We thus conclude that CD49f is a promising marker for gastric TICs, and that this culture system will be useful to find new drugs targeting gastric TICs. Materials and Methods Tumor Tissues and PDTX Lines Gastric tumor tissues were obtained Belinostat HDAC with informed consent from patients who underwent surgical resection at Tokyo Medical and Dental University Hospital and Asan Medical Center Hospital between 2008 and 2012, and the study was approved by the Medical Research Ethics Committee for Genetic Research of Tokyo Medical and Dental University, and the Institutional Review Board of Asan Medical Center. Written informed consent was obtained from each patient for the use of his/her tumor tissue for this research in both hospitals.

Freshly isolated tumor samples were cut into small pieces and transplanted subcutaneously into KSN and BALB/c nude mice at 4�C6 weeks old (Japan SCL, Inc., Shizuoka, Japan and Central Lab. Animal Inc., Seoul, Korea, respectively). The animals were housed in specific pathogen-free animal facilities in accordance with the Guideline for Care and Use of Laboratory Animals of the respective Institutional Animal Care and Use Committees, and the research was approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University, and the Institutional Animal Care and Use Committee of Asan Medical Center. When gastric tumors grew in nude mice, some tissues were stored in liquid nitrogen, and were used at early passages (p<6) because tumors grew faster and became more aggressive with the increase in passage number.

Gastric tumor tissues just after surgical operation were also used for the analysis. Dissociation and Staining of Tumor Cells for FACS Analysis Tumor tissues were disaggregated into single cells for FACS (fluorescence-activated cell sorter) analysis. Tissues were cut with scalpels into small fragments, washed thoroughly with CMF-PBS, treated with 0.05% trypsin-0.53mM EDTA with 0.01% DNase I (Sigma-Aldrich, St Louis, MO, USA) for 30 min at 37��C, and cells were disaggregated by pipetting. In some previous papers, collagenases were used for cell dissociation, but we used trypsin-EDTA because it did not affect cell surface antigen profiles and reproducible results were easily obtained, as has been shown in a previous paper [11].

Cell suspensions were filtered through 70 ��m nylon meshes (BD Biosciences, San Jose, CA, USA) to obtain single cells, which were stained with PE-labeled anti-human CD133/2 Dacomitinib (clone AC133, Miltenyi Biotec, Auburn, CA, USA), PE- or FITC-labeled rat anti-CD44 (clone IM7, BD Biosciences), FITC-labeled rat anti-human CD49f (clone GoH3, BD Biosciences) or PE-labeled anti-EpCAM (clone HEA-125, Miltenyi Biotec) antibodies. Flow cytometric analysis and cell sorting were performed by using FACS Vantage or FACS Aria II cell sorters (BD Biosciences).