Recent characterization of abnormal phosphatidylcholine metabolism in tumor cells by nuclear magnetic resonance (NMR) has identified novel fingerprints of tumor progression that are potentially useful as clinical diagnostic indicators. In the present study, we analyzed the concentrations of phosphatidylcholine metabolites, activities of phosphocholine-producing enzymes, and uptake of [methyl-¹⁴C]choline in human epithelial ovarian carcinoma cell lines (EOC) compared with normal or immortalized ovary epithelial cells (EONT). Quantification of phosphatidylcholine metabolites contributing to the ¹H NMR total choline resonance (3.20-3.24 ppm) revealed intracellular [phosphocholine] and [total choline] of 2.3 ± 0.9 and 5.2 ± 2.4 nmol/10⁶ cells, respectively, with a glycerophosphocholine/phosphocholine ratio of 0.95 ± 0.93 in EONT cells; average [phosphocholine] was 3- to 8-fold higher in EOC cells (P < 0.0001), becoming the predominant phosphatidylcholine metabolite, whereas average glycerophosphocholine/phosphocholine values decreased significantly to ≤0.2. Two-dimensional {phosphocholine/total choline, [total choline]} and {glycerophosphocholine/total choline, [total choline]} maps allowed separate clustering of EOC from EONT cells (P < 0.0001, 95% confidence limits). Rates of choline kinase activity in EOC cells were 12- to 24-fold higher (P < 0.03) than those in EONT cells (basal rate, 0.5 ± 0.1 nmol/10⁶ cells/h), accounting for a consistently elevated (5- to 15-fold) [methyl-¹⁴C]choline uptake after 1-hour incubation (P < 0.0001). The overall activity of phosphatidylcholine-specific phospholipase C and phospholipase D was also higher (∼5-fold) in EOC cells, suggesting that both biosynthetic and catabolic pathways of the phosphatidylcholine cycle likely contribute to phosphocholine accumulation. Evidence of abnormal phosphatidylcholine metabolism might have implications in EOC biology and might provide an avenue to the development of noninvasive clinical tools for EOC diagnosis and treatment follow-up.

Matrix metalloproteinase (MMP) degradation of extracellular matrix is thought to play an important role in invasion, angiogenesis, tumor growth, and metastasis. Several studies have demonstrated that CD147/extracellular MMP inducer, a membrane-spanning molecule highly expressed in tumor cells, may be involved in the progression of malignancies by regulating expression of MMP in peritumoral stromal cells. In the present study we show that CD147 is expressed in microvesicles derived from epithelial ovarian cancer cells and that CD147-positive vesicles may promote an angiogenic phenotype in endothelial cells in vitro. Vesicles shed by human ovarian carcinoma cell lines OVCAR3, SKOV3, and A2780 expressed different levels of CD147 and stimulated proangiogenic activities of human umbilical vein endothelial cells (HUVECs) in a CD147-dependent fashion (OVCAR3 > SKOV3 > A2780). Moreover, vesicles shed by ovarian carcinoma cell line CABA I with low CD147 expression had no significant effect on the development of angiogenic phenotype in HUVECs. The treatment of OVCAR3 cells with small interfering RNA against CD147 suppressed the angiogenic potential of OVCAR3-derived microvesicles. However, transfection of CD147 cDNA into the CABA I cell line enabled CABA I-derived vesicles to induce angiogenesis and to promote MMP genes expression in HUVECs. We therefore conclude that vesicles shed by ovarian cancer cells may induce proangiogenic activities of HUVECs by a CD147-mediated mechanism.

Tumor angiogenesis is regulated by a dynamic cross-talk between tumor cells and the host microenvironment. Because membrane vesicles shed by tumor cells are known to mediate several tumor-host interactions, we determined whether vesicles might also stimulate angiogenesis. Vesicles shed by human ovarian carcinoma cell lines CABA I and A2780 stimulated the motility and invasiveness of endothelial cells in vitro. Enzyme-linked immunosorbent assay and Western blot analysis revealed relevant amounts of vascular endothelial growth factor (VEGF) and the two matrix metalloproteinases MMP-2 and MMP-9, but not fibroblast growth factor-2, contained in shed vesicles. An A2780 cell-derived clone transfected to overexpress VEGF shed the same amount of vesicles as did a control clone, but contained significantly more VEGF within the vesicles. Despite a greater amount of VEGF in vesicles of the overexpressing clone, vesicles of both clones stimulated endothelial cell motility to comparable levels, suggesting that VEGF was stored within the vesicle and was unavailable. Only following vesicle burst induced by acidic pH (a characteristic of the tumor microenvironment) was VEGF released, leading to significantly higher stimulation of cell motility. Thus, tumor-shed membrane vesicles carry VEGF and release it in a bioactive form in conditions typical of the tumor microenvironment.