective cancer treatment is hindered by a small subset of cells called cancer stem cells (CSCs), which are responsible for chemotherapy resistance and metastasis. In order to study CSCs tumoursphere culture is commonly used to enrich CSCs in vitro. However, not all cells in tumourspheres are CSCs, therefore, clear characterisation and isolation methods are necessary. Importantly, due to the diversity of cancer tissues, there are no universal molecular markers for CSCs. There is also little consensus on CSC surface markers within commercially available cell lines, such as colon cancer cell line HCT116, with ambiguity surrounding the relevance of proposed CSC, markers including CD133. This uncertainty may arise from using only surface markers as primary identifiers of CSCs rather than fundamental growth characteristics of CSCs. Here we used a fluorescent label retaining assay, commonly used to identify stem cells in neurospheres, to isolate potential CSCs based on their slow growth (quiescent) characteristics. Preliminary data demonstrates negligible overlap between the label retain cells (LRCs) and the CD133 surface marker. This indicates that CD133 may not designate quiescent CSCs. Other molecular markers were explored, including the OCT4B protein. Confocal analysis demonstrated differential cellular localisation and expression levels of OCT4B in HCT116 cells. Subsequent characterisation of OCT4B expression in LRCs using flow cytometry and whole sphere confocal microscopy will determine the functionality of using OCT4B as a marker for CSC isolation in tumourspheres. Finding relevant molecular markers would enable meaningful monitoring of CSCs in tumoursphere culture and will provide more information in the development of targeted CSCs treatments.