A New Strategy for Biomarker Discovery Applied to Melanoma

Melanoma is a malignant tumor arising from melanocytes. Metastasis is associated with poor prognosis. Early detection may thus tremendously improve patient survival. We have established a novel strategy for the identification of early biomarkers. This strategy is based on proteome profiling and secretome analysis of purified primary cells. Tumor associated stroma cells eventually secrete proteins which may act as powerful tumor promoters. This cooperativity contributed by stromal cells is reversible and may thus be directly accessible to therapeutic intervention. The onset of these characteristic events seems to precede tumor progression. Thus, proteins specifically secreted by these cells may serve as early disease biomarkers. Due to the leaky nature of newly formed blood vessels and the increased hydrostatic pressure within tumors, secreted proteins are most plausibly shed into the blood. We have already identified characteristic aberrations of melanoma-associated fibroblasts including candidate biomarkers by mass spectrometry. The present proposal is based on three different model systems, i.e. established cultured cell lines, animal model systems and clinical human samples. We will analyze stroma cells in addition to tumor cells out of all three model systems. Functional studies focusing on cell cooperativity will employ co-cultures, which mimic the in vivo interaction between cancer cells and stroma cells before and after the onset of cancer invasion. In conclusion, the presently outlined proposal will identify diagnostic marker proteins aiding early detection of metastatic melanoma and improve our understanding of pathomechanisms involving the microenvironment to enable the design of novel therapeutic strategies.

The main aim of this project was the identification of early biomarkers for melanoma. A strong motivation had been the finding that genetically normal stroma cells which are in the direct neighbourhood of melanoma cells show functional aberrations which may result in characteristic protein secretion patterns. Such secreted potential marker proteins were successfully identified by us and evaluated for their potential use as biomarkers.True technical challenges of this project were to isolate and analyse the secreted proteins of primary melanoma-associated cells in a representative way. In order to achieve that, we have developed a self-designed laboratory information and management system (LIMS) recording a complete history and all information of each sample which may be or relevance for future interpretation. Furthermore, we did a lot of systematic studies giving us information about the specificity of the secreted proteins as well as the relation of functional cell states to the actual secretome of cells. To that aim we have treated normal skin fibroblasts with the inflammatory cytokine interleukin 1-beta (Il-1beta) to record proteins indicative for inflammation. The functional signature obtained from this study was used to interpret the data obtained from primary melanoma-associated cells. In order to systematically assess the potential specificity of the secreted proteins with respect to cell and tissue origin we have also analysed primary human fibroblasts from bone marrow, liver and lung in a similar fashion. All these data have been collected in a self-designed database for evaluation.The analysis of the secreted proteins using nano-flow liquid chromatography coupled to an ion trap mass spectrometer typically resulted in the identification of more than 100 different genuinely secreted proteins per sample. These proteins included extracellular matrix proteins, cytokines, growth factors and other important signalling molecules. Indeed we were able to identify secretome signatures characteristic for inflammation and, most importantly, to assess these features in tumor-associated cells. Actually we observed apparent differences in protein expression dependent on the tumor type as well as individual variations. In a consequent step we tried to assess whether some candidate proteins would be found increased in blood samples of melanoma patients. After obtaining approval from the Ethics Committee of the MUW we collected more than 100 blood samples from melanoma patients as well as patients suffering from other skin diseases for controls. In a collaboration project with the Institute of Immunology we evaluated ELISA and Luminex assays for four candidate molecules. However, critical evaluation of these assays revealed matrix-effects strongly interfering with an appropriate quantification of these molecules. Alternative techniques such as multiple reaction monitoring are currently developed and evaluated by us.