Isolation and identification of putative oral cancer stem cells (OSCCs) and changes associated with tumour progression

Abstract

Oral squamous cell carcinomas (OSCC) appear to contain a sub-population of cells endowed with indefinite self-renewal capacity, usually referred to as cancer stem cells (CSCs). These cells are clonogenic and are responsible, in all probability, for tumour initiation and propagation. Cancer cell lines consistently show morphological patterns similar to normal keratinocytes (holoclones, meroclones and paraclones) reflecting a hierarchical organization associated with stem, amplifying and differentiated cells. In-vitro studies of OSCC cell lines have revealed further phenotypic differences in colony formation between the stem and non-stem fractions and have identified cell surface proteins such as CD44, E-Cadherin, ß-catenin and Vimentin which are differentially expressed in association with stemness. Recent studies suggest tumour progression to be associated with Epithelial Mesenchymal Transition (EMT), a process occurring in SCCs which confers invasiveness and motility to CSCs and also suggests a strong involvement of external factors such as those present within the tumour-host microenvironment. Analysis of these properties may provide insight into mechanisms of stem cell fate determination and provide mechanisms for therapeutic targeting of CSCs. Methods and Materials: Immunocytochemistry was used to analyze differential expression of cell surface and internal markers for their role in stemness and EMT, FACS was used to isolate and analyze sub-fractions of cells for clonal studies. Real time videos of cell lines were used to examine cell movement in colonies and cell acquisition of a fibroblastic phenotype. As a test of whether this process is actually EMT, cells were exposed to TGF-ß, a known inducer of EMT, to determine whether this enhanced the formation of the mesenchymal-looking cells at colony margins. Results and Conclusions: Video images showed cells at the margins of holoclones acquiring a motile phenotype. Immunohistochemical analysis revealed that certain stem-cell-related cell surface molecules were expressed at higher levels in holoclones than paraclones and at different levels within holoclones. Expression of CD44 was stronger in the center than at the edge of holoclones suggesting that this molecule plays a role in maintaining a stem-like state. Some images indicated nuclear translocation of CD44 and ß-catenin at colony margins. TGF-ß increased the incidence of mesenchymal-like cells at the edges of colonies and seemed to scatter and mobilize these cells with loss of the normal colony architecture. Further, TNF-α alsogreatly enhanced this effect by working synergistically with TGF-β suggestingthe involvement of external factors such as inflammatory cytokines and other cells present within the tumour-host environment,collectivelycontributing tothe generation of EMT cells with stem potential.