Molecular characterization of signalling pathways in cancer stem cells

To avoid artefacts introduced by culturing cells for extended periods of time, it is crucial to use low-passage patient-derived tumour cells. The ability to enrich, isolate and assay sub-populations of cells that behave as cancer stem cells (CSCs) from these primary cell lines is essential before performing characterizations such as gene-expression profiling. We have isolated cells from glioblastomas which show characteristics of CSCs. Although glioblastomas contain only a relatively small amount of putative CSCs, these cells express many genes which seem to be worthy targets for future therapies.


Introduction
Al-Hajj et al were the first to identify and prospectively isolate a minority sub-population of cells from human solid tumours that contained all of the in vivo tumour-forming abilities. The tumourigenic cell population was identified based on its cell surface phenotype. This population could initiate tumours in immuno-compromised mice with as few as 200 cells, while as many as 500,000 or more of the remaining cells in the tumour did not initiate new tumours in mice [1].
Fang et al have shown that upon culturing of metastatic melanoma cell suspensions under appropriate conditions, a subset of cells could be propagated as non-adherent spheres, which could then be induced to differentiate in vitro and to generate tumours in vivo [2]. The ability to acutely isolate and assay sub-populations of cells from tumours that behave as cancer stem cells (CSCs) is essential before performing characterizations such as gene-expression profiling, to avoid artefacts introduced by culturing cells for extended periods of time.

Tissue samples
Glioblastoma (GBM) tissues were obtained intra-surgically. A trained pathologist evaluated stage and grading of the removed tumour tissue. For our experiments, we only cultivated cells from tissues containing at least 90% tumour cells as determined by H&E staining (Figure1A-C). In a more elaborate diagnosis, we determined glial origin (GFAP) (Figure1D), mitotic activity (KI-67) (Figure1E) and neoplastic character (MAP2C) (Figure1F).

In vitro generation of neurospheres
Tissues that met our criteria were used for low-passage cell culture. We enriched CSCs using embryonic stem (ES) cell medium supplemented with FGF2 [3]. Cells cultured under these conditions began to change their morphology towards a colony-forming phenotype ( Figure 2). This phenotype was similar to that of hES cells cultured under the same conditions. Further analysis showed that the phenotype remained stable for at least four weeks and was reversible by FGF withdrawal.
To show the de-differentiation towards a more primitive neural phenotype, we performed immunofluorescence microscopy of cells from the normal medium and ES medium with NESTIN.

Cell sorting
To investigate if the treated cells were enriched for putative cancer stem cells, we employed magnetic sorting with a beadcoupled anti-CD133 antibody. Enrichment of CSCs by selective cell culture methods resulted in higher yields of CD133+ cells ( Figure 4A). This enrichment was time dependent, with its maximum yield after 16 days of stimulation ( Figure 4B).
To determine the quality of enrichment, we kept the cells from the positive and negative fractions in culture for four days and stained them with a fluorescence coupled anti-CD133 antibody ( Figure 5).

Pathway analysis
To identify the mechanisms and pathways underlying CSCs, we conducted RNA-expression analysis using the Illumina platform. For expression analysis, we used CD133+, CD133− and bulk cells from the GBM1207 cell line and compared this data to the expression profile of hES lines H1/H9 ( Figure 6). Expression analysis revealed a group of 11 genes that were overexpressed and known to play a role in the progression of various malignancies (Figure 7). We validated the differential expression of eight selected marker genes from the expression analysis by real-time PCR ( Figure  8). All markers were found significantly (>1.5-fold) overexpressed in CD133+ cells compared to the respective negative fraction.

Conclusion
Still only little is understood about the origin and nature of CSCs [4]. From our findings, we conclude that although GBMs contain only a relatively small amount of putative CSCs, these cells express many genes, which seem to be worthy targets for future therapies. Among these genes, we found important signalling molecules for angiogenesis, apoptosis, multi-drug resistance and metastasis.

Figure 3: We stained both, adherent monolayer (A) and colony (B) grown cells against Nestin. Nestin is a type VI intermediate filament, which is commonly used as a marker for neuronal precursor cells. Untreated cells do not express Nestin (C), while it was expressed in all spheres (D-F).
ecancer 2008, 2:115     CD133− counterparts. Additionally, these CD133+ cells share important pathways with human ES cells, which help explaining self-renewal capacity, drug-resistance and other properties credited to stem cells and cancer.