Isolation and characterization of renal cancer stem cells from patient-derived xenografts

As rapidly developing patient-derived xenografts (PDX) could represent potential sources of cancer stem cells (CSC), we selected and characterized non-cultured PDX cell suspensions from four different renal carcinomas (RCC). Only the cell suspensions from the serial xenografts (PDX-1 and PDX-2) of an undifferentiated RCC (RCC-41) adapted to the selective CSC medium. The cell suspension derived from the original tumor specimen (RCC-41-P-0) did not adapt to the selective medium and strongly expressed CSC-like markers (CD133 and CD105) together with the non-CSC tumor marker E-cadherin. In comparison, PDX-1 and PDX-2 cells exhibited evolution in their phenotype since PDX-1 cells were CD133(high)/CD105-/Ecad(low) and PDX-2 cells were CD133(low)/CD105-/Ecad-. Both PDX subsets expressed additional stem cell markers (CD146/CD29/OCT4/NANOG/Nestin) but still contained non-CSC tumor cells. Therefore, using different cell sorting strategies, we characterized 3 different putative CSC subsets (RCC-41-PDX-1/CD132+, RCC-41-PDX-2/CD133-/EpCAM(low) and RCC-41-PDX-2/CD133(+)/EpCAM(bright)). In addition, transcriptomic analysis showed that RCC-41-PDX-2/CD133(−) over-expressed the pluripotency gene ERBB4, while RCC-41-PDX-2/CD133(+) over-expressed several tumor suppressor genes. These three CSC subsets displayed ALDH activity, formed serial spheroids and developed serial tumors in SCID mice, although RCC-41-PDX-1/CD132(+) and RCC-41-PDX-2/CD133(+) displayed less efficiently the above CSC properties. RCC-41-PDX-1/CD132(+) tumors showed vessels of human origin with CSC displaying peri-vascular distribution. By contrast, RCC-41-PDX-2 originated tumors exhibiting only vessels of mouse origin without CSC peri-vascular distribution. Altogether, our results indicate that PDX murine microenvironment promotes a continuous redesign of CSC phenotype, unmasking CSC subsets potentially present in a single RCC or generating ex novo different CSC-like subsets.