Figure 9. IL-17A/IL-17RA signaling regulates colorectal CIC growth and self-renewal. (A) Quantitative RT-PCR analysis of IL-17A in CICs over-expressing IL-17A. Data are mean ± SD (n = 3); ***, P < 0.001. Student’s t test was used to assess the significance. The experiment was performed three times and representative data are shown. (B) Quantitative RT-PCR analysis of five different shRNAs for IL-17RA in CICs. Data are presented as means ± SD (n = 3); ***, P < 0.001. Student’s t test was used to assess the significance. The experiment was performed three times and representative data are shown. (C) Effects of IL-17RA knockdown with two different shRNAs (shIL-17RA1 or shIL-17RA2) or IL-17A overexpression with IL-17A transduction on tumorsphere formation. 064 and 199 and 656 CICs transduced with two different shRNAs or shControl and with IL-17A vector or empty vector were plated in replicates in stem cell media in a limiting dilution assay, and were then analyzed for the presence of spheres after 14 d. Data are mean ± SD (n = 20); *, P < 0.05; **, P < 0.01; ***, P < 0.001. The likelihood ratio test was used to assess the significance. The experiment was performed two times and representative data are shown. (D, E) The dependence of CICs on IL-17A was evaluated in vivo. 104 CICs and 3 × 104 CAFs were injected into mice. When tumors were established to approximately 0.3 cm3 in volume, treatment was initiated. Four arms were included: isotype control (2 mg/kg antibody five times a week for two weeks and then once a week for two weeks), chemotherapy alone (Oxaliplatin: 0.25 mg/kg once a week for 4 wk; 5-FU: 15 mg/kg five times a week for 2 wk), IL-17A blocking antibody alone (2 mg/kg five times a week for 2 wk and then once a week for 2 wk) or a combination of the IL-17 antibody and chemotherapy (four mice per arm). Mice were weighed and the tumor volume was measured every other day for 30 d. Mice were sacrificed and tumors were collected. (D) Representative images of treated tumors and (E) relative tumor volume. Bar, 1 cm. Data are presented as mean ± SD (n = 4); *, P < 0.1; **, P < 0.05; ***, P < 0.001. ANOVA, followed by Bonferroni’s post-hoc test was used to assess the significance.
Figure 9.

IL-17A/IL-17RA signaling regulates colorectal CIC growth and self-renewal. (A) Quantitative RT-PCR analysis of IL-17A in CICs over-expressing IL-17A. Data are mean ± SD (n = 3); ***, P < 0.001. Student’s t test was used to assess the significance. The experiment was performed three times and representative data are shown. (B) Quantitative RT-PCR analysis of five different shRNAs for IL-17RA in CICs. Data are presented as means ± SD (n = 3); ***, P < 0.001. Student’s t test was used to assess the significance. The experiment was performed three times and representative data are shown. (C) Effects of IL-17RA knockdown with two different shRNAs (shIL-17RA1 or shIL-17RA2) or IL-17A overexpression with IL-17A transduction on tumorsphere formation. 064 and 199 and 656 CICs transduced with two different shRNAs or shControl and with IL-17A vector or empty vector were plated in replicates in stem cell media in a limiting dilution assay, and were then analyzed for the presence of spheres after 14 d. Data are mean ± SD (n = 20); *, P < 0.05; **, P < 0.01; ***, P < 0.001. The likelihood ratio test was used to assess the significance. The experiment was performed two times and representative data are shown. (D, E) The dependence of CICs on IL-17A was evaluated in vivo. 104 CICs and 3 × 104 CAFs were injected into mice. When tumors were established to approximately 0.3 cm3 in volume, treatment was initiated. Four arms were included: isotype control (2 mg/kg antibody five times a week for two weeks and then once a week for two weeks), chemotherapy alone (Oxaliplatin: 0.25 mg/kg once a week for 4 wk; 5-FU: 15 mg/kg five times a week for 2 wk), IL-17A blocking antibody alone (2 mg/kg five times a week for 2 wk and then once a week for 2 wk) or a combination of the IL-17 antibody and chemotherapy (four mice per arm). Mice were weighed and the tumor volume was measured every other day for 30 d. Mice were sacrificed and tumors were collected. (D) Representative images of treated tumors and (E) relative tumor volume. Bar, 1 cm. Data are presented as mean ± SD (n = 4); *, P < 0.1; **, P < 0.05; ***, P < 0.001. ANOVA, followed by Bonferroni’s post-hoc test was used to assess the significance.

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