Flow cytometric method for determining folate receptor expression on ovarian carcinoma cells

Abstract

The α-folate receptor (α-FR) is a folate transporter with restricted expression levels in normal tissues. It is over-expressed in several cancers, particularly epithelial carcinomas, including nonmucinous ovarian carcinoma. It offers a novel therapeutic target for selective imaging and cytotoxic agents. Measurement of the receptor could be a valuable tool in selecting patients more likely to respond to new drugs that target the α-FR, and monitoring them while on treatment. While tumor samples are often unavailable, a number of patients who relapse develop ascites, which are often rich in tumor cells. We have therefore developed a triple antibody flow cytometric method to assess α-FR expression on tumor cells from ascites. An antibody to BerEP4, an epithelial cell marker expressed on >90% ovarian cancers, labeled with fluorescein, and an α-FR antibody labeled with antimouse-phycoerythrin have been used to label tumor cells, with a CD45-phycoerythrin-cyanine5 antibody used to exclude white blood cells from the analysis. The method was optimized using human carcinoma cell lines (JEG-3, IGROV-1, and KB cells). Calibrated beads were used to quantify the number of antibodies bound per cell. The triple antibody protocol successfully measured α-FR expression levels in cell lines spiked with blood. Tumor cells were obtained from ascites in 25 patients with relapsed ovarian cancer. In each case sufficient cells were harvested to identify an epithelial cell population to estimate the number of binding sites/cell. All the samples contained a single population of BerEP4, α-FR positive cells between 5 × 103 and 5 × 105 antibody binding sites/cell. The method can be used to determine the number of anti-α-FR antibodies bound per epithelial cell in ascites from patients with ovarian carcinoma. The results obtained were reproducible and the method could be applied to specimens that had been stored at -80°C. © 2007 International Society for Analytical Cytology.