Colorectal Cancer Serum Biomarkers-serine Proteases Discovery And Verification

Colorectal cancer (CRC) is one of the most common cancers and characterized by a high incidence and mortality. Despite dramatic improvements in the five-year survival rate of CRC patients diagnosed at the early stage, most patients are diagnosed too late to receive effective medical treatment due to lack of sensitive diagnostic tools. As a result, discovery of effective serum biomarkers plays a critical role in diagonosis and therapy of CRC. Because highly abundant proteins in serum significantly interfere identification of low-abundance proteins, the tumor tissue interstitial fluid (TIF) with high concentration of disease-related proteins and no interference by high-abundance proteins has started to gain populatiry in cancer biomarker study. Moreover, to seek the serum biomarkers which accurately reflect tumor progression, dynamic study with multiple time points shall provide more information for biomarker candidates screening compared with study only at one particular stage. So in order to get samples for dynamic study, we chose ApcMin/+ CRC mouse model for discovery and screening of biomarker candidates, and then verified in the clinical samples to identify CRC serum biomarkers.According to the colon tumor features of ApcMin/+ mouse, the mice were divided into four groups,8,13,18 and 22 weeks. The age-matched wide-type (WT) mice with the same genetic background and treatment were used as the control group. iTRAQ-based quantification proteomics was conducted to survey the TIF proteins from ApcMin/+ and WT mice, A total of 120 differential proteins related to the tumor progression were screened, and by analysis of the protein change patterns during tumor progression,46 proteins that exhibited consecutive changes in abundance were identified, including six serine proteases, chymotrypsin-like elastase 1 (CELA1), chymotrypsin-like elastase 2A (CEL2A), chymopasin, chymotrypsinogen B (CTRB1), trypsin 2 (TRY2), and trypsin 4 (TRY4).Verification by multiple reaction monitor (MRM) suggested the consecutively increased change pattern of the six proteases were ubiquitous in individual TIFs of ApcMin/+ mice, and the immunohistochemistry (IHC) detection in colon tissues and MRM detection in ApcMin/+ mouse sera confirmed the same change patterns as in TIFs. Therefore, abundance changes of some proteins in TIFs could be reflected in sera, and the six serine proteases could serve as diagonistic serum biomarkers in ApcMin/+ mice.Four serine proteases, CELA1, CEL2A, CTRL (human homologue of chymopasin) and TRY2 were further successfully verified in serum of CRC patients, and the elevated levels of the four proteases could be observed in sera of CRC patients compared with the healthy controls. Receiver Operating Characteristic (ROC) analysis illustrated that the combination of CELA1 and CTRL reached the best diagnostic performance, and IHC against CELA1 and CTRL in colon tumors and their corresponding normal tissues from 80 CRC patients confirmed the high expressions in tumor tissues.Taken together, the combination of identification of TIF proteins in ApcMin/+ mouse and verification in human clinical samples demonstrated four serine proteases, CELA1, CEL2A, CTRL and TRY2, were indicative of CRC progression.