Semaphorin 3A Attenuates Melanoma Progression Figure 7. Sema 3A overexpression attenuates melanoma metastasis. (A) The mice described in Fig. 6A, were dissected ventrally and photographed. Growth of melanoma tumors were indicated by arrows (Fig. 7A, panel I). Internal organs (liver, intestine and kidney) were analyzed histopathologically and the micrographs were shown at 606 magnification (Fig. 7A, panels II–IV). Melanoma positive cells in metastatic lesions were marked by red arrows, whereas surrounding stromal regions were indicated by black arrows. Six mice were used in each group. Un-injected mice are used as control. (B) Sema 3A attenuates melanoma metastasis in mice. Control or clone 2 cells were injected intracardiaclly and mice were sacrificed, dissected ventrally and photographed (Fig. 7B, panel I). Metastasized organs were indicated by arrows. Liver and intestine of the mice were analyzed histopathologically by H&E staining (Fig. 7B, panels II & III). Micrographs were taken at 106magnification. Arrows showed the melanoma foci. Fig. 7B, panel IV shows the excised lung of respective mice. Lung sections were also analyzed histopathologically and photographs were taken at 106 magnification (Fig. 7B, panel V). Melanoma metastatic foci in lung sections are indicated by arrows. Six mice were used in each set of experiment. Un- injected mice were used as control. doi:10.1371/journal.pone.0033633.g007 diminished in vivo melanoma progression and angiogenesis in subcutaneous melanoma model, (vii) Sema 3A suppresses metastasis in melanoma models developed upon intracardiac injection and finally (viii) Sema 3A regulates melanoma cell migration and invasion through autocrine and paracrine mecha- nisms. Altogether, our experimental evidences suggested that Sema 3A acts as a potential tumor suppressor in melanoma model. Angiogenesis has been demonstrated as one of the major event during malignant progression of cancer [47]. Recent advancement of cancer therapeutics indicated that targeting tumor angiogenesis could be one of the rational and promising approaches for the treatment of cancer. Earlier studies have shown that among various members of semaphorin family, Sema 3A could act as a potential suppressor of angiogenesis [13–16,33], however the molecular mechanism underlying this process is not clearly understood and has been a field of intense investigation. In this study, using various in vitro as well as in vivo models, we have demonstrated that Sema 3A could suppress melanoma progres- sion. Our clinical data for the first time revealed that melanoma growth negatively correlated with expression profile of Sema 3A. Earlier we have reported the crucial role of NRP1 in regulation of tumor endothelial interaction which ultimately regulates tumor angiogenesis [28]. In this study, we have shown that B16F10 overexpressed Sema 3A disrupts tumor-endothelial interaction through NRP1 mediated process. Furthermore, we have detected significant reduction of tumor angiogenesis in Sema 3A overex- pressed clone when injected subcutaneously into C57BL/6 mice. In summary, our experimental observations have clearly indicated that Sema 3A may act as a potential suppressor of melanoma progression by attenuating angiogenesis. Metastasis or the distant migration of cancer cells from the site of origin is the major cause of death by cancer [48]. Metastasis is a multistep process involving motility and invasion of cancer cells, intravasation, transit through vascular and/or lymphatic system, extravasation and growth of secondary tumor at new site [49]. Therefore, prevention of migration and metastasis of cancer cells is the center of attention for researchers and oncologists [48]. In this study, we have shown that Sema 3A attenuates in vitro melanoma PLoS ONE | www.plosone.org 11 March 2012 | Volume 7 | Issue 3 | e33633