Characterization of the Hoechst 33342 side population from normal and malignant human renal epithelial cells

1 Genito-Urinary Cancer Research Group, School of Cancer and Imaging Sciences, Paterson Institute for Cancer Research, University of Manchester and 2 Department of Urology, Christie Hospital NHS Foundation Trust, Manchester; and 3 Department of Urology, Salford Royal NHS Foundation Trust, Salford, United Kingdom Submitted 1 May 2008 ; accepted in final form 2 July 2008 The fundamental changes which predispose for renal cell carcinoma (RCC) are poorly characterized. It is hypothesized that "cancer stem cells" may be influential in carcinogenesis, and the epithelial side population (SP) is enriched for stemlike cells in other epithelial cancers. In this study, we have isolated and characterized the SP and non-SP (NSP) populations from normal (NK) and malignant (RCC) human kidney tissue. NK specimens were taken from patients undergoing non-renal cancer surgery and paired malignant and macroscopically normal tissue samples were taken from patients undergoing surgery for RCC. The Hoechst 33342 dye efflux technique was used to isolate epithelial SP and NSP from normal and malignant human renal tissue. Cellular subpopulations were phenotyped for lineage, cell cycle, and putative stem cell markers, and functionally characterized using in vitro colony-forming and proliferation assays. The SP constituted 3.8 ± 0.4 and 5.9 ± 0.9% of epithelial cells in NK and RCC, respectively, of which 14.1 ± 3.5 and 13.2 ± 3.6% were shown to be in G 0 . SP cells demonstrated greater proliferative potential in colony-forming efficiency, long-term culture, and spheroids assays and were shown to be maintained upon tissue culture passage. We have shown that the renal SP is enriched for quiescent cells, with a high proliferative capacity and stemlike properties. The population is, however, heterogeneous, confirming that the terms "SP cell" and "stem cell" cannot be used interchangeably. kidney; renal cancer Address for reprint requests and other correspondence: M. D. Brown, Genito-Urinary Cancer Research Group, School of Cancer and Imaging Sciences, Paterson Institute for Cancer Research, Univ. of Manchester, Wilmslow Rd., Manchester M20 4BX, UK (e-mail: mbrown{at}picr.man.ac.uk )