Recent studies support the correlation of CSCs and angiogenesis in different carcinomas. The population of CSCs increases in hypoxia, which results in rapid tumor growth and metastasis.(18) Also, CSCs can produce higher levels of vascular endothelial growth factor (one of the most important angiogenesis inducing factors)(19) than other malignant tumoral cells.(20) Thus, combination of anti-angiogenetic drugs and anti-CSC drugs together with conventional cancer therapy is highly promising.(21)
Studies on OSCC have shown the fundamental role of GLUT1 and CD105 in hypoxic response and correlation of their expression with aggressive behavior and worse clinical outcomes.(12, 22, 23) Also, studies on OSCC demonstrated OCT3/4 as a potential prognostic marker correlated with poor overall survival rate, and suggested further studies on its expression in conjunction with other markers.(24)
Considering the overexpression of GLUT1 in pre-cancerous lesions, it may have a more prominent role in early stages of malignant transformation;(25) whereas, CD105 represents the formed vessels and is associated with late stages of angiogenesis and hypoxic response. We used these two markers in this study to elucidate the role of CSCs in early and late stages of hypoxic response.
Our results suggest that in early stage of hypoxic situation in OSCC, CSCs may be responsible for hypoxic response due to overexpression of both OCT3/4 and GLUT1. But in late stage of tumor progression and angiogenesis with higher expression of CD105 which leads to angiogenesis and blood vessel formation, the expression of CSC markers is not significant, which means a reduction in the role of CSCs in angiogenesis in the late stage of tumor progression.
Also, the current results indicated that overexpression of both OCT3/4 and GLUT1 markers was correlated with poor prognosis in OSCC. Thus, CSCs may be associated with poorer prognosis via GLUT1 expression in hypoxic conditions. This finding was in accordance with recent studies that showed tumor hypoxia was associated with a poorer prognosis in head and neck cancers.(26, 27)
Recently, CSCs have been identified as potentially initiating cells of tumor angiogenesis and neovascularization. Evidence strongly suggests that association of CSCs with tumor angiogenesis may be mediated through the induction of vascular endothelial growth factor production.(28) Also, CSCs can organize a pseudo-vascular network in some malignant tumors.(28) It might be the result of their differentiation into a borderline transitional cell type between an endothelial cell and a tumor cell among tumoral cells. Further studies are necessary to evaluate these processes. CSCs are diverse and have various markers. They vary in different grades and stages of a tumor, in different tumors, and also in different patients with the same type of tumor. We evaluated only one CSC marker (OCT3/4), which was a limitation of the present study.
In recent years, utilization of dual staining methods has considerably increased. Two or more antigens can be evaluated simultaneously by using dual staining. This method can reveal the correlation of different antigens, and may also increase the chance of cross-reactions. Various procedures have been suggested to reduce these cross-reactions such as using antibodies from different species.(29)
To our knowledge, it is the first study to evaluate the expression of GLUT1 and CD105 hypoxic marker as markers of angiogenesis in association with one CSC marker in OSCC, via dual staining immunohistochemistry. We showed that CSCs could play an important role in initial stages of tumor progression and angiogenesis. Further studies are required to discover the roles, biomarkers and associated pathways of CSCs in OSCC.