Sphere formation and self-renewal capacity of melanoma cells is affected by the microenvironment

Abstract

Melanomas contain subsets of cancer stem-like cells with tumor-initiating capacity. The frequency of these cells in the tumor is still a topic of debate. We investigated the phenotypic plasticity of cancer cells grown as melanospheres to elucidate the influence of the microenvironment on some features of melanoma stem-like cells. Cells from surgical specimens of nodular melanoma were grown as anchorage-independent melanospheres in a stem cell medium and as adherent monolayer cultures in the presence of serum. Proliferation and viability were measured by cell counting and an acid phosphatase assay; surface marker expression was evaluated by flow cytometry, and the clonogenic potential of single cells was assessed by growth in soft agar. Patient-derived melanoma cells could be maintained in cell culture for more than 16 months when grown as melanospheres. In the presence of serum, melanospheres completely changed their growth characteristics and formed adherent monolayers. The transition from melanospheres to monolayers was accompanied by an apparent loss of clonogenic potential, an increased proliferation rate, and altered expressions of cell surface markers ABCB5, CD133, and CD49f. These changes, however, were reversible. Compared with adherent monolayer cultures, melanospheres are enriched in cells with clonogenic potential, reflecting the self-renewing capacity of cancer stem-like cells. This clonogenic potential can be lost and regained depending on the growth conditions. Our results demonstrate how easily melanoma cells change their function upon exposure to external stimuli and suggest that the frequency of melanoma stem-like cells strongly depends on the microenvironment.

Phenotypic diversity of patient-derived melanoma populations in stem cell medium

“…Results of this analysis are shown in Table 1 and in our previous reports. 23,25 From markers considered previously as cancer stem cell markers, only CD133 seemed to correlate well with the ability to form melanospheres. The populations containing CD133-positive cells exerted a high melanosphere-forming capacity, whereas those lacking CD133-positive cells formed either aggregates (DMBC12) or single-cell culture (DMBC9) (Figure 2).…”

“…Melanoma specimens classified histopathologically as clinical stages III and IV and various TNM staging 23,25 were obtained during surgical procedures. The study was approved by Ethical Commission of Medical University of Lodz.…”

“…23 After several washes, tumor fragments were minced with scissors and incubated in HBSS (SigmaAldrich, St Louis, MO, USA) supplemented with 3 mM calcium chloride and 1 mg/ml collagenase IV for 2-3 h at 37°C. DNase I (10 μg/ml) was added and cells were filtered through a 70 μm pore size filter.…”

“…23,25 Using transcriptome analyses we have identified MITF and DKK1 as key regulators of microenvironment-dependent phenotypic heterogeneity in vitro. 24 We observed that the ability to form melanospheres in SCM depends on the sample, and cell aggregates and single-cell culture can arise instead of melanospheres.…”

microRNA in the control of stem-like phenotype of cancer cells

“…In the case of melanoma, although the existence of melanoma initiating cells (MICs) with the stem cell phenotype was demonstrated, data regarding typical stem cell marker(s) for melanoma are inconclusive, and no markers distinguishing tumorigenic from nontumorigenic melanoma populations have been identified so far [46]. Melanoma stem-like cells can grow nonadherently as melanospheres in vitro, and this kind of growth can be achieved by the external stimuli in a cancer microenvironment [47]. These motile and invasive cells are weakly pigmented [48] [80]; lung cancer [79] miR-128 BMI-1 glioma [83] miR-145 Oct4, Sox2, c-Myc prostate cancer [87], melanoma [88] miR-150 c-Myb, Bcl-2, cyclin D1 liver cancer [103] miR-17cluster c-Myc, E2F, Rb, cyclin D1 breast cancer [108]; lung cancer [109] miR-181 CDX2, GATA-6, NLK liver cancer [105]; breast cancer [107] miR-199 HES1 medulloblastoma [100]; ovarian cancer [101]; and hepatocellular carcinoma [102] miR-200b Suz12 breast cancer [63] miR-200c BMI-1 breast cancer [82] miR-203 ZEB1 hepatocellular carcinoma [98]; prostate cancer [99] miR-302 cyclin D1 skin cancers [91] mir-34 Notch, Bcl-2, CD44 pancreatic cancer [92]; breast cancer [93]; prostate cancer [97] Table 2. microRNA, whose aberrant expression in cancer cells leads to the acquisition of the stem-like phenotype.…”