When unsorted HGC-1 cells were cultured, the ratio of CD49fhigh cells was significantly increased about 10-times from 3 to 34%. Also it was increased about 7-times from 6 to 42% in culture then of unsorted HGC-4 cells in 2 weeks (Figure 3F). The ratio of CD49fhigh cells in total cells was similar when sorted CD49fhigh HGC-1 cells or unsorted (mostly CD49flow) HGC-1 cells were cultured. This indicates that only CD49fhigh cells could grow to form spheres when unsorted HGC-1 tumor cells were cultured. In culture of unsorted HGC-2 tumor cells, the ratio of CD49fhigh cells was significantly increased more than 10-times from 6 to 88% in 8 weeks. Thus all these tumor cells retain similar features that only sphere-forming CD49fhigh cells could grow in vitro, but their growth pattern differed depending on PDTX lines.
These sphere-forming cells as well as PDTXs strongly expressed BMI1, POU5F1 and SOX2 (Figure S3), which are reported to be strongly expressed by stem cells [30]. We then examined tumorigenicity of these sphere-forming CD49fhigh cells. We found that sphere cells obtained by primary culture of unsorted HGC-1 tumor cells were strongly tumorigenic because subcutaneously injection of 3,000 sphere cells always formed tumors and injection of 10 cells sometimes formed a tumor in NOD-SCID mice (Table 2). The tumors exhibited histological features of the parental one (Figure 4), indicating that the CD49fhigh sphere-forming cells retained differentiation potency of TICs in the original tumor.
Sphere cells obtained by culture of unsorted HGC-2 and HGC-4 cells also formed tumors with histological features of parental ones (Table 2 and Figure 4), but tumorigenicity of HGC-4 sphere cells was less than that of HGC-1 sphere cells, and that of HGC-2 sphere cells was at the intermediate between HGC-1 and HGC-4 sphere cells (Table 2). We thus concluded that only CD49fhigh sphere-forming TICs could grow when unsorted tumor cells were cultured. These results are consistent with our idea that CD49f is a useful marker for gastric TICs. Figure 4 Sphere cells form tumors with histological features of parental ones. Table 2 Tumorigenicity of sphere cells obtained by culture of unsorted cells (number of tumors formed in NOD-SCID mice/number of injection of tumor cells). Some Sphere-forming TICs are Anacetrapib Resistant to Chemotherapeutic Agents TICs have been reported to be more resistant to chemotherapy than other tumor cells [31]. We thus examined whether the CD49fhigh sphere-forming TICs were resistant to anti-tumor drugs. We found that MKN45 and MKN74 human gastric tumor cell lines attached to the collagen gel to grow rapidly in the same condition where unsorted HGC-1, HGC-2 and HGC-4 tumor cells grew to form spheres. We thus used MKN45 and MKN74 cells as controls.