Abstract
BACKGROUND: Angiogenesis is essential for tumor growth and metastasis. Although ELR(+)-CXC-chemokines and their corresponding receptor CXC-receptor 2 (CXCR2) are known mediators of angiogenesis, little is known about their biologic effects in pancreatic cancer (PaCa). AIM: The purpose of this study was to determine the role of the ELR(+)-CXC-chemokine/CXCR2 biologic axis in promoting PaCa angiogenesis. METHODS: We prospectively collected secretin-stimulated exocrine pancreatic secretions (SSEPS) from the duodenum in normal individuals (NP) and PaCa patients. ELR(+)-CXC-chemokines were assayed by ELISA. Mann-Whitney test was used for statistical analysis. RT-PCR and western blots were performed to detect CXCR2 expression and ELISA for ELR(+)-CXC-chemokines production in PaCa cell lines. Further, we performed MTS (for proliferation), Matrigel(TM) invasion, and tube formation assays using human umbilical vein endothelial cells (HUVEC). Finally, we used a neutralizing antibody against murine CXCR2 in an orthotopic PaCa (BxPC-3-GFP) model in nude mice and measured final tumor volume at the end of the treatment period (26 days/11 treatments, with n=5 per group). We also measured proliferation index (Ki-67(+)) and microvessel density (factor VIII(+)) in the tumor explants. RESULTS: To date, 14 patients with no pancreatic diseases (NP) and 35 tissue-proven PaCa patients have been enrolled. Summed concentrations of ELR(+)-CXC-chemokines (ΣELR(+)-CXC-chemokines) in SSEPS from PaCa patients were significantly higher than in those from NP (P = .0001). We measured ELR(+)-CXC-chemokines levels in supernatant from multiple PaCa cell lines and confirmed that BxPC-3, Colo-357, and Panc-28 had significantly higher expression compared to immortalized human pancreatic ductal epithelial (HPDE) cell line. After confirming lack of autocrine effects of ELR(+)-CXC-chemokines on PaCa cells (due to absence of CXCR2 expression), we investigated paracrine effects of these chemokines on HUVEC. Both recombinant ELR(+)-CXC-chemokines and co-culturing with BxPC-3 significantly enhanced proliferation, invasion, and tube formation of HUVEC (P < .05). These biologic effects were potently inhibited by treatment with a neutralizing antibody against CXCR2 (anti-CXCR2 Ab) (P < .05). Finally, anti-CXCR2 Ab significantly reduced tumor volume (P < .05), Ki-67 proliferation index (P = .043), and factor VIII(+) microvessel density (P = .004) in an orthotopic nude mouse PaCa model. CONCLUSIONS: Together, these results suggest that ELR(+)-CXC-chemokines promote PaCa tumor-associated angiogenesis through CXCR2 expressed on stromal endothelial cells. Thus, CXCR2 is a novel anti-angiogenic target in PaCa.