Characterization of de novo synthesized proteins released from human colorectal tumour explants

Tumours release many proteins into their microenvironment. These proteins may enter the blood stream and have value as cancer biomarkers. We examined the range of proteins released by colorectal cancer (CRC) liver metastasis (LM) specimens and normal colon mucosa during 16 h culture as explants in the presence of [35S]-methionine. Proteins released into the conditioned media were isolated and separated by 2-DE and detected by CBB stain and de novo synthesized proteins by autoradiography. The majority of proteins released by CRC LM explants in short-term culture were plasma proteins from tumour interstitial fluid and tissue breakdown products including mitochondrial and nuclear proteins from pre-existing necrotic cells within the tumours. De novo synthesized proteins were present at a lower abundance and included a high proportion of cytoplasmic proteins in addition to classically secreted proteins. Many cytoplasmic proteins were also present in the autoradiograph secretomes of four CRC cell lines examined, despite high cell viability (>97%), suggestive of an alternative release mechanism. The secretome profiles varied significantly between different patients, and also between different cell lines, despite low intra-experimental variation. Quantitative analysis of the autoradiograph secretome profiles prepared from tumour and normal colon mucosa tissues revealed 32 protein spots that were differentially abundant between the normal and cancer tissue secretome, including desmocollin-2 and fibrinogen gamma chain, which were upregulated and downregulated in the CRC LM secretomes, respectively. Further characterization of de novo synthesized proteins released from human tumours may help to discover a novel set of serological markers for CRC.