|Abstract: ||Several kinds of tumors and cancer cell lines may contain a subpopulation of cancer stem cells (CSCs) (Setoguchi et al., 2004). In this study, we attempted to enrich CSCs from the PTC-derived B-CPAP cell line using the sphere culture system, in order to molecularly and cytogenetically characterize them. B-CPAP cell lines were able to grow as floating spheres (thyrospheres) in serum-free medium supplemented with EGF and bFGF. Thyrospheres were able to expand for more than 20 passages, with exponentiallyincreasing sphere-forming efficiency and self-renewal, up to passage 8. RT-PCR analysis showed that stem cell markers Oct 4 and Nanog were expressed at the same level in both B-CPAP adherent cells and thyrospheres, whereas ABCG2 and ALDH1A1 were
expressed more in thyrospheres. Moreover, thyroid differentiation markers TTF1 and PAX8 were both
expressed in B-CPAP thyrospheres and adherent cells, although TTF1 expression was reduced in thyrospheres. Expression of ALDHA1 protein was present only in thyropspheres, while CK19 protein, a differentiation epithelial marker, was expressed only in adherent cells. Spectral karyotyping imaging investigation showed a more complex karyotype, with additional chromosomal rearrangements in thyrospheres as compared to adherent cells, in keeping with possible higher genomic instability of CSCs. Stem-like cells from thyrospheres were isolated by flow cytometry cell sorting assay, using the lipophylic fluorescent dye PKH26. Two cell populations were isolated: the brightest (PKH26 High) making up putative stem cells and the dimmest (PKH26 Low) representing proliferating cells. PKH26 High retained the ability to form secondary spheres and showed increased expression of stem cell markers as compared to PKH26 Low. As expected, differentiated markers were expressed more in PKH26 Low. As a whole, our data suggest that the B-CPAP cell line contains cells with stem-like features, including genomic instability.
Supported by Regione Autonoma Sardegna, POR -FSE 2007–2013.|