Detection of hepatocyte growth factor (HGF) ligand-c-MET receptor activation in formalin-fixed paraffin embedded specimens by a novel proximity assay

PLoS One. 2011 Jan 21;6(1):e15932. doi: 10.1371/journal.pone.0015932.


Aberrant activation of membrane receptors frequently occurs in human carcinomas. Detection of phosphorylated receptors is commonly used as an indicator of receptor activation in formalin-fixed paraffin embedded (FFPE) tumor specimens. FFPE is a standard method of specimen preparation used in the histological analysis of solid tumors. Due to variability in FFPE preparations and the labile nature of protein phosphorylation, measurements of phospho-proteins are unreliable and create ambiguities in clinical interpretation. Here, we describe an alternative, novel approach to measure receptor activation by detecting and quantifying ligand-receptor complexes in FFPE specimens. We used hepatocyte growth factor (HGF)-c-MET as our model ligand-receptor system. HGF is the only known ligand of the c-MET tyrosine kinase receptor and HGF binding triggers c-MET phosphorylation. Novel antibody proximity-based assays were developed and used to detect and quantify total c-MET, total HGF, and HGF-c-MET ligand-receptor interactions in FFPE cell line and tumor tissue. In glioma cells, autocrine activation of c-MET by HGF-c-MET increased basal levels of c-MET phosphorylation at tyrosine (Tyr) 1003. Furthermore, HGF-c-MET activation in glioma cell lines was verified by Surface Protein-Protein Interaction by Crosslinking ELISA (SPPICE) assay in corresponding soluble cell lysates. Finally, we profiled levels ofc-MET, HGF, and HGF-c-MET complexes in FFPE specimens of human Non-Small Cell Lung Cancer (NSCLC), Gastric Cancer, Head and Neck Squamous Cell, and Head and Neck Non-Squamous Cell carcinomas. This report describes a novel approach for the detection and quantification of ligand-receptor interactions that can be widely applied to measure receptor activation in FFPE preclinical models and archived FFPE human tissue specimens.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biopsy
  • Cross-Linking Reagents
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Formaldehyde
  • Hepatocyte Growth Factor / analysis
  • Hepatocyte Growth Factor / metabolism*
  • Humans
  • Ligands
  • Neoplasm Proteins / analysis*
  • Neoplasms / pathology*
  • Paraffin Embedding
  • Protein Binding
  • Proto-Oncogene Proteins c-met / analysis
  • Proto-Oncogene Proteins c-met / metabolism*
  • Receptors, Growth Factor / analysis
  • Receptors, Growth Factor / metabolism*


  • Cross-Linking Reagents
  • Ligands
  • Neoplasm Proteins
  • Receptors, Growth Factor
  • Formaldehyde
  • Hepatocyte Growth Factor
  • MET protein, human
  • Proto-Oncogene Proteins c-met