Isolation and culture of circulating pancreatic cancer tumor cells using a microfilter.

Abstract

e16230 Background: The presence and number of circulating tumor cells (CTCs) are reportedly associated with prognosis but the rarity of CTCs in peripheral circulation limits the ability of conventional technologies to provide fully accurate analyses. The purpose of this study was thus to establish a novel approach to capture and culture the CTCs using a microfilter device. Methods: This was a prospective study in preoperative patients with pancreatic cancer at the University of Tsukuba Hospital after approved by the Institutional Review Board. Five ml of whole blood was collected from each patient into an EDTA-treated collection tube before pumping through an 8μm pore microfilter. Cells isolated on the filter were stained with PE-conjugated anti-EpCAM, FITC-conjugated anti-CD45 and Hoechst 33342. The filter was then transferred to a 35mm dish and cultured with 20%FBS-IMDM-F12 medium containing 2%Matrigel- EGF-FGF-B27 for 4-6 weeks. Cells were visualized with an automatic fluorescence microscope. A CTC was defined as an intact round oval cell with a visible nucleus, positive staining for EpCAM and negative staining for CD45. Results: A total of 9 patients [male/female; 4/5, median age;71 (39-78), Stage 0/IA/IIA/IIB; 1/3/3/2, preoperative chemotherapy/chemoradiation; 1/2] were enrolled. CTCs were captured in 8 out of 9 cases and cultured CTCs were kept viable for 4-6 weeks in 6 out of 9 cases. CTCs formed colonies by 4-6 weeks of culture, colonies were expanding and blood cells disappeared. No cultured CTCs were observed in the two cases who received preoperative chemotherapy. Conclusions: We successfully developed a novel culture method of patient CTC using a microfilter device. Our system supports the possibility of accurate detection and proliferation of CTCs on microfilters. Cultured CTCs could be used for patient-specific drug susceptibility testing and mutational profiles for future cancer patients.