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. 2016 Dec 16;27(4):590–593. doi: 10.1038/cr.2016.150

Figure 1.

Figure 1

Identification of driver genes in renal cell carcinoma stem cells via single-cell exome sequencing. (A) Detection of somatic mutations in CD133+CD133− RCC cells and in cancer tissue. The main plot shows information for genes with mutations for 20 cells and original cancer tissue. The red color represents non-silent mutations and green color represents silent mutations. (B) Somatic mutation graph. Two substitutions (A/T>G/C and C/G>T/A) are clearly frequent. (C) Venn plots show the somatic mutations in CD133+ and CD133− RCC cells. (D) Principle component analysis (PCA) of the mutations in the CD133+ RCC cells (red), CD133− RCC cells (green) and normal cells (blue). Eigenvector is defined as the Covariance Matrix. (E) A neighbor-joining tree was constructed using the somatic mutation data set. The normal cells are labeled in green, CD133− RCC cells are labeled in blue, and CD133+ RCC cells are labeled in red. (F) The average mutation frequency of 29 genes with variations in at least 3 CD133+ RCC cells. The mutation frequency indicates the percentage of CD133+ RCC cells with the mutated gene. (G) Data points indicate the average number of spheres of RCC cells with distinct mutations in serum-free conditions. Each of the 20 mutations was tested alone (first column, 'single mutation'), in combination with a KCP mutation (second column) or in combination with KCP and LOC440040 mutations (third column). Other mutations were also tested in combination with KCP, LOC440040, and LOC440563 mutations (fourth column). Mutation combinations that enhanced the in vitro spherogenicity (blue) were selected for in vivo validation. CD133+ cells spheres served as the positive control (red). (H) Representative Sanger-sequencing data of KCP, LOC440040, and LOC440563 in wild-type (WT) and mutated (Mut) renal cancer cells are listed below. (I) Representative oncospheres in mutated (Mut) and vehicle renal cancer cells. (J) The 18-week tumor-free rate of NOD/SCID mice after subcutaneous injection at the indicated dilutions of 786-O WT, 786-O Mut, 769-P WT, and 769-P cells (left panel, n = 6 mice per group). The estimated percentage of CSCs in 786-O WT, 786-O Mut, 769-P WT, and 769-P Mut cells in xenografted mice using extreme limiting dilution analysis (n = 6 grafted tumors per dilution; right panel). (K) The CD31−CD45−CD133+ cells from 57 RCC patients were individually sorted and pooled together for the indicated targeted sequencing. The mutation rates of KCP, LOC440040, and LOC440563 are indicated. (L) The average tumor-free time of 57 renal cancer patients with or without KCP, LOC440040, and/or LOC440563 mutation(s) after primary tumor resection.