In the present study we have used a set

In the present study, we have used a set of OSCC cell lines and isolated the putative stem cell fraction (CD44highCD24low) along with CD44highCD24high and CD44lowCD24high (non-stem) sub-populations. These populations were subjected to various molecular and cellular assays to determine whether CD24 has any relevant contribution in the context of CD44high population towards defining CSC marker. We have clearly demonstrated CD44highCD24low jak kinase as CSC-like cells within oral cancers.
Methods
Results
Discussion We have done an elaborate molecular and phenotypic characterization of the small percentage of CD24low cells within the predominantly CD44 expressing OSCC cell lines. These cells showed relatively high expression of Sox2, Nanog, Oct4, the well-known embryonic stem cell markers, both at the RNA and protein level. ALdh1 and Bmi1 expression were also consistently up-regulated in CD44highCD24low cell population of all cell lines. Aldh1, Bmi1 and Nanog have already been established as prognostic stem cell markers in a closely related Esophageal Cancer (Hwang et al., 2014). Their importance in OSCC is also being recognized along with other cancers. Moreover, Nanog has been shown to be a therapeutic target controlling CSC self-renewal in various cancers including HNSCCs (Jeter et al., 2015). Studies in tongue carcinomas have denoted Bmi1 as a pro-tumorigenic factor with essential role in migration and invasion (He et al., 2015). Aldh1 activity has been found to overlap with CD44+ population regulating common metabolic pathways in HNSCC (Zou et al., 2012). Collectively, these specific stemness markers were found to be enriched in the CD44highCD24low population. We also ascertained that the descendant population derived from the continually cultured CD44highCD24low cells consisted of other cell types whereas the non-stem population failed to generate the original symmetric condition within the stipulated time. This further emphasized that CD44highCD24lowcells are indeed the CSC population in oral cancers. CD44highCD24low cell types were found to be of mesenchymal nature, as reflected by loss of E-cadherin and gain of Vimentin. This observation was also supported by global analysis of NCI-60 cell line transcriptome dataset which showed expression of CD44 and CD24 to be inversely co-related in the mesenchymal vs epitheial group of cell lines. It might be that the over-expressed Oct4, a key determinant of CSCs, promotes tumour-initiating properties in OSCC by mediating EMT (Tsai et al., 2014). In this context, it must be mentioned that CD24 expression is down-modulated by one of the known EMT factor Twist, to enhance breast Cancer Stem Cell expansion (Vesuna et al., 2009). In OSCC also, it may be speculated that CD24 depletion is a pre-requisite for induction of EMT. However, in several epithelial cancers like colorectal and nasopharyngeal, CD24+ status has been observed as the definitive stem cell marker (Yeung et al., 2010; Yang et al., 2014). In view of this fact, one of the studies in HNSCC has described CD44highCD24high as the Cancer Stem Cells (Han et al., 2014). Conversely, we have found CD44highCD24low cells to be able to form larger spheres within a few days, indicative of its ability to undergo asymmetric division. These were endowed with enhanced self-renewal powers and could be perpetuated for many generations. There are several reports implicating the role of CD24 in promoting invasion and metastasis (Baumann et al., 2005). However, in our case, CD44highCD24low cells were indeed, more migratory and invasive under in vitro conditions. Although these cells did not proliferate any faster, despite altered cell cycle, but under clonal conditions, could give rise to holoclone-like colonies, a typical characteristic of stem cells with extensive differentiation potential. On the contrary, CD44highCD24high cells were not so enriched with stem-related markers. “Side population” cells, those that effectively efflux out drugs, have always been shown to have CSC property (Golebiewska et al., 2011). In this study too, we found the presence of “SP” characteristics in the isolated CD44highCD24low cell types. This not only exemplifies the stemness characteristics of CD44highCD24low cells but also adds to the fact that these cells contribute to intrinsic chemo-resistance and hence difficult to eliminate. It eventually becomes clear with the sensitivity assays whereby CD44highCD24low cells exhibit enhanced survival upon drug treatment owing to their quiescent slow cycling state and reduced apoptosis. Furthermore, as anticipated, the CD44highCD24low cells were particularly enriched upon drug treatment confirming that the drug itself can select for the CSCs. CD24 has been found to be positively associated with tumour grade in various cancers including breast cancer where CD44+CD24− cells have already been well-characterized as CSCs (Wei et al., 2011). Though CD24 has shown prognostic value, it exerts contradictory roles particularly in response to CD44 mediated signalling (Ju et al., 2011). Moreover, in breast cancers, transitions between CD44+CD24− and CD44+CD24+ have been evident upon drug exposure, which seemingly contributes to CSC-like chemo-tolerance owing to activated SFK signalling axis (Goldman et al., 2015). Therefore, distinguishing tumourigeneic properties of these two cell types may be difficult, depending on the tumour type and micro-environment. Although Han et al. found CD44+CD24+ to be more tumorigenic than CD44+CD24− in cell lines derived from salivary gland neoplasms, we found the CD44highCD24low cell type to be CSC-like cells in cell lines primarily derived from oral cavity. Perhaps, the diverse anatomic locations of tumour along with other etiological factors account for context-dependent role of CD markers.