Soluble E-cadherin as a serum biomarker candidate: elevated levels in patients with late-stage colorectal carcinoma and FAP.

Various strategies have been tested to identify serum biomarkers in patients with cancer. Recently, the entire of proteins released by cultured tumor cells into the media, the so-called secretome, has been suggested as a promising source for biomarker discovery. Ectodomains of membrane proteins cleaved from the cell surface represent a surprisingly abundant and apparently stable subset of this subproteome. Aiming for the detection of serum biomarkers for patients with colorectal cancer (CRC), we have previously detected significant amounts of the soluble form of E-cadherin in the secretomes of CRC cells. Here, we report a comprehensive analysis of sE-cadherin levels in sera from patients with CRC, colorectal adenoma, inflammatory bowel disease and familial adenomatous polyposis (FAP). Whereas mean sE-cadherin levels in patients with inflammatory bowel disease (mean: 4.7 µg/ml, SD: 1.5 µg/ml), with adenomas (mean: 4.6 µg/ml, SD: 3.0 µg/ml) and early stage cancers (mean: 4.9 µg/ml, SD: 4.7 µg/ml) do not significantly differ from healthy controls (mean: 4.8 µg/ml, SD: 1.9 µg/ml), patients with Stage III and Stage IV carcinomas display a significant increase (mean: 6.1 µg/ml, SD: 2.6 µg/ml). In individual patients with late-stage CRC, sE-cadherin serum levels directly reflect their disease status over time. These findings suggest a potential application of sE-cadherin as an alternative diagnostic biomarker for monitoring disease particularly in patients with carcinoembryonic antigen negative tumors. In patients with FAP, on the other hand, we also detected a significant increase of serum sE-cadherin levels (mean: 5.8 µg/ml, SD: 2.8 µg/ml), but this was regardless of their tumor load and colectomy status.

Immunoscreening of the extracellular proteome of colorectal cancer cells.

BACKGROUND: The release of proteins from tumors can trigger an immune response in cancer patients involving T lymphocytes and B lymphocytes, which results in the generation of antibodies to tumor-derived proteins. Many studies aim to use humoral immune responses, namely autoantibody profiles, directly, as clinical biomarkers. Alternatively, the antibody immune response as an amplification system for tumor associated alterations may be used to indicate putative protein biomarkers with high sensitivity. Aiming at the latter approach we here have implemented an autoantibody profiling strategy which particularly focuses on proteins released by tumor cells in vitro: the so-called secretome. METHODS: For immunoscreening, the extracellular proteome of five colorectal cancer cell lines was resolved on 2D gels, immobilized on PVDF membranes and used for serological screening with individual sera from 21 colorectal cancer patients and 24 healthy controls. All of the signals from each blot were assigned to a master map, and autoantigen candidates were defined based of the pattern of immunoreactivities. The corresponding proteins were isolated from preparative gels, identified by MALDI-MS and/or by nano-HPLC/ESI-MS/MS and exemplarily confirmed by duplex Western blotting combining the human serum samples with antibodies directed against the protein(s) of interest. RESULTS: From 281 secretome proteins stained with autoantibodies in total we first defined the "background patterns" of frequently immunoreactive extracellular proteins in healthy and diseased people. An assignment of these proteins, among them many nominally intracellular proteins, to the subset of exosomal proteins within the secretomes revealed a large overlap. On this basis we defined and consequently confirmed novel biomarker candidates such as the extreme C-terminus of the extracellular matrix protein agrin within the set of cancer-enriched immunoreactivities. CONCLUSIONS: Our findings suggest, first, that autoantibody responses may be due, in large part, to cross-presentation of antigens to the immune system via exosomes, membrane vesicles released by tumor cells and constituting a significant fraction of the secretome. In addition, this immunosecretomics approach has revealed novel biomarker candidates, some of them secretome-specific, and thus serves as a promising complementary tool to the frequently reported immunoproteomic studies for biomarker discovery.