In previous experiments with mammary and brain tumor cells [18], [19], CD49fhigh cells were cultured in non-adherent conditions selleckchem MG132 to induce sphere formation. Thus attachment to ECM may not be necessary for some CD49fhigh cells to exhibit tumorigenic activity while other CD49fhigh cells were cultured just in the presence of ECM [17], [38]. In previous studies with gastric TICs, cells were cultured on non-adherent substrata to form floating spheres [12]�C[14], [32], and substratum was used to induce their differentiation into non-tumorigenic cells. In the present study, we found that CD49fhigh cells could not grow on non-adherent substrata, and they could survive only in the presence of ECM in all three cases analyzed (data not shown).
This suggests that characteristics may be different between TICs identified in the present study and those reported in previous ones though both formed spheres in vitro and exhibited strong tumorigenicity in vivo. It is well known that growth, differentiation and progression of cancer cells are severely affected by ECM [39]. The role of laminin on the progression of tumors has been intensively investigated. Woo Ho Kim, one of co-authors, has repeatedly reported in collaboration with Hynda Kleinman that laminin-adherent human colon cancer cells exhibited strong tumorigenicity, increased growth and decreased apoptosis [40], [41], and similar results have been reported in pancreatic cancers [42]. Consistent with these, gastric carcinomas have been reported to use ��6��4 integrin and newly deposited laminins to adhere to surrounding tissues during the invasion [43], and Koike et al.
[44] showed that invasive behavior of gastric cancer cells was inhibited by treatment with anti-��6 integrin antibody. These results strongly indicate that CD49f plays an important role in regulating invasiveness of human gastric cancers, but its molecular mechanism remains to be solved. Recently, Yu et al. reported that OCT4 and SOX2 directly bind to the ITGA6 promoter to induce the expression of CD49f, and that CD49f plays a pivotal role in maintaining cellular pluripotency through the PI3K/AKT/p53 pathway in mesenchymal stem cells [45]. Consistent with this, we found that SOX2, POU5F1 (a gene encoding OCT4) and ITGA6 were all strongly expressed in human gastric tumors examined, except that SOX2 was not expressed by MKN74 cell line (Figure S3).
Our culture system for CD49fhigh gastric TICs will be useful for further analysis on the role of CD49f in gastric tumorigenesis. We found in the present investigation that CD49fhigh gastric TICs formed ECM-attaching spheres. There are reports showing that substrata are essential to induce sphere-formation by TICs in brain [46], [47] and prostate Anacetrapib gland [17], [38], [48] tumors while others show that TICs can form spheres in non-adherent conditions, as described above.