The Role Of Sema4D In Colorectal Cancer-induced Tumor Angiogenesis

Objective:Semaphorins and their receptors, the plexins are originally identified based on their ability for axon guidance in the development of nervous system. Given the fact that semaphorins and plexins are expressed in a variety of tissues outside the nervous system, and during embryonic development there is a close space-time relationship between growing neurons and developing blood vessels. This suggests an intriguing link between the signal events that occur during axon guidance and those involved in angiogenesis.Semaphorin4D (Sema4D) member of class IV semaphorins, initially known as a regulator for axon guidance, but emerging evidence indicate it also possesses a previously unrecognized function:a compensatory angiogenic factor. The study designed to investigate the influence of semaphorin4D on tumor growth and vascularity of colorectal carcinoma (CRC) in nude mice.Methods:Based on endogenous Sema4D and VEGF protein level, HCT-116and Caco-2were chosen for further experiment. Because both have relatively high Sema4D level in five colorectal cancer cell lines, which are supposed to make the RNA interference bring phenotype significant enough. On the other hand, HCT-116showed the lowest VEGF level while Caco-2the maximum VEGF level among five cancer cell lines, this provided a distinctive VEGF background for observing possible phenotypic variance, when the same genetic change was made to both cell lines. In addition, they show better sensitivity and endurance to lentiviruses transduction than other three cell lines.During the followed in vitro migration assay and in vivo tumor genesis, each cell line was treated in four different groups:uninfected group or control lentivirus infected group which express eGFP, or infected by Sema4D super-expressor lentivirus that help to increase Sema4D expression on the original level, or by lentivirus coding for Sema4DshRNA, knocking down Sema4D expression. These eight groups of cells were subjected to migration assay to test their ability to induce endothelial cell migration. The appropriate cells were also injected subcutaneously into nude mice. Tumor growth was documented, and tumors harvested were processed for Immunohistochemistry and Immunofluorescence. Results:In vitro migration assay indicated:regardless of cell type, media conditioned by cancer cells infected with Sema4D shRNA lentiviruses induced the least amount of endothelial cell migration. In contrast, cancer cells super-expressing Sema4D showed enhanced ability to induce endothelial cell migration, and no apparent distinction could be found between uninfected and control infected groups.However in vivo observation was more complicated. HCT-116cells form dense solid tumor, but Caco-2cells grew into liquid containing cystadenoma. There were significant differences in tumor growth rate and vascular density. Xenografts from which Sema4D was downregulated were found with marked reduction in tumor sizes and vascularity in both cell lines. However Tumors which had Sema4D super-expressor displayed more variance. In Caco-2group, Xenografts with increased Sema4D level grew faster, larger and also had higher vascular density than both control virus infected and uninfected group. While in the HCT-116group, tumors over express Sema4D showed higher growth rate, tumor weight and more dense blood vessels, when compared with control virus infected group. The study also suggests although Sema4D makes its unique contribution to tumor growth rate and blood vessel density, the extent of such influence undoubtedly affected by other growth factors and angiogenesis inducing agents, especially endogenous VEGF level.Conclusion:In fact, semaphorin-plexin interactions have already been implicated in a variety of responses, including epithelial cell contacts modification, tissue organization and immune responses. Fast growing tumor cells could easily overwhelm the ability of existing vasculature to meet their high demands for metabolism and proliferation. Cancer cells may have more than one strategy to solve this crisis, but one crafty choice could involve expressing Sema4D at higher level in order to drive net neoangiogenesis and generate tumor blood supply system. Tumor progression and metastasis critically depend on acquiring the access to circulation, cancer cells with potent angiogenic capacity would definitively gain some advantage. Therefore Sema4D could be a potential target for anti-angiogenesis therapy in CRC patients.