Non-small cell lung cancer (NSCLC) is characterised by early dissemination of tumor cells resulting in a strikingly reduced overall survival despite complete surgical removal of the primary tumour. It has been suggested that proteases contribute to this apparent aggressiveness of lung cancer because they are involved in a variety of mechanisms associated with tumor progression such as invasion, migration and angiogenesis. To investigate the significance of protease expression and to identify potentially co-regulated molecules during early dissemination and in minimal residual disease, we performed cDNA array analysis of single disseminated cancer cells or small cell clusters isolated from bone marrow and lymph nodes of NSCLC patients.
We obtained macroscopically tumor free lymph nodes and bone marrow aspirates from patients with operable NSCLC and enriched single disseminated cancer cells and small cell clusters by density gradient centrifugation. Subsequently, the freshly prepared cell suspensions were stained with an antibody against the epithelial surface molecule EpCAM and single positive tumour cells were isolated by micromanipulation. After global amplification of the single cell cDNA and non-radioactive labelling proteinase expression was assessed using a cDNA array consisting of several matrix metalloproteases, cathepsins, caspases, kallikreins and other serine / cysteine proteases. Until now we could isolate 46 EpCAM+ cells from 72 lymph nodes and 19 EpCAM+ cells from 71 bone marrow aspirates. For 30 cells the epithelial origin could be confirmed by the co-expression of several epithelial markers or by expression of the tumor specific MAGE antigens. Hybridisation of these cells on our protease cDNA array revealed the expression of various proteases in single cells and small cell clusters. Particularly, serine proteases, cathepsins and other cysteine proteinases are frequently expressed, while, contrary to our expectations, the transcripts of matrix metalloproteases (MMP) were rarely detected in the analysed cells