Rare cancer stem cells (CSC) are proposed to be responsible for tumour propagation and re-initiation and are functionally defined by identifying tumour-initiating cells (TICs) using the xenotransplantation limiting dilution assay (LDA). While TICs in clear cell renal cell carcinoma (ccRCC) appeared rare in NOD/SCID/IL2Rγ(-/-) (NSG) mice, xenografts formed more efficiently from small tumour fragments, indicating the LDA underestimated ccRCC TIC frequency. Mechanistic interrogation of the LDA identified multiple steps that influence ccRCC TIC quantitation. For example, tissue disaggregation destroys most ccRCC cells, common assays significantly overestimate tumour cell viability, and microenvironmental supplementation with human extracellular factors or pharmacological inhibition of anoikis increase clonogenicity and tumourigenicity of ccRCC cell lines and primary tumour cells. Identification of these previously uncharacterized concerns that cumulatively lead to substantial underestimation of TICs in ccRCC provides a framework for development of more accurate TIC assays in the future, both for this disease and for other cancers.
Scientific reports. 2016 Apr 28*** epublish ***
Craig Gedye, Danylo Sirskyj, Nazleen C Lobo, Jalna Meens, Elzbieta Hyatt, Michael Robinette, Neil Fleshner, Robert J Hamilton, Girish Kulkarni, Alexandre Zlotta, Andrew Evans, Antonio Finelli, Michael A S Jewett, Laurie E Ailles
School of Biomedical Sciences and Pharmacy, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia., Princess Margaret Cancer Centre, University Health Network, Toronto, ON, M5G 1L7, Canada., Princess Margaret Cancer Centre, University Health Network, Toronto, ON, M5G 1L7, Canada., Princess Margaret Cancer Centre, University Health Network, Toronto, ON, M5G 1L7, Canada., Hospital for Sick Children, Program in Genetics and Genome Biology, Toronto, ON, M5G 0A4, Canada., Princess Margaret Cancer Centre, University Health Network, Toronto, ON, M5G 1L7, Canada., Princess Margaret Cancer Centre, University Health Network, Toronto, ON, M5G 1L7, Canada., Princess Margaret Cancer Centre, University Health Network, Toronto, ON, M5G 1L7, Canada., Princess Margaret Cancer Centre, University Health Network, Toronto, ON, M5G 1L7, Canada., Princess Margaret Cancer Centre, University Health Network, Toronto, ON, M5G 1L7, Canada., Princess Margaret Cancer Centre, University Health Network, Toronto, ON, M5G 1L7, Canada., Princess Margaret Cancer Centre, University Health Network, Toronto, ON, M5G 1L7, Canada., Princess Margaret Cancer Centre, University Health Network, Toronto, ON, M5G 1L7, Canada., Princess Margaret Cancer Centre, University Health Network, Toronto, ON, M5G 1L7, Canada.