The possibility of performing microarray analysis on limited material has been demonstrated in a number of publications. In this review we approach the technical aspects of mRNA amplification and several important implicit consequences, for both linear and exponential procedures. Amplification efficiencies clearly allow profiling of extremely small samples. The conservation of transcript abundance is the most important issue regarding the use of sample amplification in combination with microarray analysis, and this aspect has generally been found to be acceptable, although demonstrated to decrease in highly diluted samples. The fact that variability and discrepancies in microarray profiles increase with minute sample sizes has been clearly documented, but for many studies this does appear to have affected the biological conclusions. We suggest that this is due to the data analysis approach applied, and the consequence is the chance of presenting misleading results. We discuss the issue of amplification sensitivity limits in the light of reports on fidelity, published data from reviewed articles and data analysis approaches. These are important considerations to be reflected in the design of future studies and when evaluating biological conclusions from published microarray studies based on extremely low input RNA quantities.

Invasiveness is a hallmark of aggressive cancer like malignant melanoma, and factors involved in acquisition or maintenance of an invasive phenotype are attractive targets for therapy. We investigated melanoma phenotype modulation induced by the metastasis-promoting microenvironmental protein S100A4, focusing on the relationship between enhanced cellular motility, dedifferentiation and metabolic changes. In poorly motile, well-differentiated Melmet 5 cells, S100A4 stimulated migration, invasion and simultaneously down-regulated differentiation genes and modulated expression of metabolism genes. Metabolic studies confirmed suppressed mitochondrial respiration and activated glycolytic flux in the S100A4 stimulated cells, indicating a metabolic switch toward aerobic glycolysis, known as the Warburg effect. Reversal of the glycolytic switch by dichloracetate induced apoptosis and reduced cell growth, particularly in the S100A4 stimulated cells. This implies that cells with stimulated invasiveness get survival benefit from the glycolytic switch and, therefore, become more vulnerable to glycolysis inhibition. In conclusion, our data indicate that transition to the invasive phenotype in melanoma involves dedifferentiation and metabolic reprogramming from mitochondrial oxidation to glycolysis, which facilitates survival of the invasive cancer cells. Therapeutic strategies targeting the metabolic reprogramming may therefore be effective against the invasive phenotype.

Background: A limiting factor of cDNA microarray technology is the need for a substantial amount of RNA per labeling reaction. Thus, 20-200 micro-grams total RNA or 0.5-2 micro-grams poly (A) RNA is typically required for monitoring gene expression. In addition, gene expression profiles from large, heterogeneous cell populations provide complex patterns from which biological data for the target cells may be difficult to extract. In this study, we chose to investigate a widely used mRNA amplification protocol that allows gene expression studies to be performed on samples with limited starting material. We present a quantitative study of the variation and noise present in our data set obtained from experiments with either amplified or non-amplified material.

Tumor cells have the ability to exploit stromal cells to facilitate metastasis. By using malignant melanoma as a model, we show that the stroma adjacent to metastatic lesions is enriched in the known metastasis-promoting protein S100A4. S100A4 stimulates cancer cells to secrete paracrine factors, such as inflammatory cytokines IL8, CCL2 and SAA, which activate stromal cells (endothelial cells and monocytes) so that they acquire tumor-supportive properties. Our data establishes S100A4 as an inducer of a cytokine network enabling tumor cells to engage angiogenic and inflammatory stromal cells, which might contribute to pro-metastatic activity of S100A4.

Splenic marginal zone lymphoma (SMZL) is a lymphoma type of putative marginal zone B-cell origin. No specific genetic alterations have yet been demonstrated in SMZL. Clinically, SMZL is a low-grade B-cell non-Hodgkin lymphoma. However, the presence of p53 mutation, 7q22-7q32 deletion or the absence of somatic hypermutations of immunoglobulin genes has been correlated with a worse prognosis. In this study, we analyzed genome-wide gene expression of 24 cases of SMZL using the microarray technique. The AP-1 transcription factors c-jun, junD, junB, and c-fos as well as Notch2 were found to be specifically up-regulated. These data were confirmed by real-time PCR and immunohistochemical staining of tissue sections. The absence of concordant high expression of the MAP kinases, the signaling cascade leading to AP-1 up-regulation, suggests autoregulation of the AP-1 transcription factors and an important role in SMZL oncogenesis. High expression of Notch2, a transcription factor that induces marginal zone B-cell differentiation, is highly suggestive for a marginal zone B-cell origin of SMZL. In addition, SMZL with the 7q deletion showed high expression of TGF-beta1 and low expression of the DNA helicase XPB, a crucial part of the nucleotide excision repair complex, possibly explaining the more aggressive clinical course of those cases.