Molecular and functional characterization of cells with stem properties isolated by sphere forming assay from human renal cell carcinoma tissues and cell lines

Cancer stem cells (CSC) are a rare subset of malignant cells that constitute a reservoir of tumor‐initiating cells with the ability to both self‐renew and differentiate into bulk tumors. As well as for other tumors, also in Renal Cell Carcinoma (RCC) the identification of CSCs might represent a step toward the development of therapies able to totally eradicate the disease. In the present study, cells with stem properties were identified from cultures of clonal tumor spheres obtained from RCC tissues after standardization of sphere‐forming assay on RCC 786‐0 cell line. Spheres obtained from the cell line and from RCC tissues were similar in term of phenotypic features, growth kinetics and sphere forming efficiency (SFE). These spheres exhibited the expression of pluripotency genes as well as the activation of self‐renewal pathways, when compared to the cultures representative of the bulk tumor population. Moreover they overexpressed the adenosine deaminase acting on RNA (ADAR1 and ADAR2) that might be involved in the regulation of self‐renewal as demonstrated by the increase of SFE after overexpression in 786‐0 cell line. When injected in immunocompromised mice, cells from spheres had a higher ability to give rise to tumor. Moreover tumor spheres from RCC tissues, as well as from 786‐0, showed a heterogeneous composition, with different cell subpopulations, displaying diverse self‐renewal ability. These subpopulations were identified on the basis of the different intensity of fluorescence of the PKH26 dye, able to discriminate quiescent cells within a proliferating population. The ability to self‐renew of the different PKH populations depended on the grading of the tumor. Although not distinguishing CSCs from the bulk tumor, surface marker expression in combination with PKH assay further confirmed the heterogeneity of cells within the spheres and allowed to identify an enrichment of CD105+ and CD133+CD105+ cells in the self‐renewing PKHhigh population. In this study, by characterizing for the first time molecular pathways, such as Notch, JAK/STAT and RNA editing, that distinguish spheres, enriched in putative CSCs, from the bulk tumor, represented by primary cell cultures, we provided possible targets for new therapies that need to be further characterized in order to discern their role. Moreover, the combination of PKH assay and surface markers might be helpful for a better definition of the CSC population within RCC.