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.
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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: firstname.lastname@example.org
Received February 2, 2013
Accepted May 3, 2013