Tumor stem-like cells isolated from MMQ cells resist to dopamine agonist treatment
Introduction
The tumor stem-like cells (TSLCs) hypothesis supposes that neoplasms contain a small population of self-sustaining cells with the exclusive ability to self-renew and maintain the tumors. These TSLCs not only have the capacity to divide and expand TSLCs pool but also to be associated with drug resistance of tumor cells.(Clarke et al., 2006; Frank et al., 2010). Recently, more and more studies confirmed that TSLCs were present in leukemia and a range of malignant solid tumors(Kreso and Dick, 2014; Lapidot et al., 1994; Tirosh et al., 2016). Although the TSLCs have been studied extensively in malignant cancer, evidence for the presence of these cells in pituitary adenomas has only recently emerged. Xu et al. for the first time isolated the TSLCs from two cases of human pituitary adenomas in 2009(Xu et al., 2009). Recently, a few studies(Chen et al., 2014; Donangelo et al., 2014; Mertens et al., 2015; Peverelli et al., 2017; Wurth et al., 2017) further supported that the TSLCs were present in human and murine pituitary adenomas.
Prolactinoma is the most common subtype of pituitary tumor and accounts for approximately 40%–66% of all pituitary adenomas(Colao and Savastano, 2011; Gillam et al., 2006). Dopamine agonist (DA) therapy is the first choice of the treatment for prolactinoma. DAs treatment, such as bromocriptine (BRC) and cabergoline (CAB), can normalize the serum prolactin level and reduce tumor volume in approximately 75%–90% of cases with prolactinoma mainly via activating cell surface dopamine 2 receptor (D2R)(Casanueva et al., 2006; Melmed et al., 2011; Passos et al., 2009; Su et al., 2012; Verhelst et al., 1999; Wu et al., 2006). In our previous study(Gao et al., 2017), we indeed found a special subtype of tumor cells in human and rat prolactinomas, which showed positive staining of CD133, a novel five-transmembrane cell surface protein originally shown to be a hematopoietic stem cell marker(Miraglia et al., 1997), and low expression of D2R, an important dopamine receptor for DA treatment(Su et al., 2012; Wu et al., 2010). The property of these cells and the role in prolactinomas remain to be determined.
Medical treatment is the first choice for prolactinomas, and only a small group of patients with DA therapeutic intolerance or DA unresponsive tumor need surgical intervention(Casanueva et al., 2006; Colao and Savastano, 2011; Melmed et al., 2011; Verhelst et al., 1999; Wu et al., 2006), which results in less primary tumor cells can be obtained for research. Furthermore, human primary pituitary tumor cells are difficult to be expanded in vitro(Mertens et al., 2015; Wurth et al., 2017), which makes it hard to get enough cells for subsequent experiments. For this reason, MMQ rat prolactinoma cells are traditionally used as a substitute for human prolactin-secreting pituitary adenoma cells in laboratory experiments, mainly because the stable secretion of prolactin and similar biological characteristics with human prolactinomas(Lin et al., 2015). Therefore, MMQ cells were used in our experiments. In this study, we reported that a small subpopulation of tumor sphere-forming cells in MMQ cells possessed characteristics of stem cell. These sphere-forming cells resisted to DA treatment and showed low expression of D2R and PRL. On the contrary, the daughter tumor cells differentiated from these DA-resistant sphere-forming cells restored the sensitivity to DA treatment and showed high expression of D2R and PRL. Furthermore, with gradient concentration of DA treatment, the proportion of CD133 positive staining cells increased gradually while the proportion of D2R positive staining cells decreased. The lower D2R expression and DA resistance in sphere-forming cells may be due to DNA hypermethylation of D2R promoter. Our findings demonstrate that the sphere-forming cells isolated from MMQ cells possess the trait of TSLCs and resist to DA treatment, which offers the opportunity to further investigate the mechanisms underlying tumor recurrence based on TSLCs.
Section snippets
MMQ cells culture
Rat prolactinoma MMQ cell lines, purchased from the American Type Culture Collection (ATCCCRL-10609™; Manassas, VA), were grown in F12 complete medium (Gibco, USA, #11765-062) supplemented with 2.5% fetal bovine serum (Gibco, USA, #10100-147), 15% horse serum (Gibco, USA, #16050-122) and 100U/ml penicillin/streptomycin (Gibco, USA, #15070-063) in a humidified incubator at 37 °C in 5% (v/v) CO2.
MMQ tumor sphere cells culture
To obtain MMQ tumor stem-like cells (MSCs), as described in our previous report(Su et al., 2017). In
Isolation and identification of tumor spheres from MMQ cells
MMQ cells at logarithmic phase were selected to culture in serum-free DMEM/F12 medium containing EGF and bFGF. After being cultured for 2–3 weeks, a small subpopulation of cells was found to grow into spheres (Fig. 1A). Furthermore, we found that the isolated cells from primary tumor spheres could generate secondary spheres in vitro (Fig. 1B). As shown in Fig. 1C, the sphere-forming cells isolated from MMQ cells (MSCs) expressed stem cell markers CD133, Nestin, HCAM, OCT4 and SCA1, which were
Discussion
Tumor stem-like cells has been implicated as cancer-initiating cells in a range of malignant tumor. These cells do not only retain the characteristics of stem cells, but also possess its own unique property(Clarke et al., 2006; Frank et al., 2010). However, people know little about tumor stem-like cells in prolactinomas. In this study, we observed spheres formation in MMQ cells and confirmed the presence of sphere-forming cells (MSCs), which expressed stem cell markers and owned the capacity of
Conclusions
In this study, we confirmed the presence of sphere-forming cells (MSCs) in MMQ cells, which possessed the trait of TSLCs and resisted to DA treatment. The research offers the opportunity to further investigate the mechanisms underlying tumor recurrence and drug resistance in prolactinomas based on TSLCs.
Author contribution
L.C and J.C designed and performed the in vitro and in vivo experiments, analyzed the data, prepared the figures. J.L.L, Q.L, X.B.C, and L.L.Z contributed to the performance of the experiments. J.S.W and W.M.Z collected clinical information about prolactinoma patients. C.D.W and Z.P.S conceived and designed research and wrote the manuscript.
Funding
This project was supported by grants from the National Natural Science Foundation of China (81201687); Zhejiang Provincial Natural Science Foundation of China (LY17H160052, LY19C070002).
CRediT authorship contribution statement
Lin Cai: Project administration, Investigation, Conceptualization, Writing – original draft, Writing – review & editing. Jian Chen: Investigation, Writing – original draft, Visualization. Jianglong Lu: Investigation, Visualization. Qun Li: Investigation, Visualization. Xianbin Chen: Investigation. Linlin Zhang: Investigation. Jinsen Wu: Resources. Weiming Zheng: Resources. Chengde Wang: Supervision, Funding acquisition, Writing – review & editing. Zhipeng Su: Supervision, Funding acquisition,
Declaration of competing interest
The authors have nothing to disclose.
Acknowledgements
We thank Jianmin Li and Kate Huang (Department of Pathology, First Affiliated Hospital of Wenzhou Medical University, China) for assistance in pathological diagnosis of prolactinoma.
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