Comparative study of human colonic tumor-derived endothelial cells (HCTEC) and normal colonic microvascular endothelial cells (HCMEC): Hypoxia-induced sVEGFR-1 and sVEGFR-2 levels

Colorectal carcinoma growth and progression is dependent on the vasculature of the tumor microenvironment. Tumor-derived endothelial cells differ functionally from their normal counterpart. For this reason we isolated microvascular endothelial cells from human colon cancer tissue (HCTEC) and compared them with endothelial cells from normal colonic tissue (HCMEC) of the same donor. Since hypoxia is a universal hallmark of carcinomas, we examined its effects on HCTEC of five patients in comparison with the corresponding HCMEC, with respect to the secretion of the soluble form of the two important vascular endothelial growth factor (VEGF) receptors, VEGFR-1 and -2. After dissociation by dispase/collagenase of central non-necrotic tumor areas obtained from colon carcinomas, HCTEC were isolated using CD31-coated magnetic beads and cultivated as monolayers. Subsequent characterization studies demonstrated the endothelial phenotype, including VEGFR-1 and -2 mRNA and protein expression as well as E-selectin expression, up-regulated after LPS, TNFα and IL-1β stimulation. sVEGFR expression analyses were performed using ELISA. In comparison with HCMEC markedly lower sVEGFR-1 protein concentrations were found in HCTEC. These low sVEGFR-1 levels remain unchanged under hypoxia. In contrast, sVEGFR-2 was significantly decreased in both HCMEC and HCTEC under hypoxic conditions (p≤0.001). Comparative studies with endothelial cells isolated from human colorectal cancer and non-neoplastic colon will be useful for understanding the progressive behavior of colorectal cancer. The different secretion profiles of sVEGFR-1 and -2 between HCTEC and HCMEC underline the importance of using a functionally adequate and relevant tumor-derived microvasculature for in vitro studies of tumor progression. Since sVEGFR-1 can act as a natural endogenous VEGF-inhibitor, the homogeneously low sVEGFR-1 levels under normoxia and hypoxia in HCTEC could be a marker for a ‘pro-angiogenetic disposition’ of the tumor-derived endothelium. The reduced sVEGFR-2 level profiles in hypoxic HCMEC and HCTEC provide evidence for a novel microvascular endothelium-specific biomarker in hypoxia-response processes.