Fibroblast-derived CXCL12/SDF-1α promotes CXCL6 secretion and co-operatively enhances metastatic potential through the PI3K/Akt/mTOR pathway in colon cancer

Figure 5 Effect of granulocyte chemotactic protein-2, stromal cell-derived factor-1 and co-culture with colon cancer cells on angiogenesis. The treatment of CXCL6 (A) and CXCL12 (B) influence HUVEC tube formation. After incubation of the HUVEC/fibroblast co-culture system in the presence or absence of CXCL6 or anti-CXCL12 Ab, then co-culture for 7 d, the HUVEC/fibroblast co-culture system was stained with anti-CD31 antibody. Tube formation area was measured quantitatively using an image analyzer. A1: Control; A2: 1 ng/mL CXCL6; A3: 10 ng/mL CXCL6; A4: 10 ng/mL CXCL6 + 10 μg/mL of CXCL6 Ab. B1: Control; B2: 1 ng/mL CXCL12; B3: 10 ng/mL CXCL12; B4: 10 ng/mL CXCL12 + 10 μg/mL of CXCL12 Ab. Effect of colon cancer cells (DLD-1, HT-29 or CaCo-2) on HUVEC tube formation is shown (C). Angiogenesis assay by HUVEC/fibroblast co-culture with DLD-1, HT-29 or CaCo-2 cells was conducted using the double-chamber method. Detection of tube formation by HUVECs was described in the “Material and Methods” section. C1: Co-culture with DLD-1; C2: Co-culture with DLD-1 + 10 ng/mL of CXCL6; C3: Co-culture with DLD-1 + 10 ng/mL of CXCL12; C4: Co-culture with DLD-1 + 10 μg/mL of CXCL12 Ab; C5: Co-culture with HT-29 cells; C6: Co-culture with HT-29 cells pre-treated with 10 ng/mL CXCL6; C7: Co-culture with HT-29 cells pre-treated with 10 ng/mL CXCL12; C8: Co-culture with HT-29 + 10 μg/mL of CXCL12 Ab; C9: Co-culture with CaCo-2 cells; C10: Co-culture with CaCo-2 cells pre-treated with 10 ng/mL CXCL6; C11: Co-culture with CaCo-2 cells pre-treated with 10 ng/mL CXCL12; C12: Co-culture with CaCo-2 cells pre-treated with 10 μg/mL anti-CXCL12 antibody. Columns, mean pixels of HUVEC tube formation area; Bars = SD. Multiple comparisons were performed by one-way ANOVA followed by the SNK test; ^bP < 0.01 vs control. Ab: Antibody; CXCL6: Granulocyte chemotactic protein-2; CXCL12: Stromal cell-derived factor-1; HUVEC: Human umbilical vein endothelial cell.