Figure 3. Fucosylation and SLe expression - functional assays.

A. Invasion assay of orospheres vs adherent cells. Orospheres are significantly more invasive than adherent cells. B. Quantitative mRNA expression of FUTs. In both cell lines, FUT5 and FUT7 are not expressed, while FUT3 and FUT6 are more expressed in orospheres. C. Sialyl Lewis X (SLe^x) expression on orospheres and adherent cells (flow cytometry). Orospheres express a higher level of SLe^x as compared with their adherent counterparts (UMSCC14B: 93% for orospheres vs 27.2 for adherent cells; UMSCC103: 97% for orospheres vs 93% for adherent cells). Moreover, the mean fluorescence intensity for UMSCC103 is much higher than that of adherent cells, with most of the cells after the 4^th decade of fluorescence intensity. D. qPCR for FUT 3/6 expression on SLe^x sorted cells. SLe^x+ cells have significantly increased FUTs expression. E. Invasion assay for SLe^x+ vs SLe^x−. SLe^x+ cells are significantly more invasive than SLe^x− cells. F. Shear flow assay. Graphs of percentages of firmly adherent (sticking) and rolling cells. The rate of firmly adherent (sticking) cells is higher for orospheres in both cell lines, while there is no significant difference in rolling cells. Moreover, the mean velocity of rolling cells is much lower for orosphere-derived cells than for adherent cells. *P<0.05.