Figure 1.
CCR6 is Upregulated in Primary Human CRC Samples.
(A) Immunohistochemical staining of CCR6 in primary CRC derived from 191 CRC patients with clinical stage I–IV. (B) Digital image analysis was performed to count staining intensity of CCR6 area fraction (CCR6-AF) values of paired para-tumor/tumor samples in each clinical stage, Wilcoxon test.
Figure 2.
Survival Analysis of CRC Patients with Low versus High Expression of CCR6.
(A) Kaplan-Meier curves of CRC patients with low versus high expression of CCR6 (n = 191, p<0.001, log-rank test). (B) Kaplan-Meier curves of CRC patients with low versus high expression of CCR6 in clinical stage I (n = 14, p = 0.0679, log-rank test). (C) Kaplan-Meier curves of CRC patients with low versus high expression of CCR6 in clinical stage II (n = 104, p = 0.0738, log-rank test). (D) Kaplan-Meier curves of CRC patients with low versus high expression of CCR6 in clinical stage III (n = 57, p = 0.1749, log-rank test). (E) Kaplan-Meier curves of CRC patients with low versus high expression of CCR6 in clinical stage IV (n = 16, p = 0.0429, log-rank test).
Figure 3.
Knockdown of CCR6 by shRNA Inhibits CRC Cell Migration in vitro.
(A) Western blotting analysis of CCR6 levels in 7 cultured CRC cell lines. Values were expressed as fold changes relative to Caco-2, and normalized to β-actin. (B) Western blotting analysis of knockdown of CCR6 in SW480 and LoVo cells, β-actin served as a loading control. Values were expressed as fold changes relative to controls (ShCtr), and normalized to β-actin. (C) Wound-healing assays for motility of CCR6-silenced SW480 and LoVo cells and control cells. Representative pictures of one field at the beginning (t = 0 hr) (upper panel) and at the end of the recording (t = 24 hr) (lower panel) in each condition are shown. The relative cell migration in ShCtr and shCCR6 groups are shown in the right panel. (D) Representative images of transwell migrated cells in CCR6-silenced SW480 and LoVo cells (lower panel) or control cells (upper panel) cells. The numbers of migrated cells in ShCtr and shCCR6 groups are shown in the right panel. Values represent mean from triplicate wells, ± S.D. *p<0.05, **p<0.01, Wilcoxon test. Data are representative of at least three independent experiments.
Figure 4.
Enhanced Proliferation and Migration of CRC cells with Overexpressed CCR6.
(A) Western blotting analysis of ectopic expression of CCR6 in HCT116Ctr and HCT116CCR6 cells or Caco-2Ctr and Caco-2CCR6 or SW1116Ctr and SW1116CCR6 cells. β-actin served as a loading control. Values were expressed as fold changes relative to controls (Ctr), and normalized to β-actin. (B) Wound-healing assay for motility of HCT116Ctr and HCT116CCR6 or Caco-2Ctr and Caco-2CCR6 or SW1116Ctr and SW1116CCR6 cells. Representative pictures of one field at the beginning (t = 0) (upper panel) and at the end of the recording (t = 24 h) (lower panel) in each condition are shown. The relative cell migration in CCR6 and control groups are shown in the right panel. (C) Representative images of transwell migrated cells in stably transfected HCT116Ctr, Caco-2Ctr, SW1116Ctr (upper panel) or HCT116CCR6, Caco-2CCR6, SW1116CCR6 (lower panel) cells. Average number of migrated cells of HCT116Ctr and HCT116CCR6 or Caco-2Ctr and Caco-2CCR6 or SW1116Ctr and SW1116CCR6 cells are shown in the right panel. (D) Representative image of colony formation in HCT116Ctr, Caco-2Ctr, SW1116Ctr (upper panel) or HCT116CCR6, Caco-2CCR6, SW1116CCR6 cells (lower panel). Values represent mean from triplicate wells, ± S.D. *p<0.05, **p<0.01, Wilcoxon test. Data are representative of at least three independent experiments.
Figure 5.
Increased Metastasis of CRC Cells with Overexpressed CCR6 in vivo.
(A) Number of metastatic nodules (indicated by white arrows) formed in the liver of BALB/c nude mice 5 weeks after spleen injection of HCT116Ctr (upper panel) or HCT116CCR6 (lower panel) cells (six mice per group). (B) In vivo metastasis assays of Luc-HCT116Ctr and Luc-HCT116CCR6 cells by tail vein injection. The whole body metastasis burden of xenografted animals was monitored at 6 weeks after CRC cell injection using the IVIS Imaging System. Statistical analysis of luciferase intensity from mice injected with Luc-HCT116Ctr or Luc-HCT116CCR6 cells was shown in the right panel. (C) Representative images of H&E staining of lungs prepared from mice injected with Luc-HCT116Ctr or Luc-HCT116CCR6 cells at ×5 (left panel) and ×10 (right panel) magnification. Statistical analysis of the number or mitosis by mm2 in each metastatic nodule in the five lung H&E staining from mice injected with Luc-HCT116Ctr or Luc-HCT116CCR6 cells was shown in the right panel. *p<0.05, **p<0.01, U-mann whitney test.
Figure 6.
Signaling Pathway Involved in the Aggressiveness of HCT116CCR6.
(A, B) Western blotting analysis of Erk1/2 or phospho-Erk1/2 and Akt, phosphorylated Akt (Ser473) or phosphorylated Akt (Ser308) in HCT116CCR6 and HCT116Ctr cells. Values were expressed as fold changes relative to HCT116Ctr, and normalized to β-actin. (C) Upregulated (FXYD5 and SYK) or down-regulated genes (CDH1, KISS1 and TIMP2) in HCT116CCR6 cells screening with a human tumor metastasis RT2 profiler PCR Array. (D) Western blotting analysis of changed FXYD5, SYK, CDH1, KISS1 and TIMP2 genes in HCT116CCR6 and HCT116Ctr cells. Values were expressed as fold changes relative to HCT116Ctr, and normalized to β-actin.
Figure 7.
Inhibition of Mouse CRC Progression by Targeting Tumor-expressing CCR6.
(A) Western blotting analysis of CCR6 in murine CMT93 colorectal tumor cell line and CRC tissue derived from CCR6−/− mice grafted with CMT93 at day10. (B) Statistical analysis of tumor weight in each group treated with IgG or anti-CCR6. (C) Western blotting of CCR6 in murine CT26 colorectal tumor cell line and CRC tissue derived from Balb/c mice grafted with CT26 at day 10. (D) Statistical analysis of tumor weight in each group treated with IgG or anti-CCR6. *p<0.05, **p<0.01, U-mann whitney test.