Stromal Matrix Metalloproteinase-9 Regulates the Vascular Architecture in Neuroblastoma by Promoting Pericyte Recruitment

Advanced stages of neuroblastoma show increased expression of matrix metalloproteinases MMP-2 and MMP-9 (Y. Sugiura et al., Cancer Res., 58: 2209-2216, 1998) that have been implicated in many steps of tumor progression, suggesting that they play a contributory role. Using pharmacological and genetic approaches, we have examined the role of these MMPs in progression of SK-N-BE (2).10 human neuroblastoma tumors orthotopically xenotransplanted into immunodeficient mice. Mice treated with Prinomastat, a synthetic inhibitor of MMPs, showed an inhibition of tumor cell proliferation in implanted tumors and a prolonged survival (50 versus 39 days in control group, P < 0.035). Treatment with Prinomastat did not affect formation of liver metastases (P = 0.52) but inhibited intravascular colonization by the tumor cells in the lung by 73.8% (P = 0.03) and angiogenesis in both primary tumors and experimental liver metastases. The primary tumors from Prinomastat-treated mice showed a 39.3% reduction in endothelial area detected by PECAM/CD31 staining in tumor sections (P < 0.001), primarily due to the presence of smaller vessels (P = 0.004). MMP-2 is expressed by neuroblastoma tumor cells and stromal cells, whereas MMP-9 is exclusively expressed by stromal cells, particularly vascular cells. To examine the contribution of MMP-9 to tumor angiogenesis, we generated RAG1/MMP-9 double-deficient mice. We observed a significant inhibition of angiogenesis in the immunodeficient RAG1/MMP-9 double-deficient mice orthotopically implanted with tumor cells (P = 0.043) or implanted s.c. with a mixture of tumor cells and Matrigel (P < 0.001). Using an FITC-labeled lectin, we demonstrated an inhibition in the architecture of the tumor vasculature in MMP-9-deficient mice, resulting in fewer and smaller blood vessels. These changes were associated with a 48% decrease in pericytes present along microvessels. Taken together, the data demonstrate that in neuroblastoma, stromally derived MMP-9 contributes to angiogenesis by promoting blood vessel morphogenesis and pericyte recruitment.