Figure 1.
SP cells analysis and expression of CSC markers in SP and NSP cells.
(A) ACHN and KRC/Y were labeled with Hoechst 33342, and then analyzed by FCM. The SP cell rates in ACHN and KRC/Y were 1.4% (A–a) and 1.7% (A–c), respectively, which decreased significantly in the presence of reserpine (A–b, A–d). The experiment was repeated at least three times for each cell line and almost identical results were obtained. A representative figure of our experiments is shown. (B) There was no apparent difference in CD90 and EpCAM expression between SP and NSP cells in ACHN. In the KRC/Y cell line, although there was no difference in EpCAM expression, SP cells expressed a higher CD105-positive cell rate than NSP cells (SP vs NSP : 24.6% vs 4.6%). The experiments were repeated twice, and almost identical results were obtained. A representative figure of our experiments is shown.
Figure 2.
Biological features of SP and NSP cells in ACHN and KRC/Y in vitro.
(A) Growth curves of SP and NSP cells. SP cells in KRC/Y showed a higher proliferative ability compared to NSP cells (* P<0.0001). (B) The clonogenity was significantly increased in SP cells in KRC/Y (* P<0.01). (C) Sphere forming ability was significantly higher in SP cells in ACHN (* P<0.05). (D) Drug resistance of SP and NSP cells treated with Sorafenib or IFNα. SP cells in ACHN had higher IFNα resistance (* P<0.0001). The experiments were repeated twice, and almost identical results were obtained.
Table 1.
Tumorigenicity of side population (SP) and Non-SP (NSP) cells in ACHN and KRC/Y.
Figure 3.
Quantification of mRNA expression of CSC-LC property-related genes in SP and NSP cells by real-time PCR.
There were no significant differences in mRNA expressions of ABC transporter genes (ABCB1 and ABCG2), self-replication genes (BMI-1 and c-MYC), anti-apoptosis genes (BCL2 and CFLAR), hypoxia-related genes (VEGFA and HIF1α), and EMT-related genes (Snail and Twist) between SP and NSP cells in the 2 cell lines. SP cells in ACHN expressed a slightly higher level of ALDH1A1 mRNA than NSP cells, but no apparent difference was observed in KRC/Y. The experiment was repeated at least four times for each cell line and almost identical results were obtained.
Figure 4.
ALDH1 expression, and biological features of ALDH1-positive and ALDH1-negative RCC cells.
(A) The expression of ALDH1 in SP cells and NSP cells in ACHN and KRC/Y. The ALDH1-positive cell rates in ACHN and KRC/Y were 15.3% and 6.5%, respectively. (B) Comparison of cell growth among control cells, cells treated with Sorafenib or IFNα, and cells exposed to hypoxia in ACHN. Cell growth was measured at 48, 72 or 96 hours after drug treatment or exposure to hypoxia. Cell growth after drug treatment or exposure to hypoxia was significantly suppressed as compared with control (* P<0.005, ** P<0.0001 vs. control). (C) The percentage of ALDH1-positive cells in cells treated with Sorafenib or IFNα, or cells exposed to hypoxia for 48, 72 or 96 hours. The percentage of ALDH1-positive cells in cells treated with Sorafenib or IFNα, or cells exposed to hypoxia for 96 hours was higher as compared with the normal condition. The experiments were repeated twice, and almost identical results were obtained. A representative figure of our experiments is shown. (D) Sphere forming ability between ALDH1-positive cells and ALDH1-negative cells. The sphere formation of ALDH1-positive cells in ACHN and KRC/Y was higher than that of ALDH1-negative cells. The experiments were repeated twice, and almost identical results were obtained.
Figure 5.
A self-renewal capacity between ALDH1-positive and ALDH1-negative ACHN cells.
The sphere forming ability of ALDH1-positive cells in ACHN was maintained during the second and third passages (* P<0.0001).
Table 2.
Tumorigenicity of aldehyde dehydrogenase 1 (ALDH1)-positive and ALDH1-negative cells in ACHN.
Figure 6.
Quantification of mRNA expression of CSC-LC property-related genes in ALDH1-positive and ALDH1-negative ACHN cells by real-time PCR.
ALDH1-positive cells showed significantly higher mRNA expression of ALDH1A1, transporter-related genes (ABCB1 and ABCG2), self-replication genes (BMI-1 and c-MYC), anti-apoptosis genes (BCL2 and CFLAR), hypoxia-related genes (HIF1α and VEGFA) and EMT-related genes (Twist) than ALDH1-negative cells in ACHN. However, there was no significant difference in mRNA expression of Snail between ALDH1-positive and ALDH1-negative cells. The experiments were repeated at least four times, and almost identical results were obtained.