The influence of tumour angiogenesis on the metastatic potential of colorectal carcinomas

Colon cancer is the fourth most common cause of cancer death in the world. It kills approximately 529,000 people annually and around two thirds of these deaths are in the developed world. These figures can be attributed to the fact that colorectal cancers are well advanced before they are detected. Effective prevention of the disease is early detection and removal of pre-cancerous polyps. In cases where cancer has already developed, early detection still significantly improves the chances of cure by surgically removing the cancer before the disease is able to metastasise. However, in metastatic colorectal cancers, in order to ascertain an effective treatment regimen and determine the prognosis of a patient after surgery a prognostic tool that accurately portrays the extent of the disease needs to be developed. Currently, the most commonly used prognostic tool to assess colorectal cancer spread is the clinicopathological staging system. However, there is a need for additional markers of metastasis as nearly one-third of patients with a clinical diagnosis of Dukes' B, a commonly used pathological staging system for colorectal cancer prognosis, will die of the disease despite complete resection of the primary tumour. In several human cancers such as breast, prostate and the lung, tumour vascularity has been shown to be of prognostic value. However, studies correlating the significance of the number of tumour vessels to prognosis in colorectal cancers are relatively lacking or show conflicting results. Moreover, it is not yet fully understood if factors involved in the complicated cascade of tumour blood vessel formation (tumour angiogenesis), such as proteases cathepsin B and dipeptidyl peptidase IV aid colorectal cancer progression. Two of the most potent angiogenic growth factors: vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) playa significant role in the development of tumour vessels; however, their influence on the production of these proteases is not clear. Therefore, this study set out to i) establish a reliable method to quantify colorectal tumour vessels ii) to assess the correlation between vessel counts and prognostic parameters associated with metastasis iii) to ascertain the pattern of spatial distribution of tumour vessels in order to defme the relationship between the positions of the vessels in relation to areas of tumour growth iv) investigate the levels of the enzyme expression of tumour associated proteases cathepsin B and dipeptidyl peptidase IV, at both protein and mRNA level, in colorectal tumours of varying clinicopathological grades and to v) assess the influence of VEGF and bFGF on CB and DPPIV proteolytic enzyme activity in human colon, rectal cancer cells and normal human umbilical vein endothelial cells. The study established that an effective method to examine tumour vascularity, was by immunolocalising tumour endothelial cells by CD31 and quantifying all of stained vessels present within each cross-sectional area. As a result of tumour endothelial cell marker assessment it was noted that CD34 was highly specific to certain vessels and distinctly immunonegative to others and that the same vessels were positive to CD31. These vessels were morphologically assessed by using the standard histological criteria identified as lymph vessels. Tumour blood and lymph vessels were quantified and a positive correlation to prognostic parameters relating to cancer spread was identified. The pattern of distribution of colorectal tumour vessels was also examined and a distinct pattern was observed in metastatic tumours with greater numbers of vessels in the peripheral regions and the invasive fronts. Qualitative analysis of protease expression and mRNA intensity, in colorectal tumours, revealed increased levels of CB and DPPIV in tumours with clinicopathological parameters associated with metastasis. mRNA localization near tumour vessels highlighted the potential for, not only cancer cells but also tumour endothelial cells, to produce proteolytic enzymes to aid the process of tumour angiogenesis and hence tumour growth and metastasis. The study also demonstrated that the angiogenic factors VEGF and bFGF up-regulated CB and DPPIV activity in human colon, rectal cancer cells and normal human umbilical vein endothelial cells.The presence of tumour lymph vessels and their positive correlation to metastatic parameters signifies an important role for such vessels in the process of colorectal cancer spread. The positive relationship of tumour blood vessels to parameters associated with metastasis re-instates the important role of tumour angiogenesis in colorectal cancer. Other factors that were investigated such as tumour associated proteases CB and DPPIV and the influence of the potent angiogenic proteins upon their activity suggests that these factors have a direct role in colorectal cancer metastasis. Therefore, it can be concluded from the results of this thesis that tumour blood vessel counts can be used as a reliable prognostic marker in colorectal cancer. Tumour angiogenic factors as well as the colorectal tumour lymph vessels have the potential to be used as additional prognostic markers in clinical prognostic evaluations. This has important implications in allowing clinicians to accurately assess post-operative survival rates and to devise an effective treatment regimen in order to prolong the lives and possibly cure colorectal cancer patients