Rat C6 glioma cell motility and glioma growth are regulated by netrin and netrin receptors unc5B and DCC

Background: Cell migration plays a key role in tumor invasion and metastasis. Deleted in colorectal cancer (DCC) and the unc5 homologues are receptors for secreted netrins that regulate cell and axon migration, cell adhesion, and tissue morphogenesis. Altered netrin and netrin receptor expression has been reported in aggressive tumors, including glioblastoma. Here, we investigated the involvement of netrin and netrin receptors in glioma cell migration and tumor formation using rat C6 glioma, a counterpart to human glioblastoma multiforme. Methods: We used Western blot analysis and RT-PCR to characterize netrin and netrin receptor expression in rat C6 glioma. C6 motility was assessed using a transfilter chemotaxis assay. To study the effect of ectopic DCC expression on C6 glioma cells, we determined the in vivo growth of three different cell populations after intracerebral implantation in rats. Three-dimensional reconstruction of tumors was performed to calculate tumor volume. Results: We determined that rat C6 glioma cells express netrin-1, netrin-3, and the netrin receptor unc5B, but not DCC. Using transfilter migration assays we demonstrated that C6 cells migrate away from a source of netrin-1, consistent with chemorepulsion signaled by Unc5B. In the absence of a gradient of netrin, disrupting netrin protein secreted by these cells reduced C6 cell motility, suggesting that autocrine netrin promotes C6 cell motility in this assay. Ectopic expression of DCC reduced the rate of migration in the absence of any gradient, and reduced the rate of directional cell migration up a gradient of laminin-1. Intracerebral implantation of glioma cells engineered to express full-length DCC (pCEP4-DCC) resulted in reduced tumor volumes and sharp borders between tumor and brain tissue, compared to tumors arising from parental or pCEP4 vector-transfected cells. Conclusions: These results provide evidence that netrin and Unc5B may promote cell motility by weakening interactions between tumor cells and that loss of DCC promotes growth and invasion of rat glioma.