Eppenberger et al. BMC Cancer 2010, 10:93 http://www.biomedcentral.com/1471-2407/10/93 Dukes’ stage and of both VEGFR1 and VEGFR2 mRNA expression and lymph node positivity. Our findings of an increased VEGF-A expression from normal tissue to tumour, but a lack of association between expression with advanced pT stage, metastasis and survival time further support a role of VEGF-A in initiation and tumour maintenance in colorectal cancer. Furthermore, the combined analysis of VEGF-A with VEGFR1 and their correlation with features of tumour progression and adverse prognosis seem to implicate in particular VEGFR1 and VEGFR2 in the progression of colorectal cancer. Inflammatory mediators have previously been shown to have a significant effect on the process of angiogen- esis through the up-regulation of certain cytokines as well as of VEGF [10,21]. Not only does VEGF increase vascularity at sites of inflammation but its production by tumour cells results in the expression of inter-cellular adhesion molecule-1 and vascular cell adhesion mole- cule-1, thereby facilitating the adhesion of leukocytes to endothelial cells [22]. Our results highlight a relation- ship between the over-expression of VEGF-A as well as VEGFR1 and the peritumoural lymphocytic inflamma- tory response at the invasive tumour front. The inflam- matory response at the tumour border has previously been linked to the tumour border configuration, which we recently underlined as an essential prognostic factor in colorectal cancer [23]. The presence of a conspicuous band of lymphocytes, as described by Jass and colleagues is frequently associated with the presence of a pushing tumour margin, and has been related to an increased number of CD8+ tumour infiltrating lymphocytes and to an improved survival time [24,25]. In this study, we find that a greater VEGFR2 expression compared to VEGF-A is possibly linked to the presence of an infil- trating margin. Since an infiltrating tumour border con- figuration is a histomorphologic feature closely correlated to epithelial mesenchymal transition (EMT), whereby tumour cell de-differentiation and loss of cell- cell adhesion occurs at the invasive tumour front, our results may implicate VEGFR2 in this process [26]. The vast majority of the literature suggest a greater invasion and metastatic phenotype in tumours expres- sing these proteins [27-33]. In particular, several groups have suggested a VEGFR1-dependent involvement in EMT. Bates and colleagues used a spheroid culture sys- tem recapitulating the structure of the colonic epithe- lium during EMT. Their results find a significant expression of VEGFR1, but not VEGFR2 in these cells [34]. In pancreatic cancer, Yang and coworkers also describe VEGFR1 mediated EMT [35] while in head and neck squamous cell carcinoma (HNSCC), VEGFR2 expression has been linked to vasculogenesis and bud- ding of tumour cells into new vessels [36]. Our results Page 8 of 10 additionally underline not only the expression of VEGF- A as a possible step in tumour progression of colorectal cancer, but more importantly that VEGFR1 and VEGFR2 as well as their ratios with VEGF-A to play a role in the events occurring at the invasive tumour front. Although VEGF-C and VEGF-D are known primarily as lymphangiogenic proteins, less is known about their prognostic effect in patients with colorectal cancer. Hu and colleagues found that protein expression of VEGF-C and VEGF-D was significantly increased from normal to tumour tissues, a result which we confirm in our study. Furthermore, an increased expression of both these pro- teins was linked to lymph node metastasis and worse survival time [37]. Kawakami et al. report that VEGF-B and VEGF-C mRNA are significantly higher in tumours with lymph node metastases and in tumours with lym- phatic invasion [38] while Onogawa and colleagues report an increased VEGF-C and VEGF-D expression at the invasive tumour front [39]. Others have found a sig- nificant association of these proteins with venous and lymphatic invasion as well as with liver metastasis. A recent report by Moehler et al. found that VEGF-D expression correlated with lymph node metastasis and interestingly, that VEGF-D expression was significantly decreased following treatment with anti-EGFR mAb both in vitro and in mouse xenograft models [40]. In our study, a lower expression ratio of VEGF-C/VEGFR2 was linked to more advanced TNM stage. Our study has several limitations. First it is a retro- spective analysis of VEGF ligand and receptor expres- sion and therefore should be investigated in a prospective setting. Secondly, having used the tissue microarray technique, it is possible that tumour hetero- geneity was not completely taken into account. As an immunohistochemistry study, inter-laboratory variation may play a role in determining the reproducibility of these findings. Also, having considered adjustment for multiple testing in this study, the associations of VEGF ligands and their receptors fall short of significance although several strong trends were observed. Therefore, our results necessitate confirmation by other, larger study groups. Finally, we were unable to randomize our patient cohort into test and validation subgroups due to the lack of statistical power that this would elicit. Never- theless, our study may still be a basis for prospective approaches and worth to be validated in future studies. Conclusions The results of this study suggest that the VEGF ligand to receptor ratio may be an informative method for evaluating the effects of these angiogenic proteins on tumour progression. Our findings further underline a potential involvement of VEGF-A, VEGR1 and VEGFR2