Identification and characterization of the adrenal tumor side population

Recent evidence suggests the existence of a stem cell like sub-fraction of cells in solid tumors, being mainly responsible for the proliferative potential and chemotherapy resistance, thus defining the malignant phenotype. A method using the vital dye Hoechst exclusion has been utilized to successfully isolate a stem cell enriched side population (SP) from neuroblastoma tumors. We demonstrated the presence of SP cells in human adrenocortical adenomas (0.02–0.98%) and murine (Y1; 3.51–4.37%) and human (NCI h295; 12.91–19.08%) adrenocortical tumor cell lines, with a notably higher SP-proportion compared to normal mouse (0.13–0.17%) and human (0.01–0.03%) adrenals. NCI SP cells revealed an expression pattern consistent with a less differentiated phenotype with lower expression of SCC and StAR. However, proliferation was not different between SP and non-SP cells (105.6±18.1% vs. 100.0±3.5%; p=0.7). Although a survival benefit was evident for SP cells after treatment with mitoxantrone (25.5±5.3 vs. 11.5±2.6%), this could not be found for cytotoxic agents commonly used in adrenocortical carcinomas. Re-sorting experiments revealed the capacity for both cell types to give rise to the original SP and NSP containing cell population. In addition, Hoechst uptake was incubation time-dependent: After 90min, a SP fraction could be identified in NCI cells, which was lost after 130min, when all cells were stained. Confocal microscopy revealed a clear nuclear staining, which persisted over several days of culturing. The membrane pump hypothesis, explaining the weaker Hoechst staining of SP cells can therefore only be upheld, if this pump is efficient enough to prevent Hoechst dye from reaching the nucleus. Taken together, these findings indicate that SP cells are not a major tumor stem cell defining marker in NCI h295 cells and mechanisms resulting in distinct Hoechst staining properties remain to be defined. However, as NCI h295 cells represent a long-term maintained cell line, these experiments need to be complemented with SP cells from primary cultures of adrenal carcinomas.