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. 2008;3(10):e3544.
doi: 10.1371/journal.pone.0003544. Epub 2008 Oct 28.

In vitro identification and characterization of CD133(pos) cancer stem-like cells in anaplastic thyroid carcinoma cell lines

Giovanni Zito  1 , Pierina Richiusa, Alessandra Bommarito, Elvira Carissimi, Leonardo Russo, Antonina Coppola, Monica Zerilli, Vito Rodolico, Angela Criscimanna, Marco Amato, Giuseppe Pizzolanti, Aldo Galluzzo, Carla Giordano
Affiliations

In vitro identification and characterization of CD133(pos) cancer stem-like cells in anaplastic thyroid carcinoma cell lines

Giovanni Zito et al. PLoS One. 2008.

Abstract

Background: Recent publications suggest that neoplastic initiation and growth are dependent on a small subset of cells, termed cancer stem cells (CSCs). Anaplastic Thyroid Carcinoma (ATC) is a very aggressive solid tumor with poor prognosis, characterized by high dedifferentiation. The existence of CSCs might account for the heterogeneity of ATC lesions. CD133 has been identified as a stem cell marker for normal and cancerous tissues, although its biological function remains unknown.

Methodology/principal findings: ATC cell lines ARO, KAT-4, KAT-18 and FRO were analyzed for CD133 expression. Flow cytometry showed CD133(pos) cells only in ARO and KAT-4 (64+/-9% and 57+/-12%, respectively). These data were confirmed by qRT-PCR and immunocytochemistry. ARO and KAT-4 were also positive for fetal marker oncofetal fibronectin and negative for thyrocyte-specific differentiating markers thyroglobulin, thyroperoxidase and sodium/iodide symporter. Sorted ARO/CD133(pos) cells exhibited higher proliferation, self-renewal, colony-forming ability in comparison with ARO/CD133(neg). Furthermore, ARO/CD133(pos) showed levels of thyroid transcription factor TTF-1 similar to the fetal thyroid cell line TAD-2, while the expression in ARO/CD133(neg) was negligible. The expression of the stem cell marker OCT-4 detected by RT-PCR and flow cytometry was markedly higher in ARO/CD133(pos) in comparison to ARO/CD133(neg) cells. The stem cell markers c-KIT and THY-1 were negative. Sensitivity to chemotherapy agents was investigated, showing remarkable resistance to chemotherapy-induced apoptosis in ARO/CD133(pos) when compared with ARO/CD133(neg) cells.

Conclusions/significance: We describe CD133(pos) cells in ATC cell lines. ARO/CD133(pos) cells exhibit stem cell-like features--such as high proliferation, self-renewal ability, expression of OCT-4--and are characterized by higher resistance to chemotherapy. The simultaneous positivity for thyroid specific factor TTF-1 and onfFN suggest they might represent putative thyroid cancer stem-like cells. Our in vitro findings might provide new insights for novel therapeutic approaches.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression of CD133 in ATC cell lines.
(A) Flow cytometry analysis of CD133 in ARO, KAT-4, KAT-18, and FRO cell lines. Black lines represent positive staining for CD133, grey lines show negative control with matched isotype antibody. Data are representative of three independent experiments. (B) Analysis of CD133 expression in ATC cell lines by qRT-PCR. Data are represented as fold change (relative scale), considering KAT-18 = 1. (C) Immunocytochemistry of CD133 in ARO cell line. Arrows indicate apical and polarized localization of CD133 (20× magnification).
Figure 2
Figure 2. Undifferentiated status of ATC cell lines.
Expression of thyroid specific genes (Tg, TPO, NIS) (A) and fetal marker onfFN (B) in ARO, KAT-4 and normal thyroid by RT-PCR. TAD-2 cell line was used as positive control for onfFN mRNA. β-actin was used as internal control in both experiments. bp = base pairs.
Figure 3
Figure 3. Cluster-forming efficiency of sorted ARO/CD133pos cells.
(A) Purity of ARO/CD133pos cells after magnetic cell sorting assessed by flow cytometry. (B) Characteristics of sorted ARO/CD133pos and ARO/CD133neg on days 0, 3 and 10 of culture. ARO/CD133pos cells formed clusters which increased in size overtime.
Figure 4
Figure 4. Proliferation, self-renewal and colony-forming capacity of ARO/CD133 cells.
(A) Cell proliferation assay (MTT). Dotted line represents ARO/CD133neg cells, continuous line represents ARO/CD133pos cells. Data are expressed as mean values±SD and are representative of four independent experiments. p<0.03. (B) Cell proliferation assay (BrdU). Dotted line represents ARO/CD133neg cells, continuous line represents ARO/CD133pos cells. Data are expressed as mean values±SD and are representative of four independent experiments. p≤0.001. (C) Self-renewal ability of ARO/CD133pos cells. Black line represents number of ARO/CD133pos cells assessed by flow cytometry. Dotted line represents cell viability assessed by MTT assay. (D) Colony formation assay in methylcellulose medium of sorted ARO/CD133pos and CD133neg cells (40× magnification) (E) Number of colonies of ARO/CD133pos and CD133neg cells after 15 days of incubation (p<0.03).
Figure 5
Figure 5. Sensitivity of ARO/CD133pos and ARO/CD133neg cells to chemotherapy agents.
(A) MTT analysis after 48 hours of culture in the presence of 0.5, 1, 1.5 and 2 µM doxorubicin (B) after 48 hours in the presence of 5, 10, 15 and 20 µM cisplatin (C) after 48 hours in the presence of 10, 30 and 100 µM etoposide. Data are expressed as mean values±SD and are representative of three independent experiments (p≤0.05). (D) (E) (F) represent BrdU analysis in the same conditions of A, B, C (p≤0.001). Dotted line represents ARO/CD133neg cells and continuous line represents ARO/CD133pos cells in all graphs.
Figure 6
Figure 6. Flow cytometry analysis of caspase 3 in ARO/CD133pos and ARO/CD133neg cells after treatment with chemotherapy agents.
(A) Caspase 3 activity after treatment with doxorubicin. Dotted line represents ARO/CD133neg cells, continuous line represents ARO/CD133pos cells. Data are expressed as mean±SD (p≤0.001). (B) Caspase 3 activity at time point 72 hours with doxorubicin. Black line represents positive staining for caspase 3, grey line shows negative control with matched isotype antibody. Data are representative of three independent experiments.
Figure 7
Figure 7. Expression of TTF-1 and OCT-4 in ARO/CD133pos and ARO/CD133neg cells.
(A) qRT-PCR of TTF-1 mRNA expression in ARO/CD133pos and CD133neg cells; TAD-2 cell line was used as positive control. (B) Flow cytometry analysis of OCT-4A in ARO/CD133pos and ARO/CD133neg cells. Black line represents positive staining for OCT-4A, grey line shows negative control with matched isotype antibody. (C) Semiquantitative RT-PCR of OCT-4 mRNA in ARO/CD133pos and ARO/CD133neg cells. β-actin was used as internal control. nTERA2 cell line was used as positive control. Data are representative of three independent experiments.
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