Abstract 4135: Isolation of circulating tumor cells (CTCs) from breast cancer patients in EpCAM independent manner using parallel multi-orifice flow fractionation (p-MOFF).
Authors: Jung Hyo-Il , Hyun Kyung-A , Han Hyunju , Kim Seung-Il 
Published: August 2014

Abstract
It is generally accepted that circulating tumor cells (CTCs) in the peripheral blood of cancer patients are highly correlated with the "invasive behaviors" of a proportion of cancer cells. Therefore the precise measurement or isolation of CTCs can serve as a powerful tool for cancer prognosis, diagnosis of minimal residual disease, assessment of tumor sensitivity to anticancer drugs, and personalization of anticancer therapy.
In a previous study, we introduced a novel hydrodynamic method for size-based particle separation multi-orifice flow fractionation (MOFF), in which the animal cellis moved laterally according to their size due to hydrodynamic inertial force. This technique is advantageous for CTC separation because of its simple experimental set up and high operational flow rate (100-300 μL/min). However, it has a limitation that the blood sample should be diluted over 400 times to make a suitable inter-particle distance in the channel. Therefore, we designed the parallel MOFF chip (p-MOFF) which is connected by four single MOFF channel to isolate CTC from cancer patient blood. Using breast cancer cell line, we separated 93.75% of MCF-7 cells and 91.60 % of MDA_MB_231 cells, and eliminate 90.8 % of WBCs from individual experiment at 600 μL/min inlet flow rate and 240 μL/min outlet flow rate.
Based on these results, we have attempted to isolate CTCs from whole blood (7.5ml) of the breast cancer patients under the same conditions. After processing of blood from metastatic breast cancer patients on the p-MOFF device, cells were fixed on a slide glass and stained with DAPI for DNA content, PE conjugated pan-Cytokeratin for CTC, and APC conjugated CD45 for WBC. Following imaging the slide glass, captured images were examined carefully with predefined criteria. Cell size and shape were also considered in cell characterization. We found the CTCs which are positive for DAPI, negative for CD45 and positive for Cytokeratin. When the blood samples of 24 breast cancer patients were subjected to our device, 1 to 21 CTCs were identified in 19 patients.
Subsequently, we have conducted the multi-parameter analysis to investigate the EpCAM expression level of CTCs. The blood samples of 24 metastatic breast cancer patients were analyzed. With our device, 17 patients had at least one CTC in 7.5 ml of blood. Among them, when we stained the cells using multi-parameter (DAPI for DNA content, phycoerythrin conjugated pan-Cytokeratin for CTC, APC conjugated EpCAM for EpCAM positive CTC, and FITC conjugated CD45 for WBC), we observed that the 47.1% of patients had both of EpCAM positive and negative CTCs simultaneously. So,the most advantageous feature is that p-MOFF is able to detect heterogeneous CTCs because the method is based on label-free and hydrophoresis
Citation Format: Hyo-Il Jung, Kyung-A Hyun, Hyunju Han, Seung-Il Kim. Isolation of circulating tumor cells (CTCs) from breast cancer patients in EpCAM independent manner using parallel multi-orifice flow fractionation (p-MOFF). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4135. doi:10.1158/1538-7445.AM2013-4135