Fig. 3.

Inhibition of Axl signaling suppresses diffuse-invasive tumor growth. (A) Proliferation assay of transfected cell clones. Cells were left untreated (−Gas6) or treated with 200 μg/ml Gas6 (+Gas6). Analysis was performed after 48 h of culture. Growth rate is expressed in relation to unstimulated mock-transfected cells. Experiments were performed in triplicate. The mean ± SD values are represented. ∗, P < 0.05 versus mock. (B and C) Assessment of vessel density in AXL-WT and AXL-DN xenografts after s.c. implantation. Analysis of tumor blood vessel density and morphology was assessed by immunohistochemistry for CD31 (B) and quantitatively by intravital fluorescence videomicroscopy after tumor implantation into the skinfold chamber preparation (C). The mean ± SD values are represented from four animals per group. (Scale bars, 25 μm.) (D) Histomorphological images of AXL-WT tumors and AXL-DN tumors. Hematoxylin and eosin staining. Arrows indicate tumor cell invasion into the adjacent skin muscle layer resulting in displacement of the muscle tissue. Arrowheads indicate tumor cell invasion into the adjacent skin muscle layer resulting in destruction of the muscle tissue. t, tumor mass; m, skin muscle layer; sc, s.c. tissue. (Scale bars, 100 μm.) (E) Quantitative analysis of glioma cell invasion into adjacent tissue by mock, AXL-WT, and AXL-DN tumors. Linear analysis of the length of infiltrated and destroyed muscle and s.c. tissue is presented as percentage of the total cross-sectional length of tumor mass. The mean ± SD values are represented. ∗, P < 0.05 versus mock tumors. (F) Fluorescence microscopy alone (Upper) and in combination with phase contrast (Lower) of AXL-DN tumor. Tumor cells were labeled with DiI before implantation. (Scale bars, 100 μm.) All specimens (B–F) were excised on day 21 after implantation into the dorsal skinfold chamber of nude mice. t, tumor mass; m, skin muscle layer; sc, s.c. tissue.