Current Site Of Practice: Boston, MA
Hospital Affiliation: Massachusetts General Hospital
Focus of Research: Pancreatic cancer
Fellowship Year: 2008 – 2010
Attended: 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
Co-Authors Baier P, Hopt UT
Tumor vaccination, which primarily activates cellular immunity, is a promising approach in the therapy of cancer. Experimental and clinical studies have proven its efficacy. However, several tumor immune escape mechanisms limit its success. The understanding of their action and their inhibition is a key step in the improvement of tumor vaccination response rates.To characterize these immune escape mechanisms better, in this study, balb/c-mice were vaccinated with radiated CT-26-colon carcinoma cells twice 14 and 7 days before intraperitoneal injection of living tumor cells. 100% of the control (n=25), but only 14,7% of the vaccinated (n=34) group developed peritoneal carcinosis. To determine the cell lines involved in the vaccination response, CD4+-, CD8+- und NK1-cells were depleted via intraperitoneal antibody injection. 83% of the control, 50 % of the CD8+-, 100% of the NK1-depleted and 33% CD4+- depleted animals (n=6) developed peritoneal carcinosis. These results demonstrate a significant vaccination effect. Its basis is the activation of innate cellular immunity, especially of NK1+-cells.2D gel electrophoresis yielded 1958 proteins. The comparison of protein expression among the groups showed 25 proteins with significant difference (p<0,001) in their expression pattern (0,01 %). Amongst several glucose metabolism enzymes, which were upregulated in the vaccinated group, Hdgfrp2 (hepatoma-derived growth factor related protein 2) was overexpressed, whereas Hspa4l (heat shock protein 4 like protein) was reduced in the vaccinated group. The proteomic basis of this effect is the reduced expression of Hspa4l, whose expression is immunogenic. Hdgfrp2 overexpression is a marker of cell dedifferentiation. To improve vaccination results, the addition of Hspa4l to the vaccine will be tested next.
presented at Chirurgisches Forum 2007