Abstract
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.
Highlights
The most significant opportunity to improve response rates for targeted therapeutics in solid tumors resides in the ability to accurately match patient specific molecular alterations to drugs that antagonize those alterations
Using an antibody proximity-based technology (VeraTag), we describe a novel approach for the detection and quantification of the hepatocyte growth factor (HGF)-c-MET ligand-receptor complex in formalin-fixed paraffin embedded (FFPE) specimens including cell lines and human carcinoma tissues
The tissue section is illuminated with red light (670 nm) which triggers the SA-MB photosensitizer to convert dissolved O2 in the buffer to reactive singlet oxygen (1O2). This reactive state of the oxygen in turn cleaves thioether bonds linking the fluorescein- reporter to conjugated reporter antibodies that are bound in close proximity to the biotin-conjugated antibodies
Summary
The most significant opportunity to improve response rates for targeted therapeutics in solid tumors resides in the ability to accurately match patient specific molecular alterations to drugs that antagonize those alterations. Not all of the intricate details are known, there are a series of key steps leading to receptor activation and subsequent signaling of cell growth and proliferation. These key steps, ligand-receptor binding, receptor dimerization, and receptor transphosphorylation resulting in the production of substrate and adaptor protein binding sites, can be measured as potential indicators of receptor activation. One can measure total receptor expression by IHC or FISH, or total ligand levels by either IHC or ELISA, these are not direct indicators of receptor activation
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