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

Figure 4 Effect of granulocyte chemotactic protein-2, stromal cell-derived factor-1 and co-culture with fibroblasts or DLD-1 on colon cancer cell and human umbilical vein endothelial cell invasiveness. The influence of different concentrations of CXCL6 (A), CXCL12 (B) or co-culture with fibroblasts on colon cancer cell invasiveness was measured by the BD Bio-Coat Matrigel invasion assay system (BD Biosciences). HT-29 (A and B) cells and HUVECs (C and D) were pre-treated with different concentrations of CXCL6 and CXCL12, and co-culture with fibroblasts (E) or DLD-1 (F), or pre-treated with or without anti CXCL6 or CXCL12 antibody, and following a 24-h incubation. The invading cells were fixed and stained with Diff-Quick stain. Invading cells were counted in five random microscopic fields (× 200). The experiment detail is described in the “Material and Methods” section. Multiple comparisons were performed by one-way ANOVA followed by the SNK test; ^aP < 0.05, ^bP < 0.01. A1: HT-29 cells only; A2: 0.1 ng/mL of CXCL6; A3: 1 ng/mL of CXCL6; A4: 10 ng/mL of CXCL6. B1: HT-29 cells only; B2: 0.1 ng/mL of CXCL12; B3: 1 ng/mL of CXCL12; B4: 10 ng/mL of CXCL12; C1: HUVECs only; C2: 1 ng/mL of CXCL6; C3: 10 ng/mL of CXCL6; C4: 10 ng/mL of CXCL6 treated with 10 μg/mL CXCL6 Ab. D1: HUVECs only; D2: 1 ng/mL of CXCL12; D3: 10 ng/mL of CXCL12; D4: 10 ng/mL of CXCL12 treated with 10 μg/mL CXCL12 Ab. E1: HUVECs only; E2: HUVECs co-culture with fibroblasts; E3: Co-culture with fibroblasts + 10 μg/mL CXCL12 Ab; E4: Co-culture with fibroblasts + 10 ng/mL CXCL6. F1: HUVECs only; F2: HUVECs co-culture with DLD-1 cells; F3: Co-culture with DLD-1 cells + 10 μg/mL CXCL12 Ab; F4: Co-culture with CaCo-2 cells. Ab: Antibody; CXCL6: Granulocyte chemotactic protein-2; CXCL12: Stromal cell-derived factor-1; HUVEC: Human umbilical vein endothelial cell.