Detection And Molecular Phenotype Of The Circulating Tumor Cells In Peripheral Blood Of The Patients With Ovarian Carcinoma

PART I Preparation of IMB and characterizationObjective To prepare IMB targeted circulating tumor cells of ovarian cancer and characterization the beadsMethods The carboxyl beads were activated by EDC/NHS. While terminal carboxyl group of magnetic beads activated, the beads can be linked the antibody molecule via an amino intersects, NHS can stable the synthetic IMB. EPCAM is known as epithelial cell-specific cell surface molecule, as a total of epithelial cells in cell surface markers. MUC16 is ovarian cancer cell membrane surface molecule and as an ovarian cancer tissue-specific cell surface markers. After activating the carboxyl beads, EPCAM/MUC16 antibody conjugates to the beads and the ovarian cancer-specific IMB are prepared. The dispersion and immunological activity of IMB were identified through IF and FCM.Results Using anti-Fc segments fluorescent secondary antibody to analyze the IMB, the expression rate of EPCAM, MUC16 after combination with the beads were 94.17±1.05%,97.80±0.60%. The IMB presented good dispensability after combing antibodies by IF analysis.Conclusions In our study, we successfully prepared ovarian cancer-specific EPCAM/MUC16 immune beads. The beads present dispersed and high antibody activity, suggesting the immune beads can combine to the target cells.PART II The spiking experiment and immunomagnetic beads detection of circulating tumor cellsObjective Discussion the sensitivity and specificity of IMB of isolation the circulating tumor cells in peripheral blood of ovarian cancer patients.Methods Flow cytometric analysis of EPCAM/MUC16 expression on SKOV3 cells. Four-milliliter samples of blood from healthy volunteers were spiked with tumor cells (20/50/100). Immunomagneticbeads labeled EPCAM, MUC16 antibodies and mixtures of these two beads were spiked into the blood specimens to detect tumor cells.Results EPCAM/MUC16 expression on SKOV3 cell lines was 99.41%, 99.99%, respectively. When blood samples were spiked with 50 tumor cells, the cells were detectable, and when spiked with 100, the cells were easily and consistently detectable. None of leukocytes was found combined with the beads in peripheral blood samples. Statistical results of the three groups of Immunomagneticbeads labeled EPCAM, MUC16 antibodies and mixtures of these two beads had no difference (P> 0.05).Conclusions There were high sensitivity and specificity of isolation circulating tumor cells of ovarian cancer with EPCAM/MUC16 immunomagnetic beads.PART III Molecular phenotype of circulating tumor cells of ovarian cancerObjective To study the EPCAM, MUC16 molecules expression of circulating tumor cells in ovarian cancerMethods Collect 12ml blood samples of ovarian cancer patients. Immunomagneticbeads labeled EPCAM, MUC16 antibodies and mixtures of these two beads were spiked into three aliquots blood specimens to detect circulating tumor cells in patient. Three detection methods used the same amount of beads. HE staining was used to identify the count of the tumor cells after immunomagnetic enrichment.Results The detection rates were 22±11,14±6,28±12 (P<0.05). The immunomagnetic beads were around the selected CTCs by HE staining.Conclusions For the spiking experiments, the sensitivity of detection of EPCAM, MUC16 immunemagnetic beads individually and jointly applied to detect CTCs was no significant difference, while there are significant differences in the peripheral blood of patients. It show that EPCAM, MUC16 expression ratio different between SKOV3 and CTCs of ovarian cancer patients. The CTCs detection counts of using EPCAM immunomagnetic beads are more than MUC16 immunomagnetic detection value, indicating that circulating tumor cells express EPCAM higher than MUC16. But CTCs counts are higher while using the mixture IMB, suggesting that EPCAM and MUC16 expression on CTCs independent in circulating tumor cells.