Increased fucosylation has a pivotal role in invasive and metastatic properties of head and neck cancer stem cells

Abstract

Oral squamous cell carcinoma (OSCC) is an aggressive malignancy with high mortality rates. Major challenges for OSCC management include development of resistance to therapy and early formation of distant metastases. Cancer stem cells (CSCs) have emerged as important players in both pathologic mechanisms. Increased fucosylation activity and increased expression of fucosylated polysaccharides, such as Sialyl Lewis X (SLex), are associated with invasion and metastasis. However, the role of fucosylation in CSCs has not been elucidated yet. We used the spheroid culture technique to obtain a CSC-enriched population and compared orospheres with adherent cells. We found that orospheres expressed markers of CSCs and metastasis at higher levels, were more invasive and tumorigenic, and were more resistant to cisplatin/radiation than adherent counterparts. We found fucosyltransferases FUT3 and FUT6 highly up-regulated, increased SLex expression and increased adhesion by shear flow assays in orospheres. Inhibition of fucosylation negatively affected orospheres formation and invasion of oral CSCs. These results confirm that orospheres are enriched in CSCs and that fucosylation is of paramount importance for CSC invasion. In addition, SLex may play a key role in CSC metastasis. Thus, inhibition of fucosylation may be used to block CSCs and metastatic spread.

Figure 1. Cancer stem cell characterization

A. Flow cytometry analysis of CD44 and ALDH activity. The CD44⁺/ALDH⁺ subpopulation is larger in orospheres in both UMSCC14B and UMSCC103 cell lines. B. Summary of marker expression in orospheres and adherent cells. *P<0.05 C. Real time PCR for the most common stem cell-related markers (OCT3/4, SOX-2, NANOG). All stem cell-related markers are remarkably more expressed on orospheres. *P<0.05. D. Growth curve of orospheres and adherent cells implanted in immunocompromised mice. In both cell lines, the orospheres show faster growth, resulting in larger tumors at the time of sacrifice. E. Examples of explanted tumors for both cell lines. Orospheres produce remarkably larger tumors. Scale bar=4mm. F. H&E staining on sections of explanted and original primary tumors. Orospheres and adherent cells recapitulate the architecture of the original tumor. Original magnification: 100X (xenograft); 400X (original primary tumor).

Figure 2. Cisplatin and radiation resistance

A. Graphs of non-viable (apoptotic+necrotic) cells measured by Annexin V/PI staining after cisplatin (after 24 hours) or radiation (after 48 hours) treatment. Orospheres are more resistant to both treatments. *P<0.05. B. Dose-response curves for IC50 calculation (UMSCC14B adherent cells, 114 (95% CI 104-126); UMSCC14B orospheres, 144 (95% CI 134-155); UMSCC103 adherent cells, 26.5 (95% CI 18-39); UMSCC103 orospheres, 58 (95% CI 47-72). C. Colony formation was measured 15 days after cisplatin or radiation treatment. Orospheres form significantly more colonies than adherent cells. *P<0.05.

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.

Figure 4. FUT inhibition

A. Orosphere size in cells treated with 2F-peracetyl-fucose. Treated cells of both lines produced significantly smaller sized orospheres. B. Image of orospheres from 2F-peracetyl-fucose-treated and -untreated cells. C. Invasion assay of orospheres treated and untreated with 2F-peracetyl-fucose. Treated cells had significantly reduced invasion ability.