The present study attempts to find an effective method for the isola tion of cervical thenthereby CSCs. We sorted and cultured SP cells from the cervical cancer cell line HeLa, and then identi fied their stem like characteristics. This study may be helpful to elucidate novel targets for effective clinical treatments of cervical cancer. Results SP cells among HeLa cells After excluding dead cells and cellular debris based on scatter signals and propidium iodide fluorescence, the SP and non SP cells were sorted. In multiple inde pendent HeLa cultures, we detected 1. 07 0. 32% SP cells as shown in Figure 1A. After 6 hour plating, observation of the morphology of SP and non SP cells revealed that the SP cells were more adherent than non SP cells.

In addition, the sorted SP cells were smaller and rounder in shape than non SP cells, and mostly showed colony like growth. Expression of various biomarkers related to stem cells The expression of various biomarkers putatively related to CSCs was investigated in freshly sorted SP and non SP fractions of HeLa cells. Considering that the expression of Oct3/4 mainly occurs in the nucleus, hematoxylin counterstaining of the nucleus was omitted in the immunocytochemical analysis. The results showed that Oct3/4 was mainly expressed in the nucleus, and a small amount of Oct3/4 was found in the cytoplasm. The expression level of Oct3/4 in SP cells was substantially higher than that in non SP cells. CD133 and BCRP were mainly expressed in the cytoplasm, and their expression levels were substantially higher in SP cells compared with non SP cells.

Notably, BCRP was hardly expressed in non SP cells. ALDH 1 was expressed in the cytoplasm of SP and non SP cells, and the expression level was almost the same in SP and non SP cells. These results indicate that the phenotype of SP cells is closely related to the expres sion of BCRP and shows a certain correlation with stem cell related biomarkers, i. e, Oct3/4 and CD133. Cell cycle and apoptosis analyses We analyzed the cell cycle of SP and non SP cells sorted from HeLa cell line. No significant difference was ob served in the cell cycle distribution between SP and non SP cells under normal culture conditions. was significantly higher than that of SP cells and the active cells in SP cells were apparently more than non SP cells, which indicated that the anti apoptosis ability of SP cells was more efficient.

Tumor formation in NOD/SCID mice We tested the tumorigenic potential of SP and non SP cells by tumor incidence, latency, and growth rate. It was evident that with an decreasing number of injected GSK-3 cells, the tumor incidence in NOD/SCID mice decreased, while the latency of tumorigenesis was noticably pro longed and the tumor volume gradually decreased. How ever, there were no statistically significant differences in the above mentioned parameters between SP and non SP cells.