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

Abstract

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.

Gene Expression Profiling Identifies Microphthalmia-Associated Transcription Factor (MITF) and Dickkopf-1 (DKK1) as Regulators of Microenvironment-Driven Alterations in Melanoma Phenotype

“…Enhanced self-renewing capacity and tumorigenic potential in vivo have already been demonstrated for melanospheres [11], [12], [17]. High percentage of differentiated cells in melanospheres has also been shown [30], however, opposite results have been obtained as well [17], [18]. Fang et al [11] have shown that patient-derived melanoma spheres that contained tumorigenic cells can be both pigmented or nonpigmented.…”

Lactose‐Functionalized Dendrimers Arbitrate the Interaction of Galectin‐3/MUC1 Mediated Cancer Cellular Aggregation

“…Here, our focus was to use the natural affinity that galectin‐3 has for lactose‐functionalized dendrimers in order to survey galectin‐3‐mediated homotypic aggregation in three different cancer cell lines, thereby gaining insight into the mechanism by which cancer cellular aggregation occurs. A correlation between cancer cells aggregation in vitro and their metastatic potential in vivo has been demonstrated,30, 31 thus suggesting the real value of a therapeutic agent to inhibit galectin‐3‐mediated cellular aggregation. By using PAMAM dendrimers, we were able to fine‐tune the number of lactose groups on the multivalent framework, thus allowing more control over the outcome of the interaction.…”

Novel Genetic Melanoma Vaccines Based on Induced Pluripotent Stem Cells or Melanosphere-Derived Stem-Like Cells Display High Efficacy in a murine Tumor Rejection Model

“…We constructed two vaccine variants. For the first variant, as the source of MSCs, we generated melanospheres, using B16F10 cell culture under nonadherent conditions and medium for suspension culture [13,14]. Cells acquired from the melanospheres displayed a phenotype similar to that of MSCs, as revealed by the decreased expression of MITF and Tyrosinase, the increased expression of vascular endothelial growth factor (VEGF), and high ALDH activity, compared with parental B16F10 cells.…”