Figure 6. WISP-1 knockdown in OSCC decreases VEGF-A expression and angiogenesis-related tumor growth in vivo.

(A) SCC4 cells stably expressing shRNA constructs or control shRNA were seeded as monolayers and counted daily. Cells (10³) were plated in 6 well plates and grown for 2 days. Cells were trypsinized, and cell numbers was counted. (B–C) WISP-1 and VEGF-A mRNA and protein expression in SCC4 cells stably expressed a control shRNA or a WISP-1 shRNA was examined by western blot, qPCR, and ELISA. (D–E) EPCs were incubated with CM collected from control-shRNA and WISP-1-shRNA transfected SCC4 cells for 24 h and cell migration or tube formation were examined. (F) PBS, VEGF-A, control shRNA/SCC4 CM, and WISP-1 shRNA/SCC4 CM mixed in Matrigel were placed on chick chorioallantoic membranes. CAMs in each group were photographed on developmental day 12. (G) Mice were subcutaneously injected with Matrigel mixed with PBS, control shRNA/SCC4 CM or WISP-1 shRNA/SCC4 CM for seven days. Plugs excised from the mice were photographed and stained with CD31. (H) Control shRNA and WISP-1 shRNA SCC4 cells were mixed with Matrigel and injected into the flank of the mice for 28 days. Tumor growth was monitored using the IVIS Imaging System. Tumor growth was quantified by fluorescent imaging from week 0–6. (I) Tumors were paraffin embedded, and sections were immunostained using the WISP-1, VEGF-A, and CD31 antibodies. (E = epithelial, T = tumor, S = stroma). (J) Diagrammatic model for the role of WISP-1 in OSCC. (1) WISP-1 induces VEGF-A expression and secretion in OSCC cells through the integrin αvβ3/FAK/c-Src pathway, which transactivates the EGFR/ERK/HIF1-α signal pathway. (2) The WISP-1-induced secretion of VEGF-A subsequently recruiting EPCs to OSCC tumor microenvironment and promoting neoangiogenesis. Data represent the mean ± SEM *P < 0.05 compared to control shRNA/SCC4.