Semaphorin 3A Attenuates Melanoma Progression Figure 6. Overexpression of Sema 3A abrogates in vivo melanoma growth and angiogenesis in subcutaneous allograft tumor model in C57BL/6 mice. Control B16F10 and clone 2 cells (16106/mice) were injected subcutaneously into the dorsal flank region of male mice (6–8 weeks old; n = 6). In separate experiments, serum free conditioned media (CM) collected from clone 2 cells were injected intratumorally twice a week to the tumors generated by B16F10 cells upto termination of the experiments (n = 6). Mice were sacrificed after 4 weeks. (A) Typical photographs of subcutaneous melanoma in C57BL/6 mice and excised tumors of respective mice were shown. (B) Mice allograft tumors were analyzed by histopathology and immunohistochemistry using anti-vWF antibody. vWF was stained with Cy2 (green) whereas nuclei were countered stained with PI (red). (C) Weight of the excised tumors were measured, analyzed and represented in the form of bar graph (*p = 0.006, #p = 0.002). (D) Tumor volumes of the allograft melanoma tumors were measured weekly, analyzed and plotted graphically. Six mice were used in each set of experiments. doi:10.1371/journal.pone.0033633.g006 Sema 3A attenuates melanoma metastasis To determine whether Sema 3A inhibits melanoma metastasis, we implanted melanoma (control or clone 2) cells into the arterial circulation via intracardiac injection. After 18 days, mice were sacrificed, dissected and photographed (Fig. 7B, panel I). Mice with uninjected cells were used as control. The data suggested the significant melanoma metastasis in liver and intestine of control B16F10 but not in clone 2 injected mice (Fig. 7B, panels II & III). Moreover, we have detected several metastatic foci in the tissue sections of liver and intestine of control B16F10 injected mice by histopathology using H&E staining. The lungs of these mice were photographed and analyzed histopathologically. Significant lung metastasis was observed in control B16F10 but not in clone 2 cells injected mice (Fig. 7B, panels IV & V). Overall, the data clearly provided evidence that overexpression of Sema 3A significantly attenuates melanoma metastasis to lung, liver and intestine in murine melanoma model. Discussion In this study, using various in vitro as well as in vivo models, we have demonstrated for the first time that Sema 3A could act as a potential tumor suppressor in melanoma model. The data also revealed that Sema 3A could be a rational and effective therapeutic molecule for treatment of melanoma. Our conclusion is supported by several lines of evidences: (i) expression profile of Sema 3A is negatively correlated with melanoma progression in human clinical specimens; (ii) overexpression of Sema 3A attenuates in vitro cell motility, invasiveness and proliferation in highly metastasic melanoma cells; (iii) tumor derived Sema 3A abrogated tumor-endothelial interaction via NRP1 mediated paracrine mechanism; (iv) Sema 3A promotes drug sensitivity of melanoma cells; (v) Sema 3A alone is not able to induce apoptosis however it enhances the apoptotic potential of curcumin and DTIC in B16F10 cells; (vi) overexpression of Sema 3A significantly PLoS ONE | www.plosone.org 10 March 2012 | Volume 7 | Issue 3 | e33633