To characterize cetuximab resistant oral squamous cell carcinoma (OSCC) cells, we compared the proliferative properties of the OSCC cell lines. Although SAS cells were unable to proliferate in serum-free monolayer culture, they formed spheres from single cells in floating culture. HSC4 cells were unable to proliferate under serum-free culture conditions or form spheres. Thus, although cetuximab-resistant SAS cells exhibited cancer stem cell-like characteristics, cetuximab-sensitive HSC4 cells were not able to form growing aggregates. We subjected the aggregates to immunofluorescence staining using anti-Ki-67 antibody. Although many nuclei of SAS aggregates stained with this antibody, Ki-67-positive nuclei were absent in HSC4 cell aggregates. Growth of SAS aggregation was inhibited by cetuximab. Cetuximab-resistant SAS cells not only engaged in EGFR-independent growth, but also exhibited stem cell-like properties. However, growth was EGFR-dependent in aggregation culture, and the SAS cell aggregates became cetuximab-sensitive. This suggests that cetuximab sensitivity is not only cell-type-dependent, but is also affected by the growth microenvironment.

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CD44 is one of the adhesion molecules expressed on the surface of various cells. Several human cancers, such as colon cancer and breast cancer, are expressed at a high level of CD44, and it is regarded as one of the markers for stem-like cancer cells. Similarly, nuclear factor of kappa B (NFκB) is not only known to participate in inflammation and immunoresponse, but is also thought to be an important signaling pathway that maintains the breast cancer stem cell. In this study, we investigated the expression and localization of CD44 and NFκB in SAS and KB, which are oral squamous cell carcinoma (OSCC) cell lines, using western blotting and immunostaining. Western blotting analysis showed that both cell lines expressed CD44 and NFκB. In particular, the expression of CD44 variant isoform (CD44v) was higher in SAS than in KB. In addition, the expressions of NFκB and phosphate NFκB were obvious in SAS. Further-more, we observed the expression of CD44 in both OSCC cell lines by immunostaining. However, the expression levels were different. SAS expressed CD44 on the cell membrane strongly compared with KB. Moreover, sulfasalazine (SSZ), which is an anticancer drug for CD44 and NFκB, inhibited the cell proliferation of SAS. These results suggest that SAS, because it expresses CD44 and NFκB, has cancer stem cell properties. Also, we demonstrated the effect of SSZ susceptibility on 0 cancer stem cells.

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