CD166 Acts As The Cancer Stem Cell Marker Of Oral Squamous Cell Carcinoma

Background&Purpose:Accumulated evidence showed cancer stem cells(CSCs)are tumorigenic and make great contribution to tumor metastasis and recurrence.The discovery of specific membrane-associated cancer stem cell markers is crucial for developing novel therapeutic strategies to target these CSCs.Methods:Sphere cultures used CAL27,HN6,HN12 and HN13 cells were established to enrich OSCC CSCs.Immunocytochemistry,western blot and xenografts in nude mice were performed to assess the stemness characteristics of OSCC spheres.Membrane proteins were isolated and analyzed by nano HPLC-Chip Cub MS and MS/MS spectra were used to search the Swissprot human database.Two data sets for the h ESC membrane protein signature were used to identify the overlapping proteins in our data.In addition,western blot,immunohistochemistry,confocal microscope and flow cytometric analysis were used to analysis CD44 and CD166 expression in OSCC cell lines and samples.Furthermore,CD166^high and CD166^low cells in CAL27 and HN13 populations were sorted to assess the stemness characteristics by sphere-formation assay and tumorigenicity in nude mice.Cancer associated fibroblasts(CAFs)were primary cultured originating from OSCC tissues and co-cultured with OSCC cells to study the interaction between stromal cells with cancer cells.The Sonic Hedgehog signal pathway activity was analyzed in CAFs and shh was upregulated in CAFs to evaluate the role of hedgehog in stemness maintain of OSCC.Results:OSCC spheres overexpressed stem-cell-related molecules and improved in vivo tumorigenic ability.Of a total data set that included 376 identified proteins.Among them,123 proteins were up-or down-regulated in the spheres compared with the adherent cultures.These proteins included cell adhesion molecules,receptors,and transporter proteins.Some of them play key roles in wnt,integrin and TGFbsignaling pathways.Comparing our dataset with two published h ESC membrane protein signatures,we found 18 proteins common to all three of the databases.CD166 and CD44 were two of them.Patients whose tumors expressed high levels of CD166 had a significantly poorer clinical outcome than those whose tumors expressed low levels of CD166(cohort 1:96 cases,p=0.040),while the level of CD44 expression had only a marginal influence on the clinical outcome of patients with OSCC(p=0.078).The level of CD166 expression in OSCC tumors was also associated with the tumor recurrence rate(cohort 2:104 cases,p=0.016).Additionally,CD166^high cells had higher sphere-formation ability and tumorigenicity in nude mice.We also demonstrated that CAFs promoted OSCC cells proliferation,invasion,spheres formation and CD166 upregulated.Further investigation showed that hedgehog signal pathway was actived in CAFs to enhance the tumor cells stemness characteristics.Conclusion:This study demonstrates that compared with well recognized stem cell related cell surface marker CD44,membrane expression of CD166 is a more effectively stem like cell enrichment marker in OSCC.Moreover,Sonic Hedgehog pathway in CAFs plays an important role in stemness maintenance of OSCC.