Fig 2: CCL4 promotes OSCC angiogenesis in vivo. Mice were injected subcutaneously with Matrigel containing 0.2 mL CM from OSCC cells (treated with or without CCL4). On Day 14, Matrigel plugs were excised, photographed, quantified for hemoglobin content (A), then stained with CD31 (B–D). * p < 0.05 compared with controls.

Fig 3: CCL4 increases Angpt2 expression and promotes angiogenesis in OSCC cells. (A,B) OSCC cells were treated with various concentrations of CCL4 protein (0–30 µg/mL), then levels of ANGPT2 mRNA and protein expression were detected by qPCR and Western blot, respectively. (C,D) OSCC cells were treated with various concentrations of CCL4 (0–30 µg/mL) for 24 h. Culture medium was collected as CM and applied to EPCs for 16 h. The capillary-like structure formation was examined by the tube formation assay. Scale bar, 100 µm. (E,F) OSCC cells were treated with various concentrations of CCL4 (0–30 µg/mL) for 24 h. CM was applied to the EPCs for 24 h. EPC migration ability was examined by the Transwell migration assay. Scale bar, 50 µm. Each experiment was performed three times. * p < 0.05 compared with controls.; # p < 0.05 compared with the CCL4-treated group (30 µg/mL).

Fig 4: The schema depicts the involvement of signaling pathways in CCL4-induced stimulation of Angpt2 expression and the subsequent stimulation of OSCC angiogenesis.

Fig 5: Silencing CCL4 expression inhibits tumor-induced angiogenesis in vivo. (A–C) Mice were injected subcutaneously with Matrigel OSCC CM for 14 days, after which time the plugs were excised, photographed and quantified for hemoglobin content (A), then stained with CD31 (B,C). (D–F) The tumor sections were immunostained using Angpt2 and CD31 antibodies. * p < 0.05 compared with controls.