The Novel Specific Anti-Met Monoclonal Antibody For Detecting Met Expression Of Human Colorectal Cancer

The proto-oncogene MET, which encodes the hepatocyte growth factor/scatter factor (HGF/SF) receptor Met, is frequently expressed in the surface of epithelial cells. The tyrosine kinase receptor Met together with its ligand HGF/SF regulates cellular proliferation, migration and differentiation during organ development and tissue homeostasis. Overexpression of Met has been shown to be associated with poor prognosis in a number of solid tumors. In colorectal cancer(CRC), however, the relationship between Met protein level and clinical character has been controversial, which was mostly determined by the lackage of the reliable Met antibody for evaluating Met expression. In our previous study, a novel mouse monoclonal antibody Met4 was developed and validated to specifically reacts with an epitope in the extracellular domain of Met in FFPE tissues. In immunohistochemistry (IHC), immunofluorescence and Western blot analysis, Met4 may react strongly with Met as expressed on the cultured human cancer cells including human CRC cells. Met4 can be worked as a useful reagent for reproducible, specific and sensitive quantification of Met protein expression levels in all fixed mammalian cells.ObjectiveTo investigate the expression level of Met in human colorectal cancer and the relationship between Met expression levels and clinicopathological factors, and explore the effect of Met in the process of invasion and metastasis in CRC.To compare the specificity of Met4 and commercial rabbit anti human Met polyclonal antibody C28(sc-161, Santa Cruz, USA) for detecting the formalin-treated Met protein in the paraffin-embedded CRC specimens.MethodsThe expression levels of Met in formalin-fixed and paraffin-embedded (FFPB) specimens from 78 primary CRC and 6 adenoma were estimated by IHC using Met4 as well as the commercial antibody C28. The relationship between Met expression levels and clinicopathological factors was evaluated. Simultaneously we performed fluorescent-based IHC to compare the reactivity efficacy of Met4 in the colorectal cancer with that of the polyclonal antibody C28.Results1. Met cytoplasm staining results of 78 primary CRC: positive, 46 cases; moderate, 22 cases (the rates of positive expression were 59% and 28.2%, respectively); negative, 10 cases (12.8%). Met cytoplasm staining results of 6 adenoma: positive, 2 cases; moderate, 2 cases; negtive, 2 cases. Normal colon epithelium cells showed negative or weak cytoplasm staining. No significant statistical differences were observed in the cytoplasm staining intensity across clinical subtypes of colorectal cancer.2. Met membrane staining results of 78 primary CRC: When membrane immunostaining score 3 was considered as positive and≤2 as negative, Met membrane expression may be associated with the lymphatic early-stage invasion and metastasis of CRC. stageⅢand stageⅠwere tend to present higher membrane Met staining than cytoplasm Met staining, while the cytoplasm Met staining were mostly appeared in stageⅡand stageⅣpatients.3. In chemochromagen and fluorescent-based IHC analysis by Met4, Met staining exhibited a combined membranous and cytoplasmic pattern, and nuclear staining was not observed. While in the analysis by polyclonal antibody C28, Met staining revealed positive brown signals in nuclei of tumor cells, and fluorescent-based IHC results showed there was non-specificity response with C28.ConclusionThe data from this study demonstrated the specificity and reproducibility of Met4 for assessing Met in CRC primary tumor. The membrane distribution of Met in CRC primary tumor may predict tumor regional metastatic potential. The clinical application of Met4 antibody may be explored to select the potential responders who may be benefit from Met antagonistic drugs.