Focus of Research: Pancreatic cancer
Massachusetts General Hospital
Co-Authors Pausch TH, Keck T, Hopt UT
Pancreatic carcinoma shows strong tumor neoangiogenesis mediated by VEGF. Another important angiogenic co-factor are matrix metalloproteases (MMP), especially MMP-9, which are derived peritumoral stroma and inflammation mediated by polymorphonuclear neutrophils (PMN). PMN action seems to be VEGF dependant. We studied the angiogenic role of MMP-9 derived from PMN and the ability to block its action using tetracyclines as MMP inhibitors during tumor angiogenesis in an in vitro model of pancreatic cancer using a human vascular endothelial cell (HUVEC) spheroid based sprouting assay and CAPAN-1 human pancreas cancer cells.
To generate endothelial cell spheroids, HUVEC were seeded in hanging drops, harvested within 24 h and embedded into collagen I gels. The test substances and/or cells were added on top of the gels. To quantify angiogenesis, the cumulative length of all sprouts was measured after 24 h. At least 10 spheroids per experimental group and experiment were analyzed. To analyze the expression of VEGF and MMP-9 in the different cells, quantitative Western blot analysis was performed.
Western blot analysis of CAPAN-1, PMN and HUVEC for MMP-9 and VEGF demonstrated no significant MMP-9 production in HUVEC and CAPAN-1, whereas the pro-form of the protein was found in PMN. VEGF was only found in CAPAN-1. VEGF and MMP-9 alone induced significant angiogensis and their combination resulted in significantly increased angiogenesis. CAPAN-1 and PMN alone also stimulated angiogensis and their combination resulted in significantly more angiogenesis. The combination of CAPAN-1 and VEGF or PMN and MMP-9 lead to no further angiogenesis. Antibodies to VEGF were able to abolish angiogenesis in VEGF and CAPAN stimulated spheroids, but not in MMP-9 stimulated HUVECS whereas antibodies to MMP-9 were able to abolish angiogenesis stimulated by MMP-9 and PMN. Tetracycline action was similar to antibodies to MMP-9. Combination of VEGF with MMP-9 antibodies and vice-versa reduced angiogenesis to the level of a single stimulant. The same effect was observed for the combination of CAPAN-1 and PMN whereas tetracyclines acted as antibodies to MMP-9. The combination of both antibodies or the combination of tetracyclines and antibodies to VEGF completely abolished angiogenesis.
This study demonstrates that PMN derived MMP-9 plays a role in tumor angiogenesis in an in vitro model of pancreatic cancer and that a combination of different anti-angiogenic agents is feasible. A VEGF independent angiogenic effect of MMP-9 was found. Inhibition of peritumoral inflammation around pancreatic cancer cells might be efficient to reduce peritumoral stomal inflammation mediated angiogenesis.
Additionally, the MMP-9 dependent anti-angiogenic properties of tetracyclines were demonstrated.
presented at Pancreas Club 2007