Isolamento e caratterizzazione biologico-molecolare di cellule tumorali simil-staminali da una linea cellulare derivata da un carcinoma papillare tiroideo

CARIA, PAOLA

2012-03-02

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Abstract

Recent reports have shown that tumor growth is supported by a specific subpopulation of stem cells, known as cancer stem cells (CSCs) or tumor-initiating cells. This cells have stem-like features, such as self renewal,multipotency, high migration capacity, drug resistance and aberrant differentiation. CSCs have been detected in several kind of tumors and in cancer cell lines grown as non-adherent spheres in serum–free medium under intense stimulation whit growth factors. Presence of cancer stem-like cells in thyroid differentiated carcinoma has not yet fully investigated and the literature on such issue is still limited. The objective of this study was to identify and characterize CSCs from a cancer cell line from papillary thyroid carcinoma (B-CPAP) and a cell line, derived from human thyroid follicular cells (N-THY-ORI 3-1), as control. Thyrospheres from B-CPAP cells could be propagated up to ten generations, whereas those from N-THY-ORI 3-1 lasted only for four generations. The “stemness” profile was evaluated by functional assays and RT-PCR. B-CPAP sphere forming efficiency (SFE) and self renewal increased exponentially at every generation with maximum value at the 8th. By contrast, N-THY-ORI 3-1 SFE and self renewal progressively decreased along generations. RT-PCR showed mRNA expression of stem cell markers (Oct 4, Nanog, ABCG2) and early and thyroid late differentiation markers (PAX8, TTF1, Tg) in B-CPAP thyrosphere. In particular, ABCG2 expression significantly increased in thyrospheres at 9th generation. On the contrary, PAX8, TTF1 and Tg showed a decrease in thyrosphere along the generation of spheres, while p63 maintained the same expression in all samples. Thyrospheres from non tumorigenic cell line were positive for mRNA expression of stem cell markers (Oct4, Nanog, ABCG2,) and PAX8. In this cell line, Nanog and ABCG2 mRNA expression increased along the generations. To isolate stem-like cells in tumor and normal thyrospheres, we used the fluorescent dye PKH26. The single cells suspensions from thyrospheres were then FACS sorted. According to fluorescent intensity we selected two populations: the brightest fluorescent cells (PKH26 high), which constitute the putative stem cell population and the dimmest fluorescent cells (PKH26 low), which represent proliferating and differentiated cells. Both populations were able to form sphere, but only PKH26 high formed secondary spheres. The molecular analysis showed a higher stem cell marker mRNA expression in PKH26 high compare to PKH26 low cells. On the contrary TTF-1, PAX8 and Tg mRNA were more expressed in PKH26 low cells. Take together, our data show, for the first time, that B-CPAP and N-THY-ORI 3-cell lines contain cells with features and properties of stem cell.