Effect of resection of localised pancreatic cancer on tissue-factor promoted pathways of thrombosis, cell invasion and angiogenesis

Echrish, Hussein H. Jassim

Abstract

Pancreatic (PC) is the eleventh most common malignancy in the UK but it has the poorest prognosis of all human adenocarcinoma. The autopsy, epidemiological and clinical studies have consistently identified PC as one of the most highly angiogenic and invasive malignancies, with the greatest prevalence and incidence of thrombo-embolism (TE). The incidence of TE in PC has been reported as high as 57%. Tissue factor (TF) bearing microparticles (MP) have recently been shown to promote thrombosis. The biological link between cancer haemostasis, cell invasion and angiogenesis remains unclear. These three indices may be driven by PC cells directly, be a reflection of the individual tumour stromal microenvironment and/or a result of the inflammatory response of the host. The hypothesis of the thesis is that factors directly attributed to the cancer promote the observed pathophysiology and that the removal of the tumour should result in reversal of these abnormalities.

Flow cytometry was used for the evaluation of MP in plasma and quantification of surface-expressed TF, VEGFR-1 and-2 and EGFR. Cellular TF activity and pro-coagulant activity (prothrombin time) of PC patients were measured using a coagulometer. Matrigel Invasion Chambers and Boyden chambers with collagen IV were used to measure cellular invasion. A two dimensional angiogenesis assay was used to evaluate tubule formation in vitro in response to PC sera. Relative levels of protein expression of 55 angiogenic markers in the sera of PC patients were evaluated using a human angiogenesis array kit. Enzyme-linked immunosorbent assay was undertaken on VEGF, TF, TFPI, Leptin and annexin autoantibodies using sera or plasma from PC patients as appropriate. Immunohistochemical analysis of key markers of angiogenesis and thrombosis was also undertaken on resected PC samples.

The in vitro optimisation experiments revealed that the cell invasion was significantly correlated with TF antigen expression and activity on PC cell lines (MIA-PaCa-2, AsPC-1 and CFPAC1) and that blocking TF on these cells decreased cell invasion. In the same manner neutralising soluble TF in PC serum samples also significantly decreased cell invasion, as did spiking of the serum with low molecular weight heparin. Analysis of sera from patients showed that TF bearing MP, pro-coagulant activity, cell invasion and angiogenesis (total length and number of capillaries) of PC cases were significantly higher than the control. Furthermore, the post-operative median number of TF bearing MP, procoagulant activity, cell invasion and angiogenesis (total length and total number of capillaries) were all significantly lower compared with pre-operative samples. Out of 55 angiogenic markers studied in 6 PC patients, pre- and post-operatively there was a significant decrease of angiopoietin-1, angiostatin/plasminogen, PDGF-AA, PDGF-AB/PDGF-BB and VEGF post-operatively. This result was supported by ELISA analysis of 29 samples and 14 controls that also showed significantly higher levels of VEGF in pancreatic cancer sera versus control groups, and that there was a significant decrease observed post-operatively only in the cancer patients. Furthermore both angiogenesis array and ELISA showed increased leptin levels post-operatively.

Immunohistochemical analysis of the pancreatic tissue sections revealed that TF was expressed on 62 % of PC samples. There was significant correlation between TF expression on the tissue and procoagulant activity. Also, there was a significant correlation between tissue-expressed TF and in vitro angiogenesis, i.e. total length and number of capillaries. Furthermore, there was a significant correlation between TF expression on tissue with intratumoural microvascular density (MVD) and tumour-expressed with VEGFR 2. As expected, high levels of MVD correlated with high levels of tissue-expressed VEGF. Finally serum from patients who showed a high level of tissue-expressed VEGF also induced the greatest level of in vitro angiogenesis, i.e. number of capillaries.

In summary, it was shown that TF expression on cell lines was significantly correlated with TF activity and cell invasion, and that TF expression in plasma and on tissue from PC patients was significantly correlated with procoagulant activity, cell invasion and angiogenesis. PC tissue-expressed VEGF was significantly associated with the angiogenic activity of PC sera and tissue MVD. Thus, the pathophysiology represented by a high procoagulant state, elevated cell invasion and angiogenic properties seen in PC patient sera appears to be driven by the malignant cells, as removal of the tumour causes a return towards the normal state.

Citation

Echrish, H. H. J. Effect of resection of localised pancreatic cancer on tissue-factor promoted pathways of thrombosis, cell invasion and angiogenesis. (Thesis). University of Hull. https://hull-repository.worktribe.com/output/4212008

• Thesis Type: Thesis
• Deposit Date: Mar 16, 2012
• Publicly Available Date: Feb 22, 2023
• Keywords: Medicine
• Public URL: https://hull-repository.worktribe.com/output/4212008
• Additional Information: Postgraduate Medical Institute, The University of Hull
• Award Date: Oct 1, 2011