VEGFR-3, VEGF-C and VEGF-D mRNA Quantification by RT-PCR in Different Human Cell Types

The molecular events favoring lymphangiogenic pathways for tumor growth and dissemination are not perfectly understood, nor are the expression patterns of lymphangiogenic biomarkers such as the VEGFR-3 receptor and its ligands, VEGF-C and VEGF-D. In particular, VEGFR-3 expression is not restricted to the lymphatic endothelium, but is found on some cancer cells and other cell types. A quantitative RT-PCR method was set up to measure the mRNA levels of VEGFR-3, VEGF-C and VEGF-D. With this method, a very low detection threshold was obtained when tested on 17 different human cell types. It was found that, in contrast to VEGF-D mRNA, the VEGFR-3 and VEGF-C mRNAs were not expressed in all the cell types studied, and that blood cells expressed high VEGFR-3 mRNA levels compared to solid tumor cells. As a result, quantitative RT-PCR is considered to be a highly reliable and reproducible technique that could help elucidate lymphangiogenic marker patterns of expression and function in cancer. The physiological functions of the lymphatic network mainly consist of the regulation of fluid homeostasis, participation in immunological surveillance and response, and lipid absorption. During the tumoral processes, the lymphatic system is diverted by the tumor which requires endothelial microvasculature development for a nutrient supply and metastatic dissemination to distal tissues and organs (1). An important marker for the lymphatic endothelium is vascular endothelial growth factor receptor-3 (VEGFR-3 or FLT4), a tyrosine kinase receptor (2), initially thought to be expressed, in normal adults, exclusively by lymph vessel endothelium (3). However, its expression has also been reported in blood capillaries (4, 5), in some endothelial cells (6), and in a wide variety of cancer cells originating from colorectal and lung adenocarcinomas (7, 8), prostatic and breast tumors (9, 10), or myeloid leukemia (11). In addition, many tumors have been shown to express VEGF-C and/or VEGF-D, two members of the VEGF family, that bind to VEGFR-3 and then activate lymphatic endothelial cell migration to the tumor site and microvasculature establishment at the tumor periphery (12-14). The tumor lymphangiogenic processes can be inhibited through neutralization of VEGF-C and VEGF-D by a soluble form of VEGFR-3, as reported in a xenograft model of human lung cancer (15). However, if VEGF-C and/or VEGF-D expression is often associated with poor prognosis in many cancers (16, 17), the VEGFR-3, VEGF-C and VEGF-D expression panel and function in tumor processes remain unclear. Using highly accurate real-time quantitative RT-PCR (qRT-PCR), VEGF-C, VEGF-D and VEGFR-3 mRNA expressions were measured in 17 cell lines, cancer or not, of human origin. Noticeable differences were found in the VEGFR-3, VEGF-C and VEGF-D mRNA levels among the cell types studied, whether they originated from solid tumors, or from healthy or leukemic circulating blood cells.