Culture and identification of the tumor stem-like cells isolated from rat prolactinoma MMQ cell lines

Abstract

Objectives To explore the in vitro culture condition of the tumor stem-like cells isolated from rat prolactinoma MMQ cell lines and to observe their characteristics of proliferation, differentiation, and formation of planted tumor. Methods MMQ cells were cultured with serum-free medium suspension of containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), and the tumor stem cell spheres (briefly cell spheres) were obtained. Clone culture was conducted and its proliferative capacity was observed. Then flow cytometry was used to detect the CD133-positive cell ratio. Western blot was used to detect cell sphere CD133 and nestin expressions. Immunofluorescence technique was used to detect the expressions of cell sphere CD133, nestin, homing cell adhesion molecules (HCAM/CD44), octamer binding transcription factor 4 (OCT4), stem cell antigen 1 (SCA1), as well as their expressions of differentiated cell glial fibrillary acidic protein (GFAP), β-tublinIII, and S100 protein. Twenty-seven female nude mice were randomly divided into 3 groups: a cell sphere planting group, a MMQ cell sphere planting group, and a normal control group (n=9 in each group). Six weeks after subcutaneous plantation, the tumor volume and serum prolactin (PRL) levels were measured. The sections of tumor tissue were detected by the HE staining and immunohistochemistry. Results Under the condition of suspension culture of serum-free medium, only a few cells survived and formed cell spheres. After being blown away, cells could clone and proliferate into cell spheres. The proportion of CD133 positive cells was 1.10±0.17%. CD133, nestin, HCAM/CD44, OCT4, and SCA1 on the cell spheres showed positive expression. After the differentiation of cell spheres, the cells expressed GFAP, β-tublinIII, and S10 protein. The volume of tumor formation in the cell sphere planting group was 1262.2±279.1 mm3, and the MMQ cell sphere planting group was 495.9±158.9 mm3 (P<0.05). The serum PRL of the tumor-bearing nude rats in the cell sphere planting group (median, P25, P75) was 36.08 (34.23, 41.42) ng/ml, and the MMQ cell sphere planting group was 21.51 (20.39, 22.98) ng/ml respectively, and they both higher than 0.26 (1.03-0.15) ng/ml in the normal control group (P<0.05). The HE staining of tumor tissue showed the similar characteristics of the rat pituitary adenoma. Immunohistochemical staining showed that the PRL and nerve synaptophysin (Syn) expressions were positive. Conclusions In MMQ cell lines of rat pituitary prolactin adenoma, a small amount cells with tumor stem-like cell characteristics exist, and they can be cultured successfully In vitro. Key words: Prolactinoma; In vitro; In vivo; MMQ cell line; Tumor stem-like cells; Rats