Functional features of cancer stem cells in melanoma cell lines

Abstract

Background: Recent evidence suggests a subset of cells within a tumor with "stem-like" characteristics. These cells are able to transplant tumors in immunodeficient hosts. Distinct from non-malignant stem cells, cancer stem cells (CSC) show low proliferative rates, high self-renewing capacity, propensity to differentiate into actively proliferating tumor cells, and resistance to chemotherapy or radiation. They are often characterized by elevated expression of stem cell surface markers, in particular CD133, and sets of differentially expressed stem cell-associated genes. CSC are usually rare in clinical specimens and hardly amenable to functional studies and gene expression profiling. In this study, a panel of heterogenous melanoma cell lines was screened for typical CSC features.

Methods: Nine heterogeneous metastatic melanoma cell lines including D10 and WM115 were studied. Cell lines were phenotyped using flow cytometry and clonogenic assays were performed by limiting dilution analysis on magnetically sorted cells. Spheroidal growth was investigated in pretreated flasks. Gene expression profiles were assessed by using real-time rt-PCR and DNA microarrays. Magnetically sorted tumor cells were subcutaneously injected into the flanks of immunodeficient mice. Comparative immunohistochemistry was performed on xenografts and primary human melanoma sections.

Results: D10 cells expressed CD133 with a significantly higher clonogenic capacity as compared to CD133- cells. Na8, D10, and HBL cells formed spheroids on poly-HEMA-coated flasks. D10, Me39, RE, and WM115 cells expressed at least 2 of the 3 regulatory core transcription factors SOX2, NANOG, and OCT4 involved in the maintenance of stemness in mesenchymal stem cells. Gene expression profiling on CD133+ and CD133- D10 cells revealed 68 up- and 47 downregulated genes (+/-1.3 fold). Two genes, MGP and PROM1 (CD133), were outstandingly upregulated. CD133+ D10 cells formed tumors in NSG mice contrary to CD133- cells and CD133 expression was detected in xenografts and primary human melanoma sections using immunohistochemistry.

Conclusions: Established melanoma cell lines exhibit, to variable extents, the typical features of CSCs. The tumorigenic cell line D10, expressing CD133 and growing in spheroids and might qualify as a potential model of melanoma CSCs.

Figure 1

Expression of CD133 and CD117 in melanoma cell lines. Cell lines were stained with APC-and PE-conjugated monoclonal antibodies against the CD133 and CD117 epitope. The figure shows the dotplots of A: D10, B: Me39, C: RE, D: Na8, E: Me59 and F: HBL. Corresponding statistics are shown in Table 3.

Figure 2

Na8 spheroids in poly-HEMA-coated tissue culture flasks. A: 2× magnification (scale bar 1 mm). B: 4× magnification (scale bar 500 μm). C: 40× magnification (scale bar 50 μm).

Figure 3

CD133 expression in Na8 cells after 2D and 3D culture. Dotplots of CD133-Expression (PE-conjugated) after A: monolayer culture, B: 3D culture (spheroidal growth). Following 3D- culture in pre-treated tissue culture flasks, almost 5% of the Na8 cells stained positive for CD133 compared to 1%.

Figure 4

Limiting dilution analysis and clonogenic assay. A: Limiting dilution analysis. Percentage of positive wells (positive well = 1 cell colony/well) at different cell concentrations of CD133+ and CD133- D10 cells for calculating Poisson’s distribution (Figure 4B). B: Clonogenic capacity of D10 cells. Results of Poisson’s distribution with the frequency of proliferating cells in CD133+ (black column) and CD133- (white column) D10 cells, results expressed as mean percentages ± SD; (*) = p ≤ 0.001.

Figure 5

Tumorigenicity of D10 cells. A: The Change in tumor volume over a period of 12 weeks is shown in the graph. (*) = p ≤ 0.05. Bars represent SD. Tumor progression of CD133+ D10 cells significantly (p ≤ 0.05) differs from unsorted D10 tumors. B: Injection of CD133+ D10 cells into the right flank (R) leads to tumor formation (white arrows) while CD133- D10 cells are not capable of inducing tumor growth. Bilateral injection of unsorted D10 cells is followed by bilateral tumor formation.

Figure 6

Immunohistochemistry of xenografts and patient samples. CD133 expression (brown spots, black arrows) in different tissue samples: A: normal skin section showing a few brown spots. B: primary melanoma tissue section, C: melanoma lymph node metastasis and D: xenograft induced by CD133+ D10 cells, all showing intense positivity for CD133. E: xenograft induced by unsorted D10 cells with less intense CD133+ staining. Scale bar 100 μm.

Figure 7

Categorization of differentially expressed genes, detected in CD133+ D10 cells. Number of genes encompassed with a specific A: molecular function and B: biological process. Black columns: upregulated genes. Gray columns: downregulated genes.