The PCR products were inhibitor Bosutinib separated by electrophoresis on 2.0% agarose gels in 0.5��TAE buffer. Effect of Chemotherapeutic Agents on the Growth of TICs in Primary Culture HGC-1, HGC-2 and HGC-4 tumors were dissociated into single cells as described above, and seeded on collagen gel in 24-well plates at 50,000 cells per well with REBM-based culture medium, and their growth was examined after 2 weeks. MKN45 and MKN74 human gastric cancer cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum on plastic, but we found that both cells proliferated rapidly in the REBM-based serum-free condition used for culture of HGC-1, HGC-2 and HGC-4 cells. We thus used both cells as controls.
Since MKN45 cells proliferated more rapidly than MKN74 cells, we seeded MKN45 cells more sparsely (1,000 cells per well) than MKN74 cells (3,000 cells per well) to give enough space for the cells to keep growing for 2 weeks. The cells were plated with 0.5 ml media on day 0, and they were treated with doxorubicin (DXR), 5-fluorouracil (5-FU), and doxifluridine (DXF) (Wako Pure Chemical, Osaka, Japan) on day 1, by adding 0.5 ml media containing ��2 concentrated chemicals. 5-FU and DXF were dissolved in dimethyl sulfoxide (DMSO), while DXR in water. Thus DMSO (final concentration=0.1%) was added to each well when cells were treated with 5-FU or DXF. After 2 weeks, cell growth was determined by using 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as substrate as described previously . Statistical Analyses The results were statistically analyzed using the non-parametric Mann-Whitney��s U test or Student��s t-test.
For the analysis of data in tables, Chi-square test was used. A P values of <0.05 were considered statistically significant. Results CD49f is a useful Marker for Gastric TICs We first established PDTX lines by subcutaneously injecting newly-dissected human gastric tumor tissues into nude mice, because such lines have been used to identify TICs in colon ,  and lung  cancers. We established 5 PDTX lines (HGC-1 to -5, Table 1), and confirmed that histological features of parental tumors were maintained in PDTXs even when tissues were passaged several times (data not shown). Table 1 Case description and tumorigenic activity of CD49fhigh and CD49flow tumor cells. CD44 has been reported as a marker for gastric TICs �C.
We thus compared tumorigenicity AV-951 of CD44-positive and -negative cells in the PDTXs, and found that not only CD44high cells but also CD44low cells were tumorigenic in 3 PDTX lines (Figure S1 and Table S2). Rocco et al.  also reported that CD133+ and CD133+/CD44+ cells in human primary gastric cancers did not exhibit tumor-initiating activities when they were transplanted into immunodeficient mice. These results suggest that TICs in primary gastric cancers do not always express CD44.