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The in-vitro spheroid culture induces a more highly differentiated but tumorigenic population from melanoma cell lines

Mo, Jinga*; Sun, Baocuna,c,d*; Zhao, Xiulana,d*; Gu, Qianga,d; Dong, Xueyia; Liu, Zhiyongc; Ma, Yuemeib; Zhao, Nana; Liu, Yanronga; Chi, Jiadonga; Sun, Rana

doi: 10.1097/CMR.0b013e32836314e3

Cancer stem cells (CSCs) have been identified in various malignancies, and different properties have been examined to characterize CSCs: tumorigenicity in immunocompromised mice, stem cell surface markers, label-retaining properties, and proliferation as nonadherent spheres. This study explored the consistency and efficiency among these methods. Among the melanoma cell lines examined (A375, A875, MUM-2b, and MUM-2c), only A375 and MUM-2c grew as nonadherent spheres and continuously propagated in a defined serum-free medium in vitro. Flow cytometry and immunofluorescence analysis indicated that sphere-derived cells contained a smaller proportion of cells expressing the candidate surface markers of melanoma stem cells such as ABCB5, CD133, CD20 and CD271, and a larger proportion of cells expressing melanocytic differentiation markers such as HMB45 and S100 protein, compared with adherent cells. Surprisingly, the more highly differentiated sphere-derived melanoma cells exhibited increased tumorigenic potential in vivo, as indicated by shorter tumor incubation (A375) and smaller number of cells required to initiate tumor formation (A375 and MUM-2c) compared with those of parental cells. Despite the similarity in histopathological characteristics, the expression profile indicated that xenografts derived from sphere-derived melanoma cells exhibited a more tumorigenic phenotype with respect to the stem or the differentiation markers detected by immunohistochemical analysis. Therefore, sphere formation in nonadherent cultures may not be a preferred surrogate in-vitro method for enriching melanoma stem cells according to candidate markers but may be a favorable condition for activating potential CSCs.

Departments of aPathology


cDepartment of Pathology, Tianjin Cancer Hospital

dDepartment of Pathology, Tianjin General Hospital, Tianjin Medical University, Tianjin, China

*Jing Mo, Baocun Sun, and Xiulan Zhao contributed equally contribute to this study.

All supplementary digital content is available directly from the corresponding author.

Correspondence to Baocun Sun, MD, Department of Pathology, Tianjin Medical University, Qi Xiang Tai Road 22, Heping District, Tianjin 300070, People’s Republic of China Tel: +86 13602111192; fax: +86 228 333 6813; e-mail:

Received February 2, 2013

Accepted May 3, 2013

© 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins